Stability evaluation of reference genes for gene expression analysis by RT-qPCR in soybean under different conditions.

PubMed

Wan, Qiao; Chen, Shuilian; Shan, Zhihui; Yang, Zhonglu; Chen, Limiao; Zhang, Chanjuan; Yuan, Songli; Hao, Qinnan; Zhang, Xiaojuan; Qiu, Dezhen; Chen, Haifeng; Zhou, Xinan

2017-01-01

Real-time quantitative reverse transcription PCR is a sensitive and widely used technique to quantify gene expression. To achieve a reliable result, appropriate reference genes are highly required for normalization of transcripts in different samples. In this study, 9 previously published reference genes (60S, Fbox, ELF1A, ELF1B, ACT11, TUA5, UBC4, G6PD, CYP2) of soybean [Glycine max (L.) Merr.] were selected. The expression stability of the 9 genes was evaluated under conditions of biotic stress caused by infection with soybean mosaic virus, nitrogen stress, across different cultivars and developmental stages. ΔCt and geNorm algorithms were used to evaluate and rank the expression stability of the 9 reference genes. Results obtained from two algorithms showed high consistency. Moreover, results of pairwise variation showed that two reference genes were sufficient to normalize the expression levels of target genes under each experimental setting. For virus infection, ELF1A and ELF1B were the most stable reference genes for accurate normalization. For different developmental stages, Fbox and G6PD had the highest expression stability between two soybean cultivars (Tanlong No. 1 and Tanlong No. 2). ELF1B and ACT11 were identified as the most stably expressed reference genes both under nitrogen stress and among different cultivars. The results showed that none of the candidate reference genes were uniformly expressed at different conditions, and selecting appropriate reference genes was pivotal for gene expression studies with particular condition and tissue. The most stable combination of genes identified in this study will help to achieve more accurate and reliable results in a wide variety of samples in soybean.

Application of the FLP/FRT system for conditional gene deletion in yeast Saccharomyces cerevisiae.

PubMed

Park, Yang-Nim; Masison, Daniel; Eisenberg, Evan; Greene, Lois E

2011-09-01

The yeast Saccharomyces cerevisiae has proved to be an excellent model organism to study the function of proteins. One of the many advantages of yeast is the many genetic tools available to manipulate gene expression, but there are still limitations. To complement the many methods used to control gene expression in yeast, we have established a conditional gene deletion system by using the FLP/FRT system on yeast vectors to conditionally delete specific yeast genes. Expression of Flp recombinase, which is under the control of the GAL1 promoter, was induced by galactose, which in turn excised FRT sites flanked genes. The efficacy of this system was examined using the FRT site-flanked genes HSP104, URA3 and GFP. The pre-excision frequency of this system, which might be caused by the basal activity of the GAL1 promoter or by spontaneous recombination between FRT sites, was detected ca. 2% under the non-selecting condition. After inducing expression of Flp recombinase, the deletion efficiency achieved ca. 96% of cells in a population within 9 h. After conditional deletion of the specific gene, protein degradation and cell division then diluted out protein that was expressed from this gene prior to its excision. Most importantly, the specific protein to be deleted could be expressed under its own promoter, so that endogenous levels of protein expression were maintained prior to excision by the Flp recombinase. Therefore, this system provides a useful tool for the conditional deletion of genes in yeast. Published in 2011 by John Wiley & Sons, Ltd.

Characterization of Pseudomonas putida Genes Responsive to Nutrient Limitation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Syn, Chris K.; Magnuson, Jon K.; Kingsley, Mark T.

2004-06-01

The low bioavailability of nutrients and oxygen in the soil environment has hampered successful expression of biodegradation/biocontrol genes that are driven by promoters highly active during routine laboratory conditions of high nutrient- and oxygen-availability. Hence, in the present study, expression of the gus-tagged genes in 12 Tn5-gus mutants of the soil microbe Pseudomonas putida PNL-MK25 was examined under various conditions chosen to mimic the soil environment: low carbon, phosphate, nitrate, or oxygen, and in the rhizosphere. Based on their expression profiles, three nutrient-responsive mutant (NRM) strains, NRM5, NRM7, and NRM17, were selected for identification of the tagged genes. In themore » mutant strain NRM5, expression of the glutamate dehydrogenase (gdhA) gene was increased between 4.9- to 26.4-fold under various low nutrient conditions. In NRM7, expression of the novel NADPH:quinone oxidoreductase-like (nql) gene was consistently amongst the highest and was synergistically upregulated by low nutrient and anoxic conditions. The cyoD gene in NRM17, which encodes the fourth subunit of the cytochrome o ubiquinol oxidase complex, had decreased expression in low nutrient conditions but its absolute expression levels was still amongst the highest. Additionally, it was independent of oxygen availability, in contrast to that in E. coli.« less

Spectral biclustering of microarray data: coclustering genes and conditions.

PubMed

Kluger, Yuval; Basri, Ronen; Chang, Joseph T; Gerstein, Mark

2003-04-01

Global analyses of RNA expression levels are useful for classifying genes and overall phenotypes. Often these classification problems are linked, and one wants to find "marker genes" that are differentially expressed in particular sets of "conditions." We have developed a method that simultaneously clusters genes and conditions, finding distinctive "checkerboard" patterns in matrices of gene expression data, if they exist. In a cancer context, these checkerboards correspond to genes that are markedly up- or downregulated in patients with particular types of tumors. Our method, spectral biclustering, is based on the observation that checkerboard structures in matrices of expression data can be found in eigenvectors corresponding to characteristic expression patterns across genes or conditions. In addition, these eigenvectors can be readily identified by commonly used linear algebra approaches, in particular the singular value decomposition (SVD), coupled with closely integrated normalization steps. We present a number of variants of the approach, depending on whether the normalization over genes and conditions is done independently or in a coupled fashion. We then apply spectral biclustering to a selection of publicly available cancer expression data sets, and examine the degree to which the approach is able to identify checkerboard structures. Furthermore, we compare the performance of our biclustering methods against a number of reasonable benchmarks (e.g., direct application of SVD or normalized cuts to raw data).

MARQ: an online tool to mine GEO for experiments with similar or opposite gene expression signatures.

PubMed

Vazquez, Miguel; Nogales-Cadenas, Ruben; Arroyo, Javier; Botías, Pedro; García, Raul; Carazo, Jose M; Tirado, Francisco; Pascual-Montano, Alberto; Carmona-Saez, Pedro

2010-07-01

The enormous amount of data available in public gene expression repositories such as Gene Expression Omnibus (GEO) offers an inestimable resource to explore gene expression programs across several organisms and conditions. This information can be used to discover experiments that induce similar or opposite gene expression patterns to a given query, which in turn may lead to the discovery of new relationships among diseases, drugs or pathways, as well as the generation of new hypotheses. In this work, we present MARQ, a web-based application that allows researchers to compare a query set of genes, e.g. a set of over- and under-expressed genes, against a signature database built from GEO datasets for different organisms and platforms. MARQ offers an easy-to-use and integrated environment to mine GEO, in order to identify conditions that induce similar or opposite gene expression patterns to a given experimental condition. MARQ also includes additional functionalities for the exploration of the results, including a meta-analysis pipeline to find genes that are differentially expressed across different experiments. The application is freely available at http://marq.dacya.ucm.es.

Large-scale gene expression profiling data for the model moss Physcomitrella patens aid understanding of developmental progression, culture and stress conditions.

PubMed

Hiss, Manuel; Laule, Oliver; Meskauskiene, Rasa M; Arif, Muhammad A; Decker, Eva L; Erxleben, Anika; Frank, Wolfgang; Hanke, Sebastian T; Lang, Daniel; Martin, Anja; Neu, Christina; Reski, Ralf; Richardt, Sandra; Schallenberg-Rüdinger, Mareike; Szövényi, Peter; Tiko, Theodhor; Wiedemann, Gertrud; Wolf, Luise; Zimmermann, Philip; Rensing, Stefan A

2014-08-01

The moss Physcomitrella patens is an important model organism for studying plant evolution, development, physiology and biotechnology. Here we have generated microarray gene expression data covering the principal developmental stages, culture forms and some environmental/stress conditions. Example analyses of developmental stages and growth conditions as well as abiotic stress treatments demonstrate that (i) growth stage is dominant over culture conditions, (ii) liquid culture is not stressful for the plant, (iii) low pH might aid protoplastation by reduced expression of cell wall structure genes, (iv) largely the same gene pool mediates response to dehydration and rehydration, and (v) AP2/EREBP transcription factors play important roles in stress response reactions. With regard to the AP2 gene family, phylogenetic analysis and comparison with Arabidopsis thaliana shows commonalities as well as uniquely expressed family members under drought, light perturbations and protoplastation. Gene expression profiles for P. patens are available for the scientific community via the easy-to-use tool at https://www.genevestigator.com. By providing large-scale expression profiles, the usability of this model organism is further enhanced, for example by enabling selection of control genes for quantitative real-time PCR. Now, gene expression levels across a broad range of conditions can be accessed online for P. patens. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.

Integrating mean and variance heterogeneities to identify differentially expressed genes.

PubMed

Ouyang, Weiwei; An, Qiang; Zhao, Jinying; Qin, Huaizhen

2016-12-06

In functional genomics studies, tests on mean heterogeneity have been widely employed to identify differentially expressed genes with distinct mean expression levels under different experimental conditions. Variance heterogeneity (aka, the difference between condition-specific variances) of gene expression levels is simply neglected or calibrated for as an impediment. The mean heterogeneity in the expression level of a gene reflects one aspect of its distribution alteration; and variance heterogeneity induced by condition change may reflect another aspect. Change in condition may alter both mean and some higher-order characteristics of the distributions of expression levels of susceptible genes. In this report, we put forth a conception of mean-variance differentially expressed (MVDE) genes, whose expression means and variances are sensitive to the change in experimental condition. We mathematically proved the null independence of existent mean heterogeneity tests and variance heterogeneity tests. Based on the independence, we proposed an integrative mean-variance test (IMVT) to combine gene-wise mean heterogeneity and variance heterogeneity induced by condition change. The IMVT outperformed its competitors under comprehensive simulations of normality and Laplace settings. For moderate samples, the IMVT well controlled type I error rates, and so did existent mean heterogeneity test (i.e., the Welch t test (WT), the moderated Welch t test (MWT)) and the procedure of separate tests on mean and variance heterogeneities (SMVT), but the likelihood ratio test (LRT) severely inflated type I error rates. In presence of variance heterogeneity, the IMVT appeared noticeably more powerful than all the valid mean heterogeneity tests. Application to the gene profiles of peripheral circulating B raised solid evidence of informative variance heterogeneity. After adjusting for background data structure, the IMVT replicated previous discoveries and identified novel experiment-wide significant MVDE genes. Our results indicate tremendous potential gain of integrating informative variance heterogeneity after adjusting for global confounders and background data structure. The proposed informative integration test better summarizes the impacts of condition change on expression distributions of susceptible genes than do the existent competitors. Therefore, particular attention should be paid to explicitly exploit the variance heterogeneity induced by condition change in functional genomics analysis.

DigOut: viewing differential expression genes as outliers.

PubMed

Yu, Hui; Tu, Kang; Xie, Lu; Li, Yuan-Yuan

2010-12-01

With regards to well-replicated two-conditional microarray datasets, the selection of differentially expressed (DE) genes is a well-studied computational topic, but for multi-conditional microarray datasets with limited or no replication, the same task is not properly addressed by previous studies. This paper adopts multivariate outlier analysis to analyze replication-lacking multi-conditional microarray datasets, finding that it performs significantly better than the widely used limit fold change (LFC) model in a simulated comparative experiment. Compared with the LFC model, the multivariate outlier analysis also demonstrates improved stability against sample variations in a series of manipulated real expression datasets. The reanalysis of a real non-replicated multi-conditional expression dataset series leads to satisfactory results. In conclusion, a multivariate outlier analysis algorithm, like DigOut, is particularly useful for selecting DE genes from non-replicated multi-conditional gene expression dataset.

Transcriptional profiling reveals regulated genes in the hippocampus during memory formation

NASA Technical Reports Server (NTRS)

Donahue, Christine P.; Jensen, Roderick V.; Ochiishi, Tomoyo; Eisenstein, Ingrid; Zhao, Mingrui; Shors, Tracey; Kosik, Kenneth S.

2002-01-01

Transcriptional profiling (TP) offers a powerful approach to identify genes activated during memory formation and, by inference, the molecular pathways involved. Trace eyeblink conditioning is well suited for the study of regional gene expression because it requires the hippocampus, whereas the highly parallel task, delay conditioning, does not. First, we determined when gene expression was most regulated during trace conditioning. Rats were exposed to 200 trials per day of paired and unpaired stimuli each day for 4 days. Changes in gene expression were most apparent 24 h after exposure to 200 trials. Therefore, we profiled gene expression in the hippocampus 24 h after 200 trials of trace eyeblink conditioning, on multiple arrays using additional animals. Of 1,186 genes on the filter array, seven genes met the statistical criteria and were also validated by real-time polymerase chain reaction. These genes were growth hormone (GH), c-kit receptor tyrosine kinase (c-kit), glutamate receptor, metabotropic 5 (mGluR5), nerve growth factor-beta (NGF-beta), Jun oncogene (c-Jun), transmembrane receptor Unc5H1 (UNC5H1), and transmembrane receptor Unc5H2 (UNC5H2). All these genes, except for GH, were downregulated in response to trace conditioning. GH was upregulated; therefore, we also validated the downregulation of the GH inhibitor, somatostatin (SST), even though it just failed to meet criteria on the arrays. By during situ hybridization, GH was expressed throughout the cell layers of the hippocampus in response to trace conditioning. None of the genes regulated in trace eyeblink conditioning were similarly affected by delay conditioning, a task that does not require the hippocampus. These findings demonstrate that transcriptional profiling can exhibit a repertoire of genes sensitive to the formation of hippocampal-dependent associative memories.

TimesVector: a vectorized clustering approach to the analysis of time series transcriptome data from multiple phenotypes.

PubMed

Jung, Inuk; Jo, Kyuri; Kang, Hyejin; Ahn, Hongryul; Yu, Youngjae; Kim, Sun

2017-12-01

Identifying biologically meaningful gene expression patterns from time series gene expression data is important to understand the underlying biological mechanisms. To identify significantly perturbed gene sets between different phenotypes, analysis of time series transcriptome data requires consideration of time and sample dimensions. Thus, the analysis of such time series data seeks to search gene sets that exhibit similar or different expression patterns between two or more sample conditions, constituting the three-dimensional data, i.e. gene-time-condition. Computational complexity for analyzing such data is very high, compared to the already difficult NP-hard two dimensional biclustering algorithms. Because of this challenge, traditional time series clustering algorithms are designed to capture co-expressed genes with similar expression pattern in two sample conditions. We present a triclustering algorithm, TimesVector, specifically designed for clustering three-dimensional time series data to capture distinctively similar or different gene expression patterns between two or more sample conditions. TimesVector identifies clusters with distinctive expression patterns in three steps: (i) dimension reduction and clustering of time-condition concatenated vectors, (ii) post-processing clusters for detecting similar and distinct expression patterns and (iii) rescuing genes from unclassified clusters. Using four sets of time series gene expression data, generated by both microarray and high throughput sequencing platforms, we demonstrated that TimesVector successfully detected biologically meaningful clusters of high quality. TimesVector improved the clustering quality compared to existing triclustering tools and only TimesVector detected clusters with differential expression patterns across conditions successfully. The TimesVector software is available at http://biohealth.snu.ac.kr/software/TimesVector/. sunkim.bioinfo@snu.ac.kr. Supplementary data are available at Bioinformatics online. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

Using RNA-seq data to select reference genes for normalizing gene expression in apple roots.

PubMed

Zhou, Zhe; Cong, Peihua; Tian, Yi; Zhu, Yanmin

2017-01-01

Gene expression in apple roots in response to various stress conditions is a less-explored research subject. Reliable reference genes for normalizing quantitative gene expression data have not been carefully investigated. In this study, the suitability of a set of 15 apple genes were evaluated for their potential use as reliable reference genes. These genes were selected based on their low variance of gene expression in apple root tissues from a recent RNA-seq data set, and a few previously reported apple reference genes for other tissue types. Four methods, Delta Ct, geNorm, NormFinder and BestKeeper, were used to evaluate their stability in apple root tissues of various genotypes and under different experimental conditions. A small panel of stably expressed genes, MDP0000095375, MDP0000147424, MDP0000233640, MDP0000326399 and MDP0000173025 were recommended for normalizing quantitative gene expression data in apple roots under various abiotic or biotic stresses. When the most stable and least stable reference genes were used for data normalization, significant differences were observed on the expression patterns of two target genes, MdLecRLK5 (MDP0000228426, a gene encoding a lectin receptor like kinase) and MdMAPK3 (MDP0000187103, a gene encoding a mitogen-activated protein kinase). Our data also indicated that for those carefully validated reference genes, a single reference gene is sufficient for reliable normalization of the quantitative gene expression. Depending on the experimental conditions, the most suitable reference genes can be specific to the sample of interest for more reliable RT-qPCR data normalization.

Using RNA-seq data to select reference genes for normalizing gene expression in apple roots

PubMed Central

Zhou, Zhe; Cong, Peihua; Tian, Yi

2017-01-01

Gene expression in apple roots in response to various stress conditions is a less-explored research subject. Reliable reference genes for normalizing quantitative gene expression data have not been carefully investigated. In this study, the suitability of a set of 15 apple genes were evaluated for their potential use as reliable reference genes. These genes were selected based on their low variance of gene expression in apple root tissues from a recent RNA-seq data set, and a few previously reported apple reference genes for other tissue types. Four methods, Delta Ct, geNorm, NormFinder and BestKeeper, were used to evaluate their stability in apple root tissues of various genotypes and under different experimental conditions. A small panel of stably expressed genes, MDP0000095375, MDP0000147424, MDP0000233640, MDP0000326399 and MDP0000173025 were recommended for normalizing quantitative gene expression data in apple roots under various abiotic or biotic stresses. When the most stable and least stable reference genes were used for data normalization, significant differences were observed on the expression patterns of two target genes, MdLecRLK5 (MDP0000228426, a gene encoding a lectin receptor like kinase) and MdMAPK3 (MDP0000187103, a gene encoding a mitogen-activated protein kinase). Our data also indicated that for those carefully validated reference genes, a single reference gene is sufficient for reliable normalization of the quantitative gene expression. Depending on the experimental conditions, the most suitable reference genes can be specific to the sample of interest for more reliable RT-qPCR data normalization. PMID:28934340

Transposable elements contribute to activation of maize genes in response to abiotic stress.

PubMed

Makarevitch, Irina; Waters, Amanda J; West, Patrick T; Stitzer, Michelle; Hirsch, Candice N; Ross-Ibarra, Jeffrey; Springer, Nathan M

2015-01-01

Transposable elements (TEs) account for a large portion of the genome in many eukaryotic species. Despite their reputation as "junk" DNA or genomic parasites deleterious for the host, TEs have complex interactions with host genes and the potential to contribute to regulatory variation in gene expression. It has been hypothesized that TEs and genes they insert near may be transcriptionally activated in response to stress conditions. The maize genome, with many different types of TEs interspersed with genes, provides an ideal system to study the genome-wide influence of TEs on gene regulation. To analyze the magnitude of the TE effect on gene expression response to environmental changes, we profiled gene and TE transcript levels in maize seedlings exposed to a number of abiotic stresses. Many genes exhibit up- or down-regulation in response to these stress conditions. The analysis of TE families inserted within upstream regions of up-regulated genes revealed that between four and nine different TE families are associated with up-regulated gene expression in each of these stress conditions, affecting up to 20% of the genes up-regulated in response to abiotic stress, and as many as 33% of genes that are only expressed in response to stress. Expression of many of these same TE families also responds to the same stress conditions. The analysis of the stress-induced transcripts and proximity of the transposon to the gene suggests that these TEs may provide local enhancer activities that stimulate stress-responsive gene expression. Our data on allelic variation for insertions of several of these TEs show strong correlation between the presence of TE insertions and stress-responsive up-regulation of gene expression. Our findings suggest that TEs provide an important source of allelic regulatory variation in gene response to abiotic stress in maize.

Selection and evaluation of reference genes for expression studies with quantitative PCR in the model fungus Neurospora crassa under different environmental conditions in continuous culture.

PubMed

Cusick, Kathleen D; Fitzgerald, Lisa A; Pirlo, Russell K; Cockrell, Allison L; Petersen, Emily R; Biffinger, Justin C

2014-01-01

Neurospora crassa has served as a model organism for studying circadian pathways and more recently has gained attention in the biofuel industry due to its enhanced capacity for cellulase production. However, in order to optimize N. crassa for biotechnological applications, metabolic pathways during growth under different environmental conditions must be addressed. Reverse-transcription quantitative PCR (RT-qPCR) is a technique that provides a high-throughput platform from which to measure the expression of a large set of genes over time. The selection of a suitable reference gene is critical for gene expression studies using relative quantification, as this strategy is based on normalization of target gene expression to a reference gene whose expression is stable under the experimental conditions. This study evaluated twelve candidate reference genes for use with N. crassa when grown in continuous culture bioreactors under different light and temperature conditions. Based on combined stability values from NormFinder and Best Keeper software packages, the following are the most appropriate reference genes under conditions of: (1) light/dark cycling: btl, asl, and vma1; (2) all-dark growth: btl, tbp, vma1, and vma2; (3) temperature flux: btl, vma1, act, and asl; (4) all conditions combined: vma1, vma2, tbp, and btl. Since N. crassa exists as different cell types (uni- or multi-nucleated), expression changes in a subset of the candidate genes was further assessed using absolute quantification. A strong negative correlation was found to exist between ratio and threshold cycle (CT) values, demonstrating that CT changes serve as a reliable reflection of transcript, and not gene copy number, fluctuations. The results of this study identified genes that are appropriate for use as reference genes in RT-qPCR studies with N. crassa and demonstrated that even with the presence of different cell types, relative quantification is an acceptable method for measuring gene expression changes during growth in bioreactors.

Conditional clustering of temporal expression profiles

PubMed Central

Wang, Ling; Montano, Monty; Rarick, Matt; Sebastiani, Paola

2008-01-01

Background Many microarray experiments produce temporal profiles in different biological conditions but common cluster techniques are not able to analyze the data conditional on the biological conditions. Results This article presents a novel technique to cluster data from time course microarray experiments performed across several experimental conditions. Our algorithm uses polynomial models to describe the gene expression patterns over time, a full Bayesian approach with proper conjugate priors to make the algorithm invariant to linear transformations, and an iterative procedure to identify genes that have a common temporal expression profile across two or more experimental conditions, and genes that have a unique temporal profile in a specific condition. Conclusion We use simulated data to evaluate the effectiveness of this new algorithm in finding the correct number of clusters and in identifying genes with common and unique profiles. We also use the algorithm to characterize the response of human T cells to stimulations of antigen-receptor signaling gene expression temporal profiles measured in six different biological conditions and we identify common and unique genes. These studies suggest that the methodology proposed here is useful in identifying and distinguishing uniquely stimulated genes from commonly stimulated genes in response to variable stimuli. Software for using this clustering method is available from the project home page. PMID:18334028

Environmental conditions affect transcription of the pectinase genes of Erwinia chrysanthemi 3937.

PubMed Central

Hugouvieux-Cotte-Pattat, N; Dominguez, H; Robert-Baudouy, J

1992-01-01

To depolymerize plant pectin, the phytopathogenic enterobacterium Erwinia chrysanthemi produces a series of enzymes which include a pectin-methyl-esterase encoded by the pem gene and five isoenzymes of pectate lyases encoded by the five genes pelA, pelB, pelC, pelD, and pelE. We have constructed transcriptional fusions between the pectinase gene promoters and the uidA gene, encoding beta-glucuronidase, to study the regulation of these E. chrysanthemi pectinase genes individually. The transcription of the pectinase genes is dependent on many environmental conditions. All the fusions were induced by pectic catabolic products and responded, to different degrees, to growth phase, catabolite repression, temperature, and nitrogen starvation. Transcription of pelA, pelD, and pelE was also increased in anaerobic growth conditions. High osmolarity of the culture medium increased expression of pelE but decreased that of pelD; the other pectinase genes were not affected. The level of expression of each gene was different. Transcription of pelA was very low under all growth conditions. The expression of the pelB, pelC, and pem genes was intermediate. The pelE gene had a high basal level of expression. Expression of pelD was generally the most affected by changes in culture conditions and showed a low basal level but very high induced levels. These differences in the expression of the pectinase genes of E. chrysanthemi 3937 presumably reflect their role during infection of plants, because the degradation of pectic polymers of the plant cell walls is the main determinant of tissue maceration caused by soft rot erwiniae. PMID:1447147

Comparison of gene expression levels of appA, ppsR, and EL368 in Erythrobacter litoralis spheroplasts under aerobic and anaerobic conditions, and under blue light, red light, and dark conditions.

PubMed

Nishino, Koki; Takahashi, Sawako; Nishida, Hiromi

2018-03-31

We compared the gene expression levels of the blue-light-responsive genes, appA (encoding photosynthesis promoting protein AppA), ppsR (encoding photosynthesis suppressing protein PpsR), and EL368 (encoding a blue-light-activated histidine kinase with a light, oxygen, or voltage domain) between aerobic and anaerobic conditions in spheroplasts of the aerobic photosynthetic bacterium Erythrobacter litoralis. The spheroplasts conducted photosynthesis under red light but not under blue light. All three blue-light-responsive genes showed higher expression under aerobic conditions than under anaerobic conditions under blue light. In contrast, under red light, although the expression level of appA was higher in the presence of oxygen than in the absence of oxygen, the expression levels of ppsR and EL368 were similar in the presence and absence of oxygen. Our findings demonstrate that the expression of blue-light-responsive genes is strongly affected by oxygen in E. litoralis spheroplasts.

Selection of suitable reference genes for gene expression studies in Staphylococcus capitis during growth under erythromycin stress.

PubMed

Cui, Bintao; Smooker, Peter M; Rouch, Duncan A; Deighton, Margaret A

2016-08-01

Accurate and reproducible measurement of gene transcription requires appropriate reference genes, which are stably expressed under different experimental conditions to provide normalization. Staphylococcus capitis is a human pathogen that produces biofilm under stress, such as imposed by antimicrobial agents. In this study, a set of five commonly used staphylococcal reference genes (gyrB, sodA, recA, tuf and rpoB) were systematically evaluated in two clinical isolates of Staphylococcus capitis (S. capitis subspecies urealyticus and capitis, respectively) under erythromycin stress in mid-log and stationary phases. Two public software programs (geNorm and NormFinder) and two manual calculation methods, reference residue normalization (RRN) and relative quantitative (RQ), were applied. The potential reference genes selected by the four algorithms were further validated by comparing the expression of a well-studied biofilm gene (icaA) with phenotypic biofilm formation in S. capitis under four different experimental conditions. The four methods differed considerably in their ability to predict the most suitable reference gene or gene combination for comparing icaA expression under different conditions. Under the conditions used here, the RQ method provided better selection of reference genes than the other three algorithms; however, this finding needs to be confirmed with a larger number of isolates. This study reinforces the need to assess the stability of reference genes for analysis of target gene expression under different conditions and the use of more than one algorithm in such studies. Although this work was conducted using a specific human pathogen, it emphasizes the importance of selecting suitable reference genes for accurate normalization of gene expression more generally.

Impact of Ischemia and Procurement Conditions on Gene Expression in Renal Cell Carcinoma

PubMed Central

Liu, Nick W.; Sanford, Thomas; Srinivasan, Ramaprasad; Liu, Jack L.; Khurana, Kiranpreet; Aprelikova, Olga; Valero, Vladimir; Bechert, Charles; Worrell, Robert; Pinto, Peter A.; Yang, Youfeng; Merino, Maria; Linehan, W. Marston; Bratslavsky, Gennady

2013-01-01

Purpose Previous studies have shown that ischemia alters gene expression in normal and malignant tissues. There are no studies that evaluated effects of ischemia in renal tumors. This study examines the impact of ischemia and tissue procurement conditions on RNA integrity and gene expression in renal cell carcinoma. Experimental Design Ten renal tumors were resected without renal hilar clamping from 10 patients with renal clear cell carcinoma. Immediately after tumor resection, a piece of tumor was snap frozen. Remaining tumor samples were stored at 4C, 22C and 37C and frozen at 5, 30, 60, 120, and 240 minutes. Histopathologic evaluation was performed on all tissue samples, and only those with greater than 80% tumor were selected for further analysis. RNA integrity was confirmed by electropherograms and quantitated using RIN index. Altered gene expression was assessed by paired, two-sample t-test between the zero time point and aliquots from various conditions obtained from the same tumor. Results One hundred and forty microarrays were performed. Some RNA degradation was observed 240 mins after resection at 37C. The expression of over 4,000 genes was significantly altered by ischemia times or storage conditions. The greatest gene expression changes were observed with longer ischemia time and warmer tissue procurement conditions. Conclusion RNA from kidney cancer remains intact for up to 4 hours post surgical resection regardless of storage conditions. Despite excellent RNA preservation, time after resection and procurement conditions significantly influence gene expression profiles. Meticulous attention to pre-acquisition variables is of paramount importance for accurate tumor profiling. PMID:23136194

Transcriptome sequencing of Eucalyptus camaldulensis seedlings subjected to water stress reveals functional single nucleotide polymorphisms and genes under selection

PubMed Central

2012-01-01

Background Water stress limits plant survival and production in many parts of the world. Identification of genes and alleles responding to water stress conditions is important in breeding plants better adapted to drought. Currently there are no studies examining the transcriptome wide gene and allelic expression patterns under water stress conditions. We used RNA sequencing (RNA-seq) to identify the candidate genes and alleles and to explore the evolutionary signatures of selection. Results We studied the effect of water stress on gene expression in Eucalyptus camaldulensis seedlings derived from three natural populations. We used reference-guided transcriptome mapping to study gene expression. Several genes showed differential expression between control and stress conditions. Gene ontology (GO) enrichment tests revealed up-regulation of 140 stress-related gene categories and down-regulation of 35 metabolic and cell wall organisation gene categories. More than 190,000 single nucleotide polymorphisms (SNPs) were detected and 2737 of these showed differential allelic expression. Allelic expression of 52% of these variants was correlated with differential gene expression. Signatures of selection patterns were studied by estimating the proportion of nonsynonymous to synonymous substitution rates (Ka/Ks). The average Ka/Ks ratio among the 13,719 genes was 0.39 indicating that most of the genes are under purifying selection. Among the positively selected genes (Ka/Ks > 1.5) apoptosis and cell death categories were enriched. Of the 287 positively selected genes, ninety genes showed differential expression and 27 SNPs from 17 positively selected genes showed differential allelic expression between treatments. Conclusions Correlation of allelic expression of several SNPs with total gene expression indicates that these variants may be the cis-acting variants or in linkage disequilibrium with such variants. Enrichment of apoptosis and cell death gene categories among the positively selected genes reveals the past selection pressures experienced by the populations used in this study. PMID:22853646

Discovering Condition-Specific Gene Co-Expression Patterns Using Gaussian Mixture Models: A Cancer Case Study.

PubMed

Ficklin, Stephen P; Dunwoodie, Leland J; Poehlman, William L; Watson, Christopher; Roche, Kimberly E; Feltus, F Alex

2017-08-17

A gene co-expression network (GCN) describes associations between genes and points to genetic coordination of biochemical pathways. However, genetic correlations in a GCN are only detectable if they are present in the sampled conditions. With the increasing quantity of gene expression samples available in public repositories, there is greater potential for discovery of genetic correlations from a variety of biologically interesting conditions. However, even if gene correlations are present, their discovery can be masked by noise. Noise is introduced from natural variation (intrinsic and extrinsic), systematic variation (caused by sample measurement protocols and instruments), and algorithmic and statistical variation created by selection of data processing tools. A variety of published studies, approaches and methods attempt to address each of these contributions of variation to reduce noise. Here we describe an approach using Gaussian Mixture Models (GMMs) to address natural extrinsic (condition-specific) variation during network construction from mixed input conditions. To demonstrate utility, we build and analyze a condition-annotated GCN from a compendium of 2,016 mixed gene expression data sets from five tumor subtypes obtained from The Cancer Genome Atlas. Our results show that GMMs help discover tumor subtype specific gene co-expression patterns (modules) that are significantly enriched for clinical attributes.

Identification of reference genes for quantitative expression analysis using large-scale RNA-seq data of Arabidopsis thaliana and model crop plants.

PubMed

Kudo, Toru; Sasaki, Yohei; Terashima, Shin; Matsuda-Imai, Noriko; Takano, Tomoyuki; Saito, Misa; Kanno, Maasa; Ozaki, Soichi; Suwabe, Keita; Suzuki, Go; Watanabe, Masao; Matsuoka, Makoto; Takayama, Seiji; Yano, Kentaro

2016-10-13

In quantitative gene expression analysis, normalization using a reference gene as an internal control is frequently performed for appropriate interpretation of the results. Efforts have been devoted to exploring superior novel reference genes using microarray transcriptomic data and to evaluating commonly used reference genes by targeting analysis. However, because the number of specifically detectable genes is totally dependent on probe design in the microarray analysis, exploration using microarray data may miss some of the best choices for the reference genes. Recently emerging RNA sequencing (RNA-seq) provides an ideal resource for comprehensive exploration of reference genes since this method is capable of detecting all expressed genes, in principle including even unknown genes. We report the results of a comprehensive exploration of reference genes using public RNA-seq data from plants such as Arabidopsis thaliana (Arabidopsis), Glycine max (soybean), Solanum lycopersicum (tomato) and Oryza sativa (rice). To select reference genes suitable for the broadest experimental conditions possible, candidates were surveyed by the following four steps: (1) evaluation of the basal expression level of each gene in each experiment; (2) evaluation of the expression stability of each gene in each experiment; (3) evaluation of the expression stability of each gene across the experiments; and (4) selection of top-ranked genes, after ranking according to the number of experiments in which the gene was expressed stably. Employing this procedure, 13, 10, 12 and 21 top candidates for reference genes were proposed in Arabidopsis, soybean, tomato and rice, respectively. Microarray expression data confirmed that the expression of the proposed reference genes under broad experimental conditions was more stable than that of commonly used reference genes. These novel reference genes will be useful for analyzing gene expression profiles across experiments carried out under various experimental conditions.

Gene expression of Vibrio parahaemolyticus growing in laboratory isolation conditions compared to those common in its natural ocean environment.

PubMed

García, Katherine; Yáñez, Cristian; Plaza, Nicolás; Peña, Francisca; Sepúlveda, Pedro; Pérez-Reytor, Diliana; Espejo, Romilio T

2017-05-19

Vibrio parahaemolyticus is an autochthonous marine bacterial species comprising strains able to grow in broth containing bile salts at 37 °C, a condition seldom found in the ocean. However, this condition is used for isolation in the laboratory because it is considered a necessary property for pathogenesis. In this context, revealing how gene expression enables V. parahaemolyticus to adapt to this particular condition -common to almost all V. parahaemolyticus isolates- will improve our understanding of the biology of this important pathogen. To determine the genes of V. parahaemolyticus differentially expressed when growing in isolation condition (37 °C, 0.9% NaCl, and 0.04% bile salts) referred to those at the temperature and salt concentration prevailing in ocean south of Chile (marine-like condition; 12 °C, 3% NaCl, and absence of bile salts) we used high-throughput sequencing of RNA. Our results showed that in the isolation condition, among the 5034 genes annotated in the V. parahaemolyticus RIMD2210633 genome, 344 were upregulated and 433 downregulated referred to the marine-like condition, managing an adjusted P-value (Padj) < E -5 . Between the 50 more highly expressed genes, among the small RNAs (sRNA), the three carbon storage regulators B (CsrB) were up four to six times, while RyhB, related to iron metabolism besides motility control, was down about eight times. Among proteins, BfdA, a hemolysin-co-regulated protein (Hcp1) secreted by T6SS1, one of the most highly expressed genes, was about 140 times downregulated in isolation condition. The highest changes in relative expression were found among neighboring genes coding for proteins related to respiration, which were about 40 times upregulated. When V. parahaemolyticus is grown in conditions used for laboratory isolation 777 genes are up- or downregulated referred to conditions prevailing in the marine-like condition; the most significantly overrepresented categories among upregulated processes were those related to transport and localization, while secretion and pathogenesis were overrepresented among downregulated genes. Genes with the highest differential expression included the sRNAs CsrB and RhyB and the mRNAs related with secretion, nutritional upshift, respiration and rapid growing.

Root-tip-mediated inhibition of hydrotropism is accompanied with the suppression of asymmetric expression of auxin-inducible genes in response to moisture gradients in cucumber roots

PubMed Central

Miyabayashi, Sachiko; Sugita, Tomoki; Kobayashi, Akie; Yamazaki, Chiaki; Miyazawa, Yutaka; Kamada, Motoshi; Kasahara, Haruo; Osada, Ikuko; Shimazu, Toru; Fusejima, Yasuo; Higashibata, Akira; Yamazaki, Takashi; Ishioka, Noriaki; Takahashi, Hideyuki

2018-01-01

In cucumber seedlings, gravitropism interferes with hydrotropism, which results in the nearly complete inhibition of hydrotropism under stationary conditions. However, hydrotropic responses are induced when the gravitropic response in the root is nullified by clinorotation. Columella cells in the root cap sense gravity, which induces the gravitropic response. In this study, we found that removing the root tip induced hydrotropism in cucumber roots under stationary conditions. The application of auxin transport inhibitors to cucumber seedlings under stationary conditions suppressed the hydrotropic response induced by the removal of the root tip. To investigate the expression of genes related to hydrotropism in de-tipped cucumber roots, we conducted transcriptome analysis of gene expression by RNA-Seq using seedlings exhibiting hydrotropic and gravitropic responses. Of the 21 and 45 genes asymmetrically expressed during hydrotropic and gravitropic responses, respectively, five genes were identical. Gene ontology (GO) analysis indicated that the category auxin-inducible genes was significantly enriched among genes that were more highly expressed in the concave side of the root than the convex side during hydrotropic or gravitropic responses. Reverse transcription followed by quantitative polymerase chain reaction (RT-qPCR) analysis revealed that root hydrotropism induced under stationary conditions (by removing the root tip) was accompanied by the asymmetric expression of several auxin-inducible genes. However, intact roots did not exhibit the asymmetric expression patterns of auxin-inducible genes under stationary conditions, even in the presence of a moisture gradient. These results suggest that the root tip inhibits hydrotropism by suppressing the induction of asymmetric auxin distribution. Auxin transport and distribution not mediated by the root tip might play a role in hydrotropism in cucumber roots. PMID:29324818

Mining microbial metatranscriptomes for expression of antibiotic resistance genes under natural conditions.

PubMed

Versluis, Dennis; D'Andrea, Marco Maria; Ramiro Garcia, Javier; Leimena, Milkha M; Hugenholtz, Floor; Zhang, Jing; Öztürk, Başak; Nylund, Lotta; Sipkema, Detmer; van Schaik, Willem; de Vos, Willem M; Kleerebezem, Michiel; Smidt, Hauke; van Passel, Mark W J

2015-07-08

Antibiotic resistance genes are found in a broad range of ecological niches associated with complex microbiota. Here we investigated if resistance genes are not only present, but also transcribed under natural conditions. Furthermore, we examined the potential for antibiotic production by assessing the expression of associated secondary metabolite biosynthesis gene clusters. Metatranscriptome datasets from intestinal microbiota of four human adults, one human infant, 15 mice and six pigs, of which only the latter have received antibiotics prior to the study, as well as from sea bacterioplankton, a marine sponge, forest soil and sub-seafloor sediment, were investigated. We found that resistance genes are expressed in all studied ecological niches, albeit with niche-specific differences in relative expression levels and diversity of transcripts. For example, in mice and human infant microbiota predominantly tetracycline resistance genes were expressed while in human adult microbiota the spectrum of expressed genes was more diverse, and also included β-lactam, aminoglycoside and macrolide resistance genes. Resistance gene expression could result from the presence of natural antibiotics in the environment, although we could not link it to expression of corresponding secondary metabolites biosynthesis clusters. Alternatively, resistance gene expression could be constitutive, or these genes serve alternative roles besides antibiotic resistance.

Mining microbial metatranscriptomes for expression of antibiotic resistance genes under natural conditions

NASA Astrophysics Data System (ADS)

Versluis, Dennis; D'Andrea, Marco Maria; Ramiro Garcia, Javier; Leimena, Milkha M.; Hugenholtz, Floor; Zhang, Jing; Öztürk, Başak; Nylund, Lotta; Sipkema, Detmer; Schaik, Willem Van; de Vos, Willem M.; Kleerebezem, Michiel; Smidt, Hauke; Passel, Mark W. J. Van

2015-07-01

Antibiotic resistance genes are found in a broad range of ecological niches associated with complex microbiota. Here we investigated if resistance genes are not only present, but also transcribed under natural conditions. Furthermore, we examined the potential for antibiotic production by assessing the expression of associated secondary metabolite biosynthesis gene clusters. Metatranscriptome datasets from intestinal microbiota of four human adults, one human infant, 15 mice and six pigs, of which only the latter have received antibiotics prior to the study, as well as from sea bacterioplankton, a marine sponge, forest soil and sub-seafloor sediment, were investigated. We found that resistance genes are expressed in all studied ecological niches, albeit with niche-specific differences in relative expression levels and diversity of transcripts. For example, in mice and human infant microbiota predominantly tetracycline resistance genes were expressed while in human adult microbiota the spectrum of expressed genes was more diverse, and also included β-lactam, aminoglycoside and macrolide resistance genes. Resistance gene expression could result from the presence of natural antibiotics in the environment, although we could not link it to expression of corresponding secondary metabolites biosynthesis clusters. Alternatively, resistance gene expression could be constitutive, or these genes serve alternative roles besides antibiotic resistance.

VTCdb: a gene co-expression database for the crop species Vitis vinifera (grapevine).

PubMed

Wong, Darren C J; Sweetman, Crystal; Drew, Damian P; Ford, Christopher M

2013-12-16

Gene expression datasets in model plants such as Arabidopsis have contributed to our understanding of gene function and how a single underlying biological process can be governed by a diverse network of genes. The accumulation of publicly available microarray data encompassing a wide range of biological and environmental conditions has enabled the development of additional capabilities including gene co-expression analysis (GCA). GCA is based on the understanding that genes encoding proteins involved in similar and/or related biological processes may exhibit comparable expression patterns over a range of experimental conditions, developmental stages and tissues. We present an open access database for the investigation of gene co-expression networks within the cultivated grapevine, Vitis vinifera. The new gene co-expression database, VTCdb (http://vtcdb.adelaide.edu.au/Home.aspx), offers an online platform for transcriptional regulatory inference in the cultivated grapevine. Using condition-independent and condition-dependent approaches, grapevine co-expression networks were constructed using the latest publicly available microarray datasets from diverse experimental series, utilising the Affymetrix Vitis vinifera GeneChip (16 K) and the NimbleGen Grape Whole-genome microarray chip (29 K), thus making it possible to profile approximately 29,000 genes (95% of the predicted grapevine transcriptome). Applications available with the online platform include the use of gene names, probesets, modules or biological processes to query the co-expression networks, with the option to choose between Affymetrix or Nimblegen datasets and between multiple co-expression measures. Alternatively, the user can browse existing network modules using interactive network visualisation and analysis via CytoscapeWeb. To demonstrate the utility of the database, we present examples from three fundamental biological processes (berry development, photosynthesis and flavonoid biosynthesis) whereby the recovered sub-networks reconfirm established plant gene functions and also identify novel associations. Together, we present valuable insights into grapevine transcriptional regulation by developing network models applicable to researchers in their prioritisation of gene candidates, for on-going study of biological processes related to grapevine development, metabolism and stress responses.

Identification of Reference Genes and Analysis of Heat Shock Protein Gene Expression in Lingzhi or Reishi Medicinal Mushroom, Ganoderma lucidum, after Exposure to Heat Stress.

PubMed

Liu, Yong-Nan; Lu, Xiao-Xiao; Ren, Ang; Shi, Liang; Jiang, Ai-Liang; Yu, Han-Shou; Zhao, Ming-Wen

2017-01-01

Ganoderma lucidum has been considered an emerging model species for studying how environmental factors regulate the growth, development, and secondary metabolism of Basidiomycetes. Heat stress, which is one of the most important environmental abiotic stresses, seriously affects the growth, development, and yield of microorganisms. Understanding the response to heat stress has gradually become a hotspot in microorganism research. But suitable reference genes for expression analysis under heat stress have not been reported in G. lucidum. In this study, we systematically identified 11 candidate reference genes that were measured using reverse transcriptase quantitative polymerase chain reaction, and the gene expression stability was analyzed under heat stress conditions using geNorm and NormFinder. The results show that 5 reference genes-CYP and TIF, followed by UCE2, ACTIN, and UBQ1-are the most stable genes under our experimental conditions. Moreover, the relative expression levels of 3 heat stress response genes (hsp17.4, hsp70, and hsp90) were analyzed under heat stress conditions with different normalization strategies. The results show that use of a gene with unstable expression (SAND) as the reference gene leads to biased data and misinterpretations of the target gene expression level under heat stress.

Diel pattern of circadian clock and storage protein gene expression in leaves and during seed filling in cowpea (Vigna unguiculata).

PubMed

Weiss, Julia; Terry, Marta I; Martos-Fuentes, Marina; Letourneux, Lisa; Ruiz-Hernández, Victoria; Fernández, Juan A; Egea-Cortines, Marcos

2018-02-14

Cowpea (Vigna unguiculata) is an important source of protein supply for animal and human nutrition. The major storage globulins VICILIN and LEGUMIN (LEG) are synthesized from several genes including LEGA, LEGB, LEGJ and CVC (CONVICILIN). The current hypothesis is that the plant circadian core clock genes are conserved in a wide array of species and that primary metabolism is to a large extent controlled by the plant circadian clock. Our aim was to investigate a possible link between gene expression of storage proteins and the circadian clock. We identified cowpea orthologues of the core clock genes VunLHY, VunTOC1, VunGI and VunELF3, the protein storage genes VunLEG, VunLEGJ, and VunCVC as well as nine candidate reference genes used in RT-PCR. ELONGATION FACTOR 1-A (ELF1A) resulted the most suitable reference gene. The clock genes VunELF3, VunGI, VunTOC1 and VunLHY showed a rhythmic expression profile in leaves with a typical evening/night and morning/midday phased expression. The diel patterns were not completely robust and only VungGI and VungELF3 retained a rhythmic pattern under free running conditions of darkness. Under field conditions, rhythmicity and phasing apparently faded during early pod and seed development and was regained in ripening pods for VunTOC1 and VunLHY. Mature seeds showed a rhythmic expression of VunGI resembling leaf tissue under controlled growth chamber conditions. Comparing time windows during developmental stages we found that VunCVC and VunLEG were significantly down regulated during the night in mature pods as compared to intermediate ripe pods, while changes in seeds were non-significant due to high variance. The rhythmic expression under field conditions was lost under growth chamber conditions. The core clock gene network is conserved in cowpea leaves showing a robust diel expression pattern except VunELF3 under growth chamber conditions. There appears to be a clock transcriptional reprogramming in pods and seeds compared to leaves. Storage protein deposition may be circadian regulated under field conditions but the strong environmental signals are not met under artificial growth conditions. Diel expression pattern in field conditions may result in better usage of energy for protein storage.

Gene expression profile of the plant pathogen Xylella fastidiosa during biofilm formation in vitro.

PubMed

de Souza, Alessandra A; Takita, Marco A; Coletta-Filho, Helvécio D; Caldana, Camila; Yanai, Giane M; Muto, Nair H; de Oliveira, Regina C; Nunes, Luiz R; Machado, Marcos A

2004-08-15

A biofilm is a community of microorganisms attached to a solid surface. Cells within biofilms differ from planktonic cells, showing higher resistance to biocides, detergent, antibiotic treatments and host defense responses. Even though there are a number of gene expression studies in bacterial biofilm formation, limited information is available concerning plant pathogen. It was previously demonstrated that the plant pathogen Xylella fastidiosa could grow as a biofilm, a possibly important factor for its pathogenicity. In this study we utilized analysis of microarrays to specifically identify genes expressed in X. fastidiosa cells growing in a biofilm, when compared to planktonic cells. About half of the differentially expressed genes encode hypothetical proteins, reflecting the large number of ORFs with unknown functions in bacterial genomes. However, under the biofilm condition we observed an increase in the expression of some housekeeping genes responsible for metabolic functions. We also found a large number of genes from the pXF51 plasmid being differentially expressed. Some of the overexpressed genes in the biofilm condition encode proteins involved in attachment to surfaces. Other genes possibly confer advantages to the bacterium in the environment that it colonizes. This study demonstrates that the gene expression in the biofilm growth condition of the plant pathogen X. fastidiosa is quite similar to other characterized systems.

The expression of native and cultured human retinal pigment epithelial cells grown in different culture conditions.

PubMed

Tian, J; Ishibashi, K; Honda, S; Boylan, S A; Hjelmeland, L M; Handa, J T

2005-11-01

To determine the transcriptional proximity of retinal pigment epithelium (RPE) cells grown under different culture conditions and native RPE. ARPE-19 cells were grown under five conditions in 10% CO(2): "subconfluent" in DMEM/F12+10% FBS, "confluent" in serum and serum withdrawn, and "differentiated" for 2.5 months in serum and serum withdrawn medium. Native RPE was laser microdissected. Total RNA was extracted, reverse transcribed, and radiolabelled probes were hybridised to an array containing 5,353 genes. Arrays were evaluated by hierarchical cluster analysis and significance analysis of microarrays. 78% of genes were expressed by native RPE while 45.3--47.7% were expressed by ARPE-19 cells, depending on culture condition. While the most abundant genes were expressed by native and cultured cells, significant differences in low abundance genes were seen. Hierarchical cluster analysis showed that confluent and differentiated, serum withdrawn cultures clustered closest to native RPE, and that serum segregated cultured cells from native RPE. The number of differentially expressed genes and their function, and profile of expressed and unexpressed genes, demonstrate differences between native and cultured cells. While ARPE-19 cells have significant value for studying RPE behaviour, investigators must be aware of how culture conditions can influence the mRNA phenotype of the cell.

Spectral Biclustering of Microarray Data: Coclustering Genes and Conditions

PubMed Central

Kluger, Yuval; Basri, Ronen; Chang, Joseph T.; Gerstein, Mark

2003-01-01

Global analyses of RNA expression levels are useful for classifying genes and overall phenotypes. Often these classification problems are linked, and one wants to find “marker genes” that are differentially expressed in particular sets of “conditions.” We have developed a method that simultaneously clusters genes and conditions, finding distinctive “checkerboard” patterns in matrices of gene expression data, if they exist. In a cancer context, these checkerboards correspond to genes that are markedly up- or downregulated in patients with particular types of tumors. Our method, spectral biclustering, is based on the observation that checkerboard structures in matrices of expression data can be found in eigenvectors corresponding to characteristic expression patterns across genes or conditions. In addition, these eigenvectors can be readily identified by commonly used linear algebra approaches, in particular the singular value decomposition (SVD), coupled with closely integrated normalization steps. We present a number of variants of the approach, depending on whether the normalization over genes and conditions is done independently or in a coupled fashion. We then apply spectral biclustering to a selection of publicly available cancer expression data sets, and examine the degree to which the approach is able to identify checkerboard structures. Furthermore, we compare the performance of our biclustering methods against a number of reasonable benchmarks (e.g., direct application of SVD or normalized cuts to raw data). PMID:12671006

DEEP--a tool for differential expression effector prediction.

PubMed

Degenhardt, Jost; Haubrock, Martin; Dönitz, Jürgen; Wingender, Edgar; Crass, Torsten

2007-07-01

High-throughput methods for measuring transcript abundance, like SAGE or microarrays, are widely used for determining differences in gene expression between different tissue types, dignities (normal/malignant) or time points. Further analysis of such data frequently aims at the identification of gene interaction networks that form the causal basis for the observed properties of the systems under examination. To this end, it is usually not sufficient to rely on the measured gene expression levels alone; rather, additional biological knowledge has to be taken into account in order to generate useful hypotheses about the molecular mechanism leading to the realization of a certain phenotype. We present a method that combines gene expression data with biological expert knowledge on molecular interaction networks, as described by the TRANSPATH database on signal transduction, to predict additional--and not necessarily differentially expressed--genes or gene products which might participate in processes specific for either of the examined tissues or conditions. In a first step, significance values for over-expression in tissue/condition A or B are assigned to all genes in the expression data set. Genes with a significance value exceeding a certain threshold are used as starting points for the reconstruction of a graph with signaling components as nodes and signaling events as edges. In a subsequent graph traversal process, again starting from the previously identified differentially expressed genes, all encountered nodes 'inherit' all their starting nodes' significance values. In a final step, the graph is visualized, the nodes being colored according to a weighted average of their inherited significance values. Each node's, or sub-network's, predominant color, ranging from green (significant for tissue/condition A) over yellow (not significant for either tissue/condition) to red (significant for tissue/condition B), thus gives an immediate visual clue on which molecules--differentially expressed or not--may play pivotal roles in the tissues or conditions under examination. The described method has been implemented in Java as a client/server application and a web interface called DEEP (Differential Expression Effector Prediction). The client, which features an easy-to-use graphical interface, can freely be downloaded from the following URL: http://deep.bioinf.med.uni-goettingen.de.

Hypergravity-induced changes in gene expression in Arabidopsis hypocotyls

NASA Astrophysics Data System (ADS)

Yoshioka, R.; Soga, K.; Wakabayashi, K.; Takeba, G.; Hoson, T.

2003-05-01

Under hypergravity conditions, the cell wall of stem organs becomes mechanically rigid and elongation growth is suppressed, which can be recognized as the mechanism for plants to resist gravitational force. The changes in gene expression by hypergravity treatment were analyzed in Arabidopsis hypocotyls by the differential display method, for identifying genes involved in hypergravity-induced growth suppression. Sixty-two cDNA clones were expressed differentially between the control and 300 g conditions: the expression levels of 39 clones increased, whereas those of 23 clones decreased under hypergravity conditions. Sequence analysis and database searching revealed that 12 clones, 9 up-regulated and 3 down-regulated, have homology to known proteins. The expression of these genes was further analyzed using RT-PCR. Finally, six genes were confirmed to be up-regulated by hypergravity. One of such genes encoded 3-hydroxy-3-methylglutaryl-Coenzyme A reductase (HMGR), which catalyzes a reaction producing mevalonic acid, a key precursor ofterpenoids such as membrane sterols and several types of hormones. The expression of HMGR gene increased within several hours after hypergravity treatment. Also, compactin, an inhibitor of HMGR, prevented hypergravity-induced growth suppression, suggesting that HMGR is involved in suppression of Arabidopsis hypocotyl growth by hypergravity. In addition, hypergravity increased the expression levels of genes encoding CCR1 and ERD15, which were shown to take part in the signaling pathway of environmental stimuli such as temperature and water, and those of the α-tubulin gene. These genes may be involved in a series of cellular events leading to growth suppression of stem organs under hypergravity conditions.

Validation of reference genes for RT-qPCR studies of gene expression in banana fruit under different experimental conditions.

PubMed

Chen, Lei; Zhong, Hai-ying; Kuang, Jian-fei; Li, Jian-guo; Lu, Wang-jin; Chen, Jian-ye

2011-08-01

Reverse transcription quantitative real-time PCR (RT-qPCR) is a sensitive technique for quantifying gene expression, but its success depends on the stability of the reference gene(s) used for data normalization. Only a few studies on validation of reference genes have been conducted in fruit trees and none in banana yet. In the present work, 20 candidate reference genes were selected, and their expression stability in 144 banana samples were evaluated and analyzed using two algorithms, geNorm and NormFinder. The samples consisted of eight sample sets collected under different experimental conditions, including various tissues, developmental stages, postharvest ripening, stresses (chilling, high temperature, and pathogen), and hormone treatments. Our results showed that different suitable reference gene(s) or combination of reference genes for normalization should be selected depending on the experimental conditions. The RPS2 and UBQ2 genes were validated as the most suitable reference genes across all tested samples. More importantly, our data further showed that the widely used reference genes, ACT and GAPDH, were not the most suitable reference genes in many banana sample sets. In addition, the expression of MaEBF1, a gene of interest that plays an important role in regulating fruit ripening, under different experimental conditions was used to further confirm the validated reference genes. Taken together, our results provide guidelines for reference gene(s) selection under different experimental conditions and a foundation for more accurate and widespread use of RT-qPCR in banana.

Using RNA-Seq data to select refence genes for normalizing gene expression in apple roots

USDA-ARS?s Scientific Manuscript database

Gene expression in apple roots in response to various stress conditions is a less-explored research subject. Reliable reference genes for normalizing quantitative gene expression data have not been carefully investigated. In this study, the suitability of a set of 15 apple genes were evaluated for t...

Daytime soybean transcriptome fluctuations during water deficit stress.

PubMed

Rodrigues, Fabiana Aparecida; Fuganti-Pagliarini, Renata; Marcolino-Gomes, Juliana; Nakayama, Thiago Jonas; Molinari, Hugo Bruno Correa; Lobo, Francisco Pereira; Harmon, Frank G; Nepomuceno, Alexandre Lima

2015-07-07

Since drought can seriously affect plant growth and development and little is known about how the oscillations of gene expression during the drought stress-acclimation response in soybean is affected, we applied Illumina technology to sequence 36 cDNA libraries synthesized from control and drought-stressed soybean plants to verify the dynamic changes in gene expression during a 24-h time course. Cycling variables were measured from the expression data to determine the putative circadian rhythm regulation of gene expression. We identified 4866 genes differentially expressed in soybean plants in response to water deficit. Of these genes, 3715 were differentially expressed during the light period, from which approximately 9.55% were observed in both light and darkness. We found 887 genes that were either up- or down-regulated in different periods of the day. Of 54,175 predicted soybean genes, 35.52% exhibited expression oscillations in a 24 h period. This number increased to 39.23% when plants were submitted to water deficit. Major differences in gene expression were observed in the control plants from late day (ZT16) until predawn (ZT20) periods, indicating that gene expression oscillates during the course of 24 h in normal development. Under water deficit, dissimilarity increased in all time-periods, indicating that the applied stress influenced gene expression. Such differences in plants under stress were primarily observed in ZT0 (early morning) to ZT8 (late day) and also from ZT4 to ZT12. Stress-related pathways were triggered in response to water deficit primarily during midday, when more genes were up-regulated compared to early morning. Additionally, genes known to be involved in secondary metabolism and hormone signaling were also expressed in the dark period. Gene expression networks can be dynamically shaped to acclimate plant metabolism under environmental stressful conditions. We have identified putative cycling genes that are expressed in soybean leaves under normal developmental conditions and genes whose expression oscillates under conditions of water deficit. These results suggest that time of day, as well as light and temperature oscillations that occur considerably affect the regulation of water deficit stress response in soybean plants.

Substrate-Specific Differential Gene Expression and RNA editing in the Brown Rot Fungus Fomitopsis pinicola.

PubMed

Wu, Baojun; Gaskell, Jill; Held, Benjamin W; Toapanta, Cristina; Vuong, Thu; Ahrendt, Steven; Lipzen, Anna; Zhang, Jiwei; Schilling, Jonathan S; Master, Emma; Grigoriev, Igor V; Blanchette, Robert A; Cullen, Dan; Hibbett, David S

2018-06-08

Wood-decaying fungi tend to have characteristic substrate ranges that partly define their ecological niche. Fomitopsis pinicola is a brown rot species of Polyporales that is reported on 82 species of softwoods and 42 species of hardwoods. We analyzed gene expression levels and RNA editing profiles of F. pinicola from submerged cultures with ground wood powder (sampled at five days) or solid wood wafers (sampled at ten and thirty days), using aspen, pine, and spruce substrates (aspen was used only in submerged cultures). Fomitopsis pinicola expressed similar sets of wood-degrading enzymes typical of brown rot fungi across all culture conditions and timepoints. Nevertheless, differential gene expression and RNA editing were observed across all pairwise comparisons of substrates and timepoints. Genes exhibiting differential expression and RNA editing encode diverse enzymes with known or potential function in brown rot decay, including laccase, benzoquinone reductase, aryl alcohol oxidase, cytochrome P450s, and various glycoside hydrolases. There was no overlap between differentially expressed and differentially edited genes, suggesting that these may provide F. pinicola with independent mechanisms for responding to different conditions. Comparing transcriptomes from submerged cultures and wood wafers, we found that culture conditions had a greater impact on global expression profiles than substrate wood species. In contrast, the suites of genes subject to RNA editing were much less affected by culture conditions. These findings highlight the need for standardization of culture conditions in studies of gene expression in wood-decaying fungi. IMPORTANCE All species of wood-decaying fungi occur on a characteristic range of substrates (host plants), which may be broad or narrow. Understanding the mechanisms that allow fungi to grow on particular substrates is important for both fungal ecology and applied uses of different feedstocks in industrial processes. We grew the wood-decaying polypore Fomitopsis pinicola on three different wood species, aspen, pine and spruce, under various culture conditions. We examined both gene expression (transcription levels) and RNA editing (post-transcriptional modification of RNA, which can potentially yield different proteins from the same gene). We found that F. pinicola is able to modify both gene expression and RNA editing profiles across different substrate species and culture conditions. Many of the genes involved encode enzymes with known or predicted functions in wood decay. This work provides clues to how wood-decaying fungi may adjust their arsenal of decay enzymes to accommodate different host substrates. Copyright © 2018 American Society for Microbiology.

Validation of reference genes for quantitative RT-PCR studies of gene expression in perennial ryegrass (Lolium perenne L.)

PubMed Central

2010-01-01

Background Perennial ryegrass (Lolium perenne L.) is an important pasture and turf crop. Biotechniques such as gene expression studies are being employed to improve traits in this temperate grass. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) is among the best methods available for determining changes in gene expression. Before analysis of target gene expression, it is essential to select an appropriate normalisation strategy to control for non-specific variation between samples. Reference genes that have stable expression at different biological and physiological states can be effectively used for normalisation; however, their expression stability must be validated before use. Results Existing Serial Analysis of Gene Expression data were queried to identify six moderately expressed genes that had relatively stable gene expression throughout the year. These six candidate reference genes (eukaryotic elongation factor 1 alpha, eEF1A; TAT-binding protein homolog 1, TBP-1; eukaryotic translation initiation factor 4 alpha, eIF4A; YT521-B-like protein family protein, YT521-B; histone 3, H3; ubiquitin-conjugating enzyme, E2) were validated for qRT-PCR normalisation in 442 diverse perennial ryegrass (Lolium perenne L.) samples sourced from field- and laboratory-grown plants under a wide range of experimental conditions. Eukaryotic EF1A is encoded by members of a multigene family exhibiting differential expression and necessitated the expression analysis of different eEF1A encoding genes; a highly expressed eEF1A (h), a moderately, but stably expressed eEF1A (s), and combined expression of multigene eEF1A (m). NormFinder identified eEF1A (s) and YT521-B as the best combination of two genes for normalisation of gene expression data in perennial ryegrass following different defoliation management in the field. Conclusions This study is unique in the magnitude of samples tested with the inclusion of numerous field-grown samples, helping pave the way to conduct gene expression studies in perennial biomass crops under field-conditions. From our study several stably expressed reference genes have been validated. This provides useful candidates for reference gene selection in perennial ryegrass under conditions other than those tested here. PMID:20089196

Evaluation of Reference Genes for Gene Expression Analysis Using Quantitative RT-PCR in Azospirillum brasilense

PubMed Central

McMillan, Mary; Pereg, Lily

2014-01-01

Azospirillum brasilense is a nitrogen fixing bacterium that has been shown to have various beneficial effects on plant growth and yield. Under normal conditions A. brasilense exists in a motile flagellated form, which, under starvation or stress conditions, can undergo differentiation into an encapsulated, cyst-like form. Quantitative RT-PCR can be used to analyse changes in gene expression during this differentiation process. The accuracy of quantification of mRNA levels by qRT-PCR relies on the normalisation of data against stably expressed reference genes. No suitable set of reference genes has yet been described for A. brasilense. Here we evaluated the expression of ten candidate reference genes (16S rRNA, gapB, glyA, gyrA, proC, pykA, recA, recF, rpoD, and tpiA) in wild-type and mutant A. brasilense strains under different culture conditions, including conditions that induce differentiation. Analysis with the software programs BestKeeper, NormFinder and GeNorm indicated that gyrA, glyA and recA are the most stably expressed reference genes in A. brasilense. The results also suggested that the use of two reference genes (gyrA and glyA) is sufficient for effective normalisation of qRT-PCR data. PMID:24841066

Evaluation of reference genes for gene expression analysis using quantitative RT-PCR in Azospirillum brasilense.

PubMed

McMillan, Mary; Pereg, Lily

2014-01-01

Azospirillum brasilense is a nitrogen fixing bacterium that has been shown to have various beneficial effects on plant growth and yield. Under normal conditions A. brasilense exists in a motile flagellated form, which, under starvation or stress conditions, can undergo differentiation into an encapsulated, cyst-like form. Quantitative RT-PCR can be used to analyse changes in gene expression during this differentiation process. The accuracy of quantification of mRNA levels by qRT-PCR relies on the normalisation of data against stably expressed reference genes. No suitable set of reference genes has yet been described for A. brasilense. Here we evaluated the expression of ten candidate reference genes (16S rRNA, gapB, glyA, gyrA, proC, pykA, recA, recF, rpoD, and tpiA) in wild-type and mutant A. brasilense strains under different culture conditions, including conditions that induce differentiation. Analysis with the software programs BestKeeper, NormFinder and GeNorm indicated that gyrA, glyA and recA are the most stably expressed reference genes in A. brasilense. The results also suggested that the use of two reference genes (gyrA and glyA) is sufficient for effective normalisation of qRT-PCR data.

rpb2 is a reliable reference gene for quantitative gene expression analysis in the dermatophyte Trichophyton rubrum.

PubMed

Jacob, Tiago R; Peres, Nalu T A; Persinoti, Gabriela F; Silva, Larissa G; Mazucato, Mendelson; Rossi, Antonio; Martinez-Rossi, Nilce M

2012-05-01

The selection of reference genes used for data normalization to quantify gene expression by real-time PCR amplifications (qRT-PCR) is crucial for the accuracy of this technique. In spite of this, little information regarding such genes for qRT-PCR is available for gene expression analyses in pathogenic fungi. Thus, we investigated the suitability of eight candidate reference genes in isolates of the human dermatophyte Trichophyton rubrum subjected to several environmental challenges, such as drug exposure, interaction with human nail and skin, and heat stress. The stability of these genes was determined by geNorm, NormFinder and Best-Keeper programs. The gene with the most stable expression in the majority of the conditions tested was rpb2 (DNA-dependent RNA polymerase II), which was validated in three T. rubrum strains. Moreover, the combination of rpb2 and chs1 (chitin synthase) genes provided for the most reliable qRT-PCR data normalization in T. rubrum under a broad range of biological conditions. To the best of our knowledge this is the first report on the selection of reference genes for qRT-PCR data normalization in dermatophytes and the results of these studies should permit further analysis of gene expression under several experimental conditions, with improved accuracy and reliability.

Natural variation in gene expression in the early development of dauer larvae of Caenorhabditis elegans.

PubMed

Harvey, Simon C; Barker, Gary L A; Shorto, Alison; Viney, Mark E

2009-07-18

The free-living nematode Caenorhabditis elegans makes a developmental decision based on environmental conditions: larvae either arrest as dauer larva, or continue development into reproductive adults. There is natural variation among C. elegans lines in the sensitivity of this decision to environmental conditions; that is, there is variation in the phenotypic plasticity of dauer larva development. We hypothesised that these differences may be transcriptionally controlled in early stage larvae. We investigated this by microarray analysis of different C. elegans lines under different environmental conditions, specifically the presence and absence of dauer larva-inducing pheromone. There were substantial transcriptional differences between four C. elegans lines under the same environmental conditions. The expression of approximately 2,000 genes differed between genetically different lines, with each line showing a largely line-specific transcriptional profile. The expression of genes that are markers of larval moulting suggested that the lines may be developing at different rates. The expression of a total of 89 genes was putatively affected by dauer larva or non-dauer larva-inducing conditions. Among the upstream regions of these genes there was an over-representation of DAF-16-binding motifs. Under the same environmental conditions genetically different lines of C. elegans had substantial transcriptional differences. This variation may be due to differences in the developmental rates of the lines. Different environmental conditions had a rather smaller effect on transcription. The preponderance of DAF-16-binding motifs upstream of these genes was consistent with these genes playing a key role in the decision between development into dauer or into non-dauer larvae. There was little overlap between the genes whose expression was affected by environmental conditions and previously identified loci involved in the plasticity of dauer larva development.

Mining microbial metatranscriptomes for expression of antibiotic resistance genes under natural conditions

PubMed Central

Versluis, Dennis; D’Andrea, Marco Maria; Ramiro Garcia, Javier; Leimena, Milkha M.; Hugenholtz, Floor; Zhang, Jing; Öztürk, Başak; Nylund, Lotta; Sipkema, Detmer; Schaik, Willem van; de Vos, Willem M.; Kleerebezem, Michiel; Smidt, Hauke; Passel, Mark W.J. van

2015-01-01

Antibiotic resistance genes are found in a broad range of ecological niches associated with complex microbiota. Here we investigated if resistance genes are not only present, but also transcribed under natural conditions. Furthermore, we examined the potential for antibiotic production by assessing the expression of associated secondary metabolite biosynthesis gene clusters. Metatranscriptome datasets from intestinal microbiota of four human adults, one human infant, 15 mice and six pigs, of which only the latter have received antibiotics prior to the study, as well as from sea bacterioplankton, a marine sponge, forest soil and sub-seafloor sediment, were investigated. We found that resistance genes are expressed in all studied ecological niches, albeit with niche-specific differences in relative expression levels and diversity of transcripts. For example, in mice and human infant microbiota predominantly tetracycline resistance genes were expressed while in human adult microbiota the spectrum of expressed genes was more diverse, and also included β-lactam, aminoglycoside and macrolide resistance genes. Resistance gene expression could result from the presence of natural antibiotics in the environment, although we could not link it to expression of corresponding secondary metabolites biosynthesis clusters. Alternatively, resistance gene expression could be constitutive, or these genes serve alternative roles besides antibiotic resistance. PMID:26153129

Differential Anoxic Expression of Sugar-Regulated Genes Reveals Diverse Interactions between Sugar and Anaerobic Signaling Systems in Rice

PubMed Central

Lim, Mi-na; Lee, Sung-eun; Yim, Hui-kyeong; Kim, Jeong Hoe; Yoon, In Sun; Hwang, Yong-sic

2013-01-01

The interaction between the dual roles of sugar as a metabolic fuel and a regulatory molecule was unveiled by examining the changes in sugar signaling upon oxygen deprivation, which causes the drastic alteration in the cellular energy status. In our study, the expression of anaerobically induced genes is commonly responsive to sugar, either under the control of hexokinase or non-hexokinase mediated signaling cascades. Only sugar regulation via the hexokinase pathway was susceptible for O2 deficiency or energy deficit conditions evoked by uncoupler. Examination of sugar regulation of those genes under anaerobic conditions revealed the presence of multiple paths underlying anaerobic induction of gene expression in rice, subgrouped into three distinct types. The first of these, which was found in type-1 genes, involved neither sugar regulation nor additional anaerobic induction under anoxia, indicating that anoxic induction is a simple result from the release of sugar repression by O2-deficient conditions. In contrast, type-2 genes also showed no sugar regulation, albeit with enhanced expression under anoxia. Lastly, expression of type-3 genes is highly enhanced with sugar regulation sustained under anoxia. Intriguingly, the inhibition of the mitochondrial ATP synthesis can reproduce expression pattern of a specific set of anaerobically induced genes, implying that rice cells may sense O2 deprivation, partly via perception of the perturbed cellular energy status. Our study of interaction between sugar signaling and anaerobic conditions has revealed that sugar signaling and the cellular energy status are likely to communicate with each other and influence anaerobic induction of gene expression in rice. PMID:23852132

Exploring candidate biomarkers for lung and prostate cancers using gene expression and flux variability analysis.

PubMed

Asgari, Yazdan; Khosravi, Pegah; Zabihinpour, Zahra; Habibi, Mahnaz

2018-02-19

Genome-scale metabolic models have provided valuable resources for exploring changes in metabolism under normal and cancer conditions. However, metabolism itself is strongly linked to gene expression, so integration of gene expression data into metabolic models might improve the detection of genes involved in the control of tumor progression. Herein, we considered gene expression data as extra constraints to enhance the predictive powers of metabolic models. We reconstructed genome-scale metabolic models for lung and prostate, under normal and cancer conditions to detect the major genes associated with critical subsystems during tumor development. Furthermore, we utilized gene expression data in combination with an information theory-based approach to reconstruct co-expression networks of the human lung and prostate in both cohorts. Our results revealed 19 genes as candidate biomarkers for lung and prostate cancer cells. This study also revealed that the development of a complementary approach (integration of gene expression and metabolic profiles) could lead to proposing novel biomarkers and suggesting renovated cancer treatment strategies which have not been possible to detect using either of the methods alone.

Validation of reference genes for normalization of qPCR mRNA expression levels in Staphylococcus aureus exposed to osmotic and lactic acid stress conditions encountered during food production and preservation.

PubMed

Sihto, Henna-Maria; Tasara, Taurai; Stephan, Roger; Johler, Sophia

2014-07-01

Staphylococcus aureus represents the most prevalent cause of food-borne intoxications worldwide. While being repressed by competing bacteria in most matrices, this pathogen exhibits crucial competitive advantages during growth at high salt concentrations or low pH, conditions frequently encountered in food production and preservation. We aimed to identify reference genes that could be used to normalize qPCR mRNA expression levels during growth of S. aureus in food-related osmotic (NaCl) and acidic (lactic acid) stress adaptation models. Expression stability of nine housekeeping genes was evaluated in full (LB) and nutrient-deficient (CYGP w/o glucose) medium under conditions of osmotic (4.5% NaCl) and acidic stress (lactic acid, pH 6.0) after 2-h exposure. Among the set of candidate reference genes investigated, rplD, rpoB,gyrB, and rho were most stably expressed in LB and thus represent the most suitable reference genes for normalization of qPCR data in osmotic or lactic acid stress models in a rich medium. Under nutrient-deficient conditions, expression of rho and rpoB was highly stable across all tested conditions. The presented comprehensive data on changes in expression of various S. aureus housekeeping genes under conditions of osmotic and lactic acid stress facilitate selection of reference genes for qPCR-based stress response models. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Reference genes for measuring mRNA expression.

PubMed

Dundas, Jitesh; Ling, Maurice

2012-12-01

The aim of this review is to find answers to some of the questions surrounding reference genes and their reliability for quantitative experiments. Reference genes are assumed to be at a constant expression level, over a range of conditions such as temperature. These genes, such as GADPH and beta-actin, are used extensively for gene expression studies using techniques like quantitative PCR. There have been several studies carried out on identifying reference genes. However, a lot of evidence indicates issues to the general suitability of these genes. Recent studies had shown that different factors, including the environment and methods, play an important role in changing the expression levels of the reference genes. Thus, we conclude that there is no reference gene that can deemed suitable for all the experimental conditions. In addition, we believe that every experiment will require the scientific evaluation and selection of the best candidate gene for use as a reference gene to obtain reliable scientific results.

Annotation of gene function in citrus using gene expression information and co-expression networks

PubMed Central

2014-01-01

Background The genus Citrus encompasses major cultivated plants such as sweet orange, mandarin, lemon and grapefruit, among the world’s most economically important fruit crops. With increasing volumes of transcriptomics data available for these species, Gene Co-expression Network (GCN) analysis is a viable option for predicting gene function at a genome-wide scale. GCN analysis is based on a “guilt-by-association” principle whereby genes encoding proteins involved in similar and/or related biological processes may exhibit similar expression patterns across diverse sets of experimental conditions. While bioinformatics resources such as GCN analysis are widely available for efficient gene function prediction in model plant species including Arabidopsis, soybean and rice, in citrus these tools are not yet developed. Results We have constructed a comprehensive GCN for citrus inferred from 297 publicly available Affymetrix Genechip Citrus Genome microarray datasets, providing gene co-expression relationships at a genome-wide scale (33,000 transcripts). The comprehensive citrus GCN consists of a global GCN (condition-independent) and four condition-dependent GCNs that survey the sweet orange species only, all citrus fruit tissues, all citrus leaf tissues, or stress-exposed plants. All of these GCNs are clustered using genome-wide, gene-centric (guide) and graph clustering algorithms for flexibility of gene function prediction. For each putative cluster, gene ontology (GO) enrichment and gene expression specificity analyses were performed to enhance gene function, expression and regulation pattern prediction. The guide-gene approach was used to infer novel roles of genes involved in disease susceptibility and vitamin C metabolism, and graph-clustering approaches were used to investigate isoprenoid/phenylpropanoid metabolism in citrus peel, and citric acid catabolism via the GABA shunt in citrus fruit. Conclusions Integration of citrus gene co-expression networks, functional enrichment analysis and gene expression information provide opportunities to infer gene function in citrus. We present a publicly accessible tool, Network Inference for Citrus Co-Expression (NICCE, http://citrus.adelaide.edu.au/nicce/home.aspx), for the gene co-expression analysis in citrus. PMID:25023870

Gene expression analysis of E. coli strains provides insights into the role of gene regulation in diversification

PubMed Central

Vital, Marius; Chai, Benli; Østman, Bjørn; Cole, James; Konstantinidis, Konstantinos T; Tiedje, James M

2015-01-01

Escherichia coli spans a genetic continuum from enteric strains to several phylogenetically distinct, atypical lineages that are rare in humans, but more common in extra-intestinal environments. To investigate the link between gene regulation, phylogeny and diversification in this species, we analyzed global gene expression profiles of four strains representing distinct evolutionary lineages, including a well-studied laboratory strain, a typical commensal (enteric) strain and two environmental strains. RNA-Seq was employed to compare the whole transcriptomes of strains grown under batch, chemostat and starvation conditions. Highly differentially expressed genes showed a significantly lower nucleotide sequence identity compared with other genes, indicating that gene regulation and coding sequence conservation are directly connected. Overall, distances between the strains based on gene expression profiles were largely dependent on the culture condition and did not reflect phylogenetic relatedness. Expression differences of commonly shared genes (all four strains) and E. coli core genes were consistently smaller between strains characterized by more similar primary habitats. For instance, environmental strains exhibited increased expression of stress defense genes under carbon-limited growth and entered a more pronounced survival-like phenotype during starvation compared with other strains, which stayed more alert for substrate scavenging and catabolism during no-growth conditions. Since those environmental strains show similar genetic distance to each other and to the other two strains, these findings cannot be simply attributed to genetic relatedness but suggest physiological adaptations. Our study provides new insights into ecologically relevant gene-expression and underscores the role of (differential) gene regulation for the diversification of the model bacterial species. PMID:25343512

Analysis of the Salmonella regulatory network suggests involvement of SsrB and H-NS in σ E-regulated SPI-2 gene expression

DOE PAGES

Li, Jie; Overall, Christopher C.; Nakayasu, Ernesto S.; ...

2015-02-10

The extracytoplasmic functioning sigma factor σ E is known to play an essential role for Salmonella enterica serovar Typhimurium to survive and proliferate in macrophages and mice. However, its regulatory network is not well characterized, especially during infection. Here we used microarray to identify genes regulated by σ E in Salmonella grown in three conditions: a nutrient-rich condition and two others that mimic early and late intracellular infection. We found that in each condition σ E regulated different sets of genes, and notably, several global regulators. When comparing nutrient-rich and infection-like conditions, large changes were observed in the expression ofmore » genes involved in Salmonella pathogenesis island (SPI)-1 type-three secretion system (TTSS), SPI-2 TTSS, protein synthesis, and stress responses. In total, the expression of 58% of Salmonella genes was affected by σ E in at least one of the three conditions. An important finding is that σ E up-regulates SPI-2 genes, which are essential for Salmonella intracellular survival, by up-regulating SPI-2 activator ssrB expression at the early stage of infection and down-regulating SPI-2 repressor hns expression at a later stage. Moreover, σ E is capable of countering the silencing of H-NS, releasing the expression of SPI-2 genes. This connection between σ E and SPI-2 genes, combined with the global regulatory effect of σ E, may account for the lethality of rpoE-deficient Salmonella in murine infection.« less

Analysis of the Salmonella regulatory network suggests involvement of SsrB and H-NS in σ E-regulated SPI-2 gene expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Jie; Overall, Christopher C.; Nakayasu, Ernesto S.

The extracytoplasmic functioning sigma factor σ E is known to play an essential role for Salmonella enterica serovar Typhimurium to survive and proliferate in macrophages and mice. However, its regulatory network is not well characterized, especially during infection. Here we used microarray to identify genes regulated by σ E in Salmonella grown in three conditions: a nutrient-rich condition and two others that mimic early and late intracellular infection. We found that in each condition σ E regulated different sets of genes, and notably, several global regulators. When comparing nutrient-rich and infection-like conditions, large changes were observed in the expression ofmore » genes involved in Salmonella pathogenesis island (SPI)-1 type-three secretion system (TTSS), SPI-2 TTSS, protein synthesis, and stress responses. In total, the expression of 58% of Salmonella genes was affected by σ E in at least one of the three conditions. An important finding is that σ E up-regulates SPI-2 genes, which are essential for Salmonella intracellular survival, by up-regulating SPI-2 activator ssrB expression at the early stage of infection and down-regulating SPI-2 repressor hns expression at a later stage. Moreover, σ E is capable of countering the silencing of H-NS, releasing the expression of SPI-2 genes. This connection between σ E and SPI-2 genes, combined with the global regulatory effect of σ E, may account for the lethality of rpoE-deficient Salmonella in murine infection.« less

Effects of Simulated Microgravity on the Expression Profile of Microrna in Human Lymphoblastoid Cells

NASA Astrophysics Data System (ADS)

Zhang, Ye; Wu, Honglu; Ramesh, Govindarajan; Rohde, Larry; Story, Michael; Mangala, Lingegowda

2012-07-01

EFFECTS OF SIMULATED MICROGRAVITY ON THE EXPRESSION PROFILE OF MICRORNA IN HUMAN LYMPHOBLASTOID CELLS Lingegowda S. Mangala1,2, Ye Zhang1,3, Zhenhua He2, Kamal Emami1, Govindarajan T. Ramesh4, Michael Story 5, Larry H. Rohde2, and Honglu Wu1 1 NASA Johnson Space Center, Houston, Texas, USA 2 University of Houston Clear Lake, Houston, Texas, USA 3 Wyle Integrated Science and Engineering Group, Houston, Texas, USA 4 Norfolk State University, Norfolk, VA, USA 5 University of Texas, Southwestern Medical Center, Dallas, Texas, USA This study explores the changes in expression of microRNA (miRNA) and related genes under simulated microgravity conditions. In comparison to static 1g, microgravity has been shown to alter global gene expression patterns and protein levels in cultured cells or animals. miRNA has recently emerged as an important regulator of gene expression, possibly regulating as many as one-third of all human genes. However, very little is known about the effect of altered gravity on miRNA expression. To test the hypothesis that the miRNA expression profile would be altered in zero gravity resulting in altered regulation of gene expression leading to metabolic or functional changes in cells, we cultured TK6 human lymphoblastoid cells in a High Aspect Ratio Vessel (HARV; bioreactor) for 72 h either in the rotating condition to model microgravity in space or in the static condition as a control. Expression of several miRNA was changed significantly in the simulated microgravity condition including miR-150, miR-34a, miR-423-5p, miR-22 and miR-141, miR-618 and miR-222. To confirm whether this altered miRNA expression correlates with gene expression and functional changes of the cells, we performed DNA microarray and validated the related genes using q-RT PCR. Network and pathway analysis of gene and miRNA expression profiles indicates that the regulation of cell communication and catalytic activities, as well as pathways involved in immune response_IL-15 signaling and NGF mediated NF-kB activation were significantly altered under the simulated microgravity condition.

Dynamic processes at stress promoters regulate the bimodal expression of HOG response genes

PubMed Central

2011-01-01

Osmotic stress triggers the activation of the HOG (high osmolarity glycerol) pathway in Saccharomyces cerevisiae. This signaling cascade culminates in the activation of the MAPK (mitogen-activated protein kinase) Hog1. Quantitative single cell measurements revealed a discrepancy between kinase- and transcriptional activities of Hog1. While kinase activity increases proportionally to stress stimulus, gene expression is inhibited under low stress conditions. Interestingly, a slow stochastic gene activation process is responsible for setting a tunable threshold for gene expression under basal or low stress conditions, which generates a bimodal expression pattern at intermediate stress levels. PMID:22446531

Plasticity-Related Gene Expression During Eszopiclone-Induced Sleep.

PubMed

Gerashchenko, Dmitry; Pasumarthi, Ravi K; Kilduff, Thomas S

2017-07-01

Experimental evidence suggests that restorative processes depend on synaptic plasticity changes in the brain during sleep. We used the expression of plasticity-related genes to assess synaptic plasticity changes during drug-induced sleep. We first characterized sleep induced by eszopiclone in mice during baseline conditions and during the recovery from sleep deprivation. We then compared the expression of 18 genes and two miRNAs critically involved in synaptic plasticity in these mice. Gene expression was assessed in the cerebral cortex and hippocampus by the TaqMan reverse transcription polymerase chain reaction and correlated with sleep parameters. Eszopiclone reduced the latency to nonrapid eye movement (NREM) sleep and increased NREM sleep amounts. Eszopiclone had no effect on slow wave activity (SWA) during baseline conditions but reduced the SWA increase during recovery sleep (RS) after sleep deprivation. Gene expression analyses revealed three distinct patterns: (1) four genes had higher expression either in the cortex or hippocampus in the group of mice with increased amounts of wakefulness; (2) a large proportion of plasticity-related genes (7 out of 18 genes) had higher expression during RS in the cortex but not in the hippocampus; and (3) six genes and the two miRNAs showed no significant changes across conditions. Even at a relatively high dose (20 mg/kg), eszopiclone did not reduce the expression of plasticity-related genes during RS period in the cortex. These results indicate that gene expression associated with synaptic plasticity occurs in the cortex in the presence of a hypnotic medication. © Sleep Research Society 2017. Published by Oxford University Press on behalf of the Sleep Research Society. All rights reserved. For permissions, please e-mail journals.permissions@oup.com.

Effect of simulated microgravity on oxidation-sensitive gene expression in PC12 cells

NASA Astrophysics Data System (ADS)

Kwon, Ohwon; Sartor, Maureen; Tomlinson, Craig R.; Millard, Ronald W.; Olah, Mark E.; Sankovic, John M.; Banerjee, Rupak K.

2006-01-01

Oxygen utilization by and oxygen dependence of cellular processes may be different in biological systems that are exposed to microgravity (micro-g). A baseline in which cellular changes in oxygen sensitive molecular processes occur during micro-g conditions would be important to pursue this question. The objective of this research is to analyze oxidation-sensitive gene expression in a model cell line [rat pheochromocytoma (PC12)] under simulated micro-g conditions. The PC12 cell line is well characterized in its response to oxygen, and is widely recognized as a sensitive model for studying the responses of oxygen-sensitive molecular and cellular processes. This study uses the rotating wall vessel bioreactor (RWV) designed at NASA to simulate micro-g. Gene expression in PC12 cells in response to micro-g was analyzed by DNA microarray technology. The microarray analysis of PC12 cells cultured for 4 days under simulated micro-g under standardized oxygen environment conditions revealed more than 100 genes whose expression levels were changed at least twofold (up-regulation of 65 genes and down-regulation of 39 genes) compared with those from cells in the unit gravity (unit-g) control. This study observed that genes involved in the oxidoreductase activity category were most significantly differentially expressed under micro-g conditions. Also, known oxidation-sensitive transcription factors such as hypoxia-inducible factor-2α, c-myc, and the peroxisome proliferator-activated receptor-γ were changed significantly. Our initial results from the gene expression microarray studies may provide a context in which to evaluate the effect of varying oxygen environments on the background of differential gene regulation of biological processes under variable gravity conditions.

Effect of simulated microgravity on oxidation-sensitive gene expression in PC12 cells

PubMed Central

Kwon, Ohwon; Sartor, Maureen; Tomlinson, Craig R.; Millard, Ronald W.; Olah, Mark E.; Sankovic, John M.; Banerjee, Rupak K.

2008-01-01

Oxygen utilization by and oxygen dependence of cellular processes may be different in biological systems that are exposed to microgravity (micro-g). A baseline in which cellular changes in oxygen sensitive molecular processes occur during micro-g conditions would be important to pursue this question. The objective of this research is to analyze oxidation-sensitive gene expression in a model cell line [rat pheochromocytoma (PC12)] under simulated micro-g conditions. The PC12 cell line is well characterized in its response to oxygen, and is widely recognized as a sensitive model for studying the responses of oxygen-sensitive molecular and cellular processes. This study uses the rotating wall vessel bioreactor (RWV) designed at NASA to simulate micro-g. Gene expression in PC12 cells in response to micro-g was analyzed by DNA microarray technology. The microarray analysis of PC12 cells cultured for 4 days under simulated micro-g under standardized oxygen environment conditions revealed more than 100 genes whose expression levels were changed at least twofold (up-regulation of 65 genes and down-regulation of 39 genes) compared with those from cells in the unit gravity (unit-g) control. This study observed that genes involved in the oxidoreductase activity category were most significantly differentially expressed under micro-g conditions. Also, known oxidation-sensitive transcription factors such as hypoxia-inducible factor-2α, c-myc, and the peroxisome proliferator-activated receptor-γ were changed significantly. Our initial results from the gene expression microarray studies may provide a context in which to evaluate the effect of varying oxygen environments on the background of differential gene regulation of biological processes under variable gravity conditions. PMID:19081771

Validation of Reference Genes for RT-qPCR Studies of Gene Expression in Preharvest and Postharvest Longan Fruits under Different Experimental Conditions

PubMed Central

Wu, Jianyang; Zhang, Hongna; Liu, Liqin; Li, Weicai; Wei, Yongzan; Shi, Shengyou

2016-01-01

Reverse transcription quantitative PCR (RT-qPCR) as the accurate and sensitive method is use for gene expression analysis, but the veracity and reliability result depends on whether select appropriate reference gene or not. To date, several reliable reference gene validations have been reported in fruits trees, but none have been done on preharvest and postharvest longan fruits. In this study, 12 candidate reference genes, namely, CYP, RPL, GAPDH, TUA, TUB, Fe-SOD, Mn-SOD, Cu/Zn-SOD, 18SrRNA, Actin, Histone H3, and EF-1a, were selected. Expression stability of these genes in 150 longan samples was evaluated and analyzed using geNorm and NormFinder algorithms. Preharvest samples consisted of seven experimental sets, including different developmental stages, organs, hormone stimuli (NAA, 2,4-D, and ethephon) and abiotic stresses (bagging and girdling with defoliation). Postharvest samples consisted of different temperature treatments (4 and 22°C) and varieties. Our findings indicate that appropriate reference gene(s) should be picked for each experimental condition. Our data further showed that the commonly used reference gene Actin does not exhibit stable expression across experimental conditions in longan. Expression levels of the DlACO gene, which is a key gene involved in regulating fruit abscission under girdling with defoliation treatment, was evaluated to validate our findings. In conclusion, our data provide a useful framework for choice of suitable reference genes across different experimental conditions for RT-qPCR analysis of preharvest and postharvest longan fruits. PMID:27375640

Maximizing capture of gene co-expression relationships through pre-clustering of input expression samples: an Arabidopsis case study.

PubMed

Feltus, F Alex; Ficklin, Stephen P; Gibson, Scott M; Smith, Melissa C

2013-06-05

In genomics, highly relevant gene interaction (co-expression) networks have been constructed by finding significant pair-wise correlations between genes in expression datasets. These networks are then mined to elucidate biological function at the polygenic level. In some cases networks may be constructed from input samples that measure gene expression under a variety of different conditions, such as for different genotypes, environments, disease states and tissues. When large sets of samples are obtained from public repositories it is often unmanageable to associate samples into condition-specific groups, and combining samples from various conditions has a negative effect on network size. A fixed significance threshold is often applied also limiting the size of the final network. Therefore, we propose pre-clustering of input expression samples to approximate condition-specific grouping of samples and individual network construction of each group as a means for dynamic significance thresholding. The net effect is increase sensitivity thus maximizing the total co-expression relationships in the final co-expression network compendium. A total of 86 Arabidopsis thaliana co-expression networks were constructed after k-means partitioning of 7,105 publicly available ATH1 Affymetrix microarray samples. We term each pre-sorted network a Gene Interaction Layer (GIL). Random Matrix Theory (RMT), an un-supervised thresholding method, was used to threshold each of the 86 networks independently, effectively providing a dynamic (non-global) threshold for the network. The overall gene count across all GILs reached 19,588 genes (94.7% measured gene coverage) and 558,022 unique co-expression relationships. In comparison, network construction without pre-sorting of input samples yielded only 3,297 genes (15.9%) and 129,134 relationships. in the global network. Here we show that pre-clustering of microarray samples helps approximate condition-specific networks and allows for dynamic thresholding using un-supervised methods. Because RMT ensures only highly significant interactions are kept, the GIL compendium consists of 558,022 unique high quality A. thaliana co-expression relationships across almost all of the measurable genes on the ATH1 array. For A. thaliana, these networks represent the largest compendium to date of significant gene co-expression relationships, and are a means to explore complex pathway, polygenic, and pleiotropic relationships for this focal model plant. The networks can be explored at sysbio.genome.clemson.edu. Finally, this method is applicable to any large expression profile collection for any organism and is best suited where a knowledge-independent network construction method is desired.

Maximizing capture of gene co-expression relationships through pre-clustering of input expression samples: an Arabidopsis case study

PubMed Central

2013-01-01

Background In genomics, highly relevant gene interaction (co-expression) networks have been constructed by finding significant pair-wise correlations between genes in expression datasets. These networks are then mined to elucidate biological function at the polygenic level. In some cases networks may be constructed from input samples that measure gene expression under a variety of different conditions, such as for different genotypes, environments, disease states and tissues. When large sets of samples are obtained from public repositories it is often unmanageable to associate samples into condition-specific groups, and combining samples from various conditions has a negative effect on network size. A fixed significance threshold is often applied also limiting the size of the final network. Therefore, we propose pre-clustering of input expression samples to approximate condition-specific grouping of samples and individual network construction of each group as a means for dynamic significance thresholding. The net effect is increase sensitivity thus maximizing the total co-expression relationships in the final co-expression network compendium. Results A total of 86 Arabidopsis thaliana co-expression networks were constructed after k-means partitioning of 7,105 publicly available ATH1 Affymetrix microarray samples. We term each pre-sorted network a Gene Interaction Layer (GIL). Random Matrix Theory (RMT), an un-supervised thresholding method, was used to threshold each of the 86 networks independently, effectively providing a dynamic (non-global) threshold for the network. The overall gene count across all GILs reached 19,588 genes (94.7% measured gene coverage) and 558,022 unique co-expression relationships. In comparison, network construction without pre-sorting of input samples yielded only 3,297 genes (15.9%) and 129,134 relationships. in the global network. Conclusions Here we show that pre-clustering of microarray samples helps approximate condition-specific networks and allows for dynamic thresholding using un-supervised methods. Because RMT ensures only highly significant interactions are kept, the GIL compendium consists of 558,022 unique high quality A. thaliana co-expression relationships across almost all of the measurable genes on the ATH1 array. For A. thaliana, these networks represent the largest compendium to date of significant gene co-expression relationships, and are a means to explore complex pathway, polygenic, and pleiotropic relationships for this focal model plant. The networks can be explored at sysbio.genome.clemson.edu. Finally, this method is applicable to any large expression profile collection for any organism and is best suited where a knowledge-independent network construction method is desired. PMID:23738693

Abiotic conditions leading to FUM gene expression and fumonisin accumulation by Fusarium proliferatum strains grown on a wheat-based substrate.

PubMed

Cendoya, Eugenia; Pinson-Gadais, Laetitia; Farnochi, María C; Ramirez, María L; Chéreau, Sylvain; Marcheguay, Giselè; Ducos, Christine; Barreau, Christian; Richard-Forget, Florence

2017-07-17

Fusarium proliferatum produces fumonisins B not only on maize but also on diverse crops including wheat. Using a wheat-based medium, the effects of abiotic factors, temperature and water activity (a W ), on growth, fumonisin biosynthesis, and expression of FUM genes were compared for three F. proliferatum strains isolated from durum wheat in Argentina. Although all isolates showed similar profiles of growth, the fumonisin production profiles were slightly different. Regarding FUM gene transcriptional control, both FUM8 and FUM19 expression showed similar behavior in all tested conditions. For both genes, expression at 25°C correlated with fumonisin production, regardless of the a w conditions. However, at 15°C, these two genes were as highly expressed as at 25°C although the amounts of toxin were very weak, suggesting that the kinetics of fumonisin production was slowed at 15°C. This study provides useful baseline data on conditions representing a low or a high risk for contamination of wheat kernels with fumonisins. Copyright © 2017 Elsevier B.V. All rights reserved.

Regulation of endogenous human gene expression by ligand-inducible TALE transcription factors.

PubMed

Mercer, Andrew C; Gaj, Thomas; Sirk, Shannon J; Lamb, Brian M; Barbas, Carlos F

2014-10-17

The construction of increasingly sophisticated synthetic biological circuits is dependent on the development of extensible tools capable of providing specific control of gene expression in eukaryotic cells. Here, we describe a new class of synthetic transcription factors that activate gene expression in response to extracellular chemical stimuli. These inducible activators consist of customizable transcription activator-like effector (TALE) proteins combined with steroid hormone receptor ligand-binding domains. We demonstrate that these ligand-responsive TALE transcription factors allow for tunable and conditional control of gene activation and can be used to regulate the expression of endogenous genes in human cells. Since TALEs can be designed to recognize any contiguous DNA sequence, the conditional gene regulatory system described herein will enable the design of advanced synthetic gene networks.

A Hybrid One-Way ANOVA Approach for the Robust and Efficient Estimation of Differential Gene Expression with Multiple Patterns

PubMed Central

Mollah, Mohammad Manir Hossain; Jamal, Rahman; Mokhtar, Norfilza Mohd; Harun, Roslan; Mollah, Md. Nurul Haque

2015-01-01

Background Identifying genes that are differentially expressed (DE) between two or more conditions with multiple patterns of expression is one of the primary objectives of gene expression data analysis. Several statistical approaches, including one-way analysis of variance (ANOVA), are used to identify DE genes. However, most of these methods provide misleading results for two or more conditions with multiple patterns of expression in the presence of outlying genes. In this paper, an attempt is made to develop a hybrid one-way ANOVA approach that unifies the robustness and efficiency of estimation using the minimum β-divergence method to overcome some problems that arise in the existing robust methods for both small- and large-sample cases with multiple patterns of expression. Results The proposed method relies on a β-weight function, which produces values between 0 and 1. The β-weight function with β = 0.2 is used as a measure of outlier detection. It assigns smaller weights (≥ 0) to outlying expressions and larger weights (≤ 1) to typical expressions. The distribution of the β-weights is used to calculate the cut-off point, which is compared to the observed β-weight of an expression to determine whether that gene expression is an outlier. This weight function plays a key role in unifying the robustness and efficiency of estimation in one-way ANOVA. Conclusion Analyses of simulated gene expression profiles revealed that all eight methods (ANOVA, SAM, LIMMA, EBarrays, eLNN, KW, robust BetaEB and proposed) perform almost identically for m = 2 conditions in the absence of outliers. However, the robust BetaEB method and the proposed method exhibited considerably better performance than the other six methods in the presence of outliers. In this case, the BetaEB method exhibited slightly better performance than the proposed method for the small-sample cases, but the the proposed method exhibited much better performance than the BetaEB method for both the small- and large-sample cases in the presence of more than 50% outlying genes. The proposed method also exhibited better performance than the other methods for m > 2 conditions with multiple patterns of expression, where the BetaEB was not extended for this condition. Therefore, the proposed approach would be more suitable and reliable on average for the identification of DE genes between two or more conditions with multiple patterns of expression. PMID:26413858

Effect of culturing conditions on the expression of key enzymes in the proteolytic system of Lactobacillus bulgaricus *

PubMed Central

Hou, Jun-cai; Liu, Fei; Ren, Da-xi; Han, Wei-wei; Du, Yue-ou

2015-01-01

The proteolytic system of Lactobacillus bulgaricus breaks down milk proteins into peptides and amino acids, which are essential for the growth of the bacteria. The aim of this study was to determine the expressions of seven key genes in the proteolytic system under different culturing conditions (different phases, initial pH values, temperatures, and nitrogen sources) using real-time polymerase chain reaction (RT-PCR). The transcriptions of the seven genes were reduced by 30-fold on average in the stationary phase compared with the exponential growth phase. The transcriptions of the seven genes were reduced by 62.5-, 15.0-, and 59.0-fold in the strains KLDS 08006, KLDS 08007, and KLDS 08012, respectively, indicating that the expressions of the seven genes were significantly different among strains. In addition, the expressions of the seven genes were repressed in the MRS medium containing casein peptone. The effect of peptone supply on PepX transcription was the weakest compared with the other six genes, and the impact on OppD transcription was the strongest. Moreover, the expressions of the seven genes were significantly different among different strains (P<0.05). All these results indicated that the culturing conditions affected the expression of the proteolytic system genes in Lactobacillus bulgaricus at the transcription level. PMID:25845365

Diversity in Expression of Phosphorus (P) Responsive Genes in Cucumis melo L

PubMed Central

Fita, Ana; Bowen, Helen C.; Hayden, Rory M.; Nuez, Fernando; Picó, Belén; Hammond, John P.

2012-01-01

Background Phosphorus (P) is a major limiting nutrient for plant growth in many soils. Studies in model species have identified genes involved in plant adaptations to low soil P availability. However, little information is available on the genetic bases of these adaptations in vegetable crops. In this respect, sequence data for melon now makes it possible to identify melon orthologues of candidate P responsive genes, and the expression of these genes can be used to explain the diversity in the root system adaptation to low P availability, recently observed in this species. Methodology and Findings Transcriptional responses to P starvation were studied in nine diverse melon accessions by comparing the expression of eight candidate genes (Cm-PAP10.1, Cm-PAP10.2, Cm-RNS1, Cm-PPCK1, Cm-transferase, Cm-SQD1, Cm-DGD1 and Cm-SPX2) under P replete and P starved conditions. Differences among melon accessions were observed in response to P starvation, including differences in plant morphology, P uptake, P use efficiency (PUE) and gene expression. All studied genes were up regulated under P starvation conditions. Differences in the expression of genes involved in P mobilization and remobilization (Cm-PAP10.1, Cm-PAP10.2 and Cm-RNS1) under P starvation conditions explained part of the differences in P uptake and PUE among melon accessions. The levels of expression of the other studied genes were diverse among melon accessions, but contributed less to the phenotypical response of the accessions. Conclusions This is the first time that these genes have been described in the context of P starvation responses in melon. There exists significant diversity in gene expression levels and P use efficiency among melon accessions as well as significant correlations between gene expression levels and phenotypical measurements. PMID:22536378

Suboptimal evolutionary novel environments promote singular altered gravity responses of transcriptome during Drosophila metamorphosis

PubMed Central

2013-01-01

Background Previous experiments have shown that the reduced gravity aboard the International Space Station (ISS) causes important alterations in Drosophila gene expression. These changes were shown to be intimately linked to environmental space-flight related constraints. Results Here, we use an array of different techniques for ground-based simulation of microgravity effects to assess the effect of suboptimal environmental conditions on the gene expression of Drosophila in reduced gravity. A global and integrative analysis, using “gene expression dynamics inspector” (GEDI) self-organizing maps, reveals different degrees in the responses of the transcriptome when using different environmental conditions or microgravity/hypergravity simulation devices. Although the genes that are affected are different in each simulation technique, we find that the same gene ontology groups, including at least one large multigene family related with behavior, stress response or organogenesis, are over represented in each case. Conclusions These results suggest that the transcriptome as a whole can be finely tuned to gravity force. In optimum environmental conditions, the alteration of gravity has only mild effects on gene expression but when environmental conditions are far from optimal, the gene expression must be tuned greatly and effects become more robust, probably linked to the lack of experience of organisms exposed to evolutionary novel environments such as a gravitational free one. PMID:23806134

Auxins upregulate nif and fix genes.

PubMed

Bianco, Carmen; Defez, Roberto

2010-10-01

In a recent publication we analyzed the global effects triggered by IAA overproduction in S. meliloti RD64 under free-living conditions by comparing the gene expression pattern of wild type 1021 with that of RD64 and 1021 treated with IAA and other four chemically or functionally related molecules. Among the genes differentially expressed in RD64 and IAA-treated 1021 cells we found two genes of pho operon, phoT and phoC. Based on this finding we examined the mechanisms for mineral P solubilization in RD64 and the potential ability of this strain to improve Medicago growth under P-starved conditions. Here, we further analyze the expression profiles obtained in microarray analysis and evaluate the specificity and the extent of overlap between all treatments. Venn diagrams indicated that IAA- and 2,4-D-regulated genes were closely related. Furthermore, most differentially expressed genes from pSymA were induced in 1021 cells treated with 2,4-D, ICA, IND and Trp as compared to the untreated 1021 cells. RT-PCR analysis was employed to analyze the differential expression patterns of nitrogen fixation genes under free-living and symbiotic conditions. Under symbiotic condition, the relative expression levels of nif and fix genes were significantly induced in Mt- RD64 plants and in Mt-1021 plants treated with IAA and 2,4-D whereas they were unchanged or repressed in Mt-1021 plants treated with the other selected compounds when compared to the untreated Mt-1021 plants. © 2010 Landes Bioscience

Transcriptome Analysis of Plant Hormone-Related Tomato (Solanum lycopersicum) Genes in a Sunlight-Type Plant Factory.

PubMed

Tanigaki, Yusuke; Higashi, Takanobu; Takayama, Kotaro; Nagano, Atsushi J; Honjo, Mie N; Fukuda, Hirokazu

2015-01-01

In plant factories, measurements of plant conditions are necessary at an early stage of growth to predict harvest times of high value-added crops. Moreover, harvest qualities depend largely on environmental stresses that elicit plant hormone responses. However, the complexities of plant hormone networks have not been characterized under nonstress conditions. In the present study, we determined temporal expression profiles of all genes and then focused on plant hormone pathways using RNA-Seq analyses of gene expression in tomato leaves every 2 h for 48 h. In these experiments, temporally expressed genes were found in the hormone synthesis pathways for salicylic acid, abscisic acid, ethylene, and jasmonic acid. The timing of CAB expression 1 (TOC1) and abscisic acid insensitive 1 (ABA1) and open stomata 1 (OST1) control gating stomata. In this study, compare with tomato and Arabidopsis thaliana, expression patterns of TOC1 have similarity. In contrast, expression patterns of tomato ABI1 and OST1 had expression peak at different time. These findings suggest that the regulation of gating stomata does not depend predominantly on TOC1 and significantly reflects the extracellular environment. The present data provide new insights into relationships between temporally expressed plant hormone-related genes and clock genes under normal sunlight conditions.

Transcriptome Analysis of Plant Hormone-Related Tomato (Solanum lycopersicum) Genes in a Sunlight-Type Plant Factory

PubMed Central

Tanigaki, Yusuke; Higashi, Takanobu; Takayama, Kotaro; Nagano, Atsushi J.; Honjo, Mie N.; Fukuda, Hirokazu

2015-01-01

In plant factories, measurements of plant conditions are necessary at an early stage of growth to predict harvest times of high value-added crops. Moreover, harvest qualities depend largely on environmental stresses that elicit plant hormone responses. However, the complexities of plant hormone networks have not been characterized under nonstress conditions. In the present study, we determined temporal expression profiles of all genes and then focused on plant hormone pathways using RNA-Seq analyses of gene expression in tomato leaves every 2 h for 48 h. In these experiments, temporally expressed genes were found in the hormone synthesis pathways for salicylic acid, abscisic acid, ethylene, and jasmonic acid. The timing of CAB expression 1 (TOC1) and abscisic acid insensitive 1 (ABA1) and open stomata 1 (OST1) control gating stomata. In this study, compare with tomato and Arabidopsis thaliana, expression patterns of TOC1 have similarity. In contrast, expression patterns of tomato ABI1 and OST1 had expression peak at different time. These findings suggest that the regulation of gating stomata does not depend predominantly on TOC1 and significantly reflects the extracellular environment. The present data provide new insights into relationships between temporally expressed plant hormone-related genes and clock genes under normal sunlight conditions. PMID:26624004

Identification of suitable qPCR reference genes in leaves of Brassica oleracea under abiotic stresses.

PubMed

Brulle, Franck; Bernard, Fabien; Vandenbulcke, Franck; Cuny, Damien; Dumez, Sylvain

2014-04-01

Real-time quantitative PCR is nowadays a standard method to study gene expression variations in various samples and experimental conditions. However, to interpret results accurately, data normalization with appropriate reference genes appears to be crucial. The present study describes the identification and the validation of suitable reference genes in Brassica oleracea leaves. Expression stability of eight candidates was tested following drought and cold abiotic stresses by using three different softwares (BestKeeper, NormFinder and geNorm). Four genes (BolC.TUB6, BolC.SAND1, BolC.UBQ2 and BolC.TBP1) emerged as the most stable across the tested conditions. Further gene expression analysis of a drought- and a cold-responsive gene (BolC.DREB2A and BolC.ELIP, respectively), confirmed the stability and the reliability of the identified reference genes when used for normalization in the leaves of B. oleracea. These four genes were finally tested upon a benzene exposure and all appeared to be useful reference genes along this toxicological condition. These results provide a good starting point for future studies involving gene expression measurement on leaves of B. oleracea exposed to environmental modifications.

A condition-specific codon optimization approach for improved heterologous gene expression in Saccharomyces cerevisiae

PubMed Central

2014-01-01

Background Heterologous gene expression is an important tool for synthetic biology that enables metabolic engineering and the production of non-natural biologics in a variety of host organisms. The translational efficiency of heterologous genes can often be improved by optimizing synonymous codon usage to better match the host organism. However, traditional approaches for optimization neglect to take into account many factors known to influence synonymous codon distributions. Results Here we define an alternative approach for codon optimization that utilizes systems level information and codon context for the condition under which heterologous genes are being expressed. Furthermore, we utilize a probabilistic algorithm to generate multiple variants of a given gene. We demonstrate improved translational efficiency using this condition-specific codon optimization approach with two heterologous genes, the fluorescent protein-encoding eGFP and the catechol 1,2-dioxygenase gene CatA, expressed in S. cerevisiae. For the latter case, optimization for stationary phase production resulted in nearly 2.9-fold improvements over commercial gene optimization algorithms. Conclusions Codon optimization is now often a standard tool for protein expression, and while a variety of tools and approaches have been developed, they do not guarantee improved performance for all hosts of applications. Here, we suggest an alternative method for condition-specific codon optimization and demonstrate its utility in Saccharomyces cerevisiae as a proof of concept. However, this technique should be applicable to any organism for which gene expression data can be generated and is thus of potential interest for a variety of applications in metabolic and cellular engineering. PMID:24636000

Environmental history impacts gene expression during diapause development in the alfalfa leafcutting bee, Megachile rotundata.

PubMed

Yocum, George D; Childers, Anna K; Rinehart, Joseph P; Rajamohan, Arun; Pitts-Singer, Theresa L; Greenlee, Kendra J; Bowsher, Julia H

2018-05-10

Our understanding of the mechanisms controlling insect diapause has increased dramatically with the introduction of global gene expression techniques, such as RNA-seq. However, little attention has been given to how ecologically relevant field conditions may affect gene expression during diapause development because previous studies have focused on laboratory reared and maintained insects. To determine whether gene expression differs between laboratory and field conditions, prepupae of the alfalfa leafcutting bee, Megachile rotundata , entering diapause early or late in the growing season were collected. These two groups were further subdivided in early autumn into laboratory and field maintained groups, resulting in four experimental treatments of diapausing prepupae: early and late field, and early and late laboratory. RNA-seq and differential expression analyses were performed on bees from the four treatment groups in November, January, March and May. The number of treatment-specific differentially expressed genes (97 to 1249) outnumbered the number of differentially regulated genes common to all four treatments (14 to 229), indicating that exposure to laboratory or field conditions had a major impact on gene expression during diapause development. Principle component analysis and hierarchical cluster analysis yielded similar grouping of treatments, confirming that the treatments form distinct clusters. Our results support the conclusion that gene expression during the course of diapause development is not a simple ordered sequence, but rather a highly plastic response determined primarily by the environmental history of the individual insect. © 2018. Published by The Company of Biologists Ltd.

Comparative transcriptome analysis of pepper (Capsicum annuum) revealed common regulons in multiple stress conditions and hormone treatments.

PubMed

Lee, Sanghyeob; Choi, Doil

2013-09-01

Global transcriptome analysis revealed common regulons for biotic/abiotic stresses, and some of these regulons encoding signaling components in both stresses were newly identified in this study. In this study, we aimed to identify plant responses to multiple stress conditions and discover the common regulons activated under a variety of stress conditions. Global transcriptome analysis revealed that salicylic acid (SA) may affect the activation of abiotic stress-responsive genes in pepper. Our data indicate that methyl jasmonate (MeJA) and ethylene (ET)-responsive genes were primarily activated by biotic stress, while abscisic acid (ABA)-responsive genes were activated under both types of stresses. We also identified differentially expressed gene (DEG) responses to specific stress conditions. Biotic stress induces more DEGs than those induced by abiotic and hormone applications. The clustering analysis using DEGs indicates that there are common regulons for biotic or abiotic stress conditions. Although SA and MeJA have an antagonistic effect on gene expression levels, SA and MeJA show a largely common regulation as compared to the regulation at the DEG expression level induced by other hormones. We also monitored the expression profiles of DEG encoding signaling components. Twenty-two percent of these were commonly expressed in both stress conditions. The importance of this study is that several genes commonly regulated by both stress conditions may have future applications for creating broadly stress-tolerant pepper plants. This study revealed that there are complex regulons in pepper plant to both biotic and abiotic stress conditions.

Transcription profile of brewery yeast under fermentation conditions.

PubMed

James, T C; Campbell, S; Donnelly, D; Bond, U

2003-01-01

Yeast strains, used in the brewing industry, experience distinctive physiological conditions. During a brewing fermentation, yeast are exposed to anaerobic conditions, high pressure, high specific gravity and low temperatures. The purpose of this study was to examine the global gene expression profile of yeast subjected to brewing stress. We have carried out a microarray analysis of a typical brewer's yeast during the course of an 8-day fermentation in 15 degrees P wort. We used the probes derived from Saccharomyces cerevisiae genomic DNA on the chip and RNA isolated from three stages of brewing. This analysis shows a high level of expression of genes involved in fatty acid and ergosterol biosynthesis early in fermentation. Furthermore, genes involved in respiration and mitochondrial protein synthesis also show higher levels of expression. Surprisingly, we observed a complete repression of many stress response genes and genes involved in protein synthesis throughout the 8-day period compared with that at the start of fermentation. This microarray data set provides an analysis of gene expression under brewing fermentation conditions. The data provide an insight into the various metabolic processes altered or activated by brewing conditions of growth. This study leads to future experiments whereby selective alterations in brewing conditions could be introduced to take advantage of the changing transcript profile to improve the quality of the brew.

Identification of a Novel Reference Gene for Apple Transcriptional Profiling under Postharvest Conditions

PubMed Central

Storch, Tatiane Timm; Pegoraro, Camila; Finatto, Taciane; Quecini, Vera; Rombaldi, Cesar Valmor; Girardi, César Luis

2015-01-01

Reverse Transcription quantitative PCR (RT-qPCR) is one of the most important techniques for gene expression profiling due to its high sensibility and reproducibility. However, the reliability of the results is highly dependent on data normalization, performed by comparisons between the expression profiles of the genes of interest against those of constitutively expressed, reference genes. Although the technique is widely used in fruit postharvest experiments, the transcription stability of reference genes has not been thoroughly investigated under these experimental conditions. Thus, we have determined the transcriptional profile, under these conditions, of three genes commonly used as reference—ACTIN (MdACT), PROTEIN DISULPHIDE ISOMERASE (MdPDI) and UBIQUITIN-CONJUGATING ENZYME E2 (MdUBC)—along with two novel candidates—HISTONE 1 (MdH1) and NUCLEOSSOME ASSEMBLY 1 PROTEIN (MdNAP1). The expression profile of the genes was investigated throughout five experiments, with three of them encompassing the postharvest period and the other two, consisting of developmental and spatial phases. The transcriptional stability was comparatively investigated using four distinct software packages: BestKeeper, NormFinder, geNorm and DataAssist. Gene ranking results for transcriptional stability were similar for the investigated software packages, with the exception of BestKeeper. The classic reference gene MdUBC ranked among the most stably transcribed in all investigated experimental conditions. Transcript accumulation profiles for the novel reference candidate gene MdH1 were stable throughout the tested conditions, especially in experiments encompassing the postharvest period. Thus, our results present a novel reference gene for postharvest experiments in apple and reinforce the importance of checking the transcription profile of reference genes under the experimental conditions of interest. PMID:25774904

Identification of a novel reference gene for apple transcriptional profiling under postharvest conditions.

PubMed

Storch, Tatiane Timm; Pegoraro, Camila; Finatto, Taciane; Quecini, Vera; Rombaldi, Cesar Valmor; Girardi, César Luis

2015-01-01

Reverse Transcription quantitative PCR (RT-qPCR) is one of the most important techniques for gene expression profiling due to its high sensibility and reproducibility. However, the reliability of the results is highly dependent on data normalization, performed by comparisons between the expression profiles of the genes of interest against those of constitutively expressed, reference genes. Although the technique is widely used in fruit postharvest experiments, the transcription stability of reference genes has not been thoroughly investigated under these experimental conditions. Thus, we have determined the transcriptional profile, under these conditions, of three genes commonly used as reference--ACTIN (MdACT), PROTEIN DISULPHIDE ISOMERASE (MdPDI) and UBIQUITIN-CONJUGATING ENZYME E2 (MdUBC)--along with two novel candidates--HISTONE 1 (MdH1) and NUCLEOSSOME ASSEMBLY 1 PROTEIN (MdNAP1). The expression profile of the genes was investigated throughout five experiments, with three of them encompassing the postharvest period and the other two, consisting of developmental and spatial phases. The transcriptional stability was comparatively investigated using four distinct software packages: BestKeeper, NormFinder, geNorm and DataAssist. Gene ranking results for transcriptional stability were similar for the investigated software packages, with the exception of BestKeeper. The classic reference gene MdUBC ranked among the most stably transcribed in all investigated experimental conditions. Transcript accumulation profiles for the novel reference candidate gene MdH1 were stable throughout the tested conditions, especially in experiments encompassing the postharvest period. Thus, our results present a novel reference gene for postharvest experiments in apple and reinforce the importance of checking the transcription profile of reference genes under the experimental conditions of interest.

Reference Genes for Accurate Transcript Normalization in Citrus Genotypes under Different Experimental Conditions

PubMed Central

Mafra, Valéria; Kubo, Karen S.; Alves-Ferreira, Marcio; Ribeiro-Alves, Marcelo; Stuart, Rodrigo M.; Boava, Leonardo P.; Rodrigues, Carolina M.; Machado, Marcos A.

2012-01-01

Real-time reverse transcription PCR (RT-qPCR) has emerged as an accurate and widely used technique for expression profiling of selected genes. However, obtaining reliable measurements depends on the selection of appropriate reference genes for gene expression normalization. The aim of this work was to assess the expression stability of 15 candidate genes to determine which set of reference genes is best suited for transcript normalization in citrus in different tissues and organs and leaves challenged with five pathogens (Alternaria alternata, Phytophthora parasitica, Xylella fastidiosa and Candidatus Liberibacter asiaticus). We tested traditional genes used for transcript normalization in citrus and orthologs of Arabidopsis thaliana genes described as superior reference genes based on transcriptome data. geNorm and NormFinder algorithms were used to find the best reference genes to normalize all samples and conditions tested. Additionally, each biotic stress was individually analyzed by geNorm. In general, FBOX (encoding a member of the F-box family) and GAPC2 (GAPDH) was the most stable candidate gene set assessed under the different conditions and subsets tested, while CYP (cyclophilin), TUB (tubulin) and CtP (cathepsin) were the least stably expressed genes found. Validation of the best suitable reference genes for normalizing the expression level of the WRKY70 transcription factor in leaves infected with Candidatus Liberibacter asiaticus showed that arbitrary use of reference genes without previous testing could lead to misinterpretation of data. Our results revealed FBOX, SAND (a SAND family protein), GAPC2 and UPL7 (ubiquitin protein ligase 7) to be superior reference genes, and we recommend their use in studies of gene expression in citrus species and relatives. This work constitutes the first systematic analysis for the selection of superior reference genes for transcript normalization in different citrus organs and under biotic stress. PMID:22347455

A RNA-Seq Analysis of the Rat Supraoptic Nucleus Transcriptome: Effects of Salt Loading on Gene Expression

PubMed Central

Salinas, Yasmmyn D.; Shi, YiJun; Greenwood, Michael; Hoe, See Ziau; Murphy, David; Gainer, Harold

2015-01-01

Magnocellular neurons (MCNs) in the hypothalamo-neurohypophysial system (HNS) are highly specialized to release large amounts of arginine vasopressin (Avp) or oxytocin (Oxt) into the blood stream and play critical roles in the regulation of body fluid homeostasis. The MCNs are osmosensory neurons and are excited by exposure to hypertonic solutions and inhibited by hypotonic solutions. The MCNs respond to systemic hypertonic and hypotonic stimulation with large changes in the expression of their Avp and Oxt genes, and microarray studies have shown that these osmotic perturbations also cause large changes in global gene expression in the HNS. In this paper, we examine gene expression in the rat supraoptic nucleus (SON) under normosmotic and chronic salt-loading SL) conditions by the first time using “new-generation”, RNA sequencing (RNA-Seq) methods. We reliably detect 9,709 genes as present in the SON by RNA-Seq, and 552 of these genes were changed in expression as a result of chronic SL. These genes reflect diverse functions, and 42 of these are involved in either transcriptional or translational processes. In addition, we compare the SON transcriptomes resolved by RNA-Seq methods with the SON transcriptomes determined by Affymetrix microarray methods in rats under the same osmotic conditions, and find that there are 6,466 genes present in the SON that are represented in both data sets, although 1,040 of the expressed genes were found only in the microarray data, and 2,762 of the expressed genes are selectively found in the RNA-Seq data and not the microarray data. These data provide the research community a comprehensive view of the transcriptome in the SON under normosmotic conditions and the changes in specific gene expression evoked by salt loading. PMID:25897513

Identification of suitable internal control genes for expression studies in Coffea arabica under different experimental conditions

PubMed Central

Barsalobres-Cavallari, Carla F; Severino, Fábio E; Maluf, Mirian P; Maia, Ivan G

2009-01-01

Background Quantitative data from gene expression experiments are often normalized by transcription levels of reference or housekeeping genes. An inherent assumption for their use is that the expression of these genes is highly uniform in living organisms during various phases of development, in different cell types and under diverse environmental conditions. To date, the validation of reference genes in plants has received very little attention and suitable reference genes have not been defined for a great number of crop species including Coffea arabica. The aim of the research reported herein was to compare the relative expression of a set of potential reference genes across different types of tissue/organ samples of coffee. We also validated the expression profiles of the selected reference genes at various stages of development and under a specific biotic stress. Results The expression levels of five frequently used housekeeping genes (reference genes), namely alcohol dehydrogenase (adh), 14-3-3, polyubiquitin (poly), β-actin (actin) and glyceraldehyde-3-phosphate dehydrogenase (gapdh) was assessed by quantitative real-time RT-PCR over a set of five tissue/organ samples (root, stem, leaf, flower, and fruits) of Coffea arabica plants. In addition to these commonly used internal controls, three other genes encoding a cysteine proteinase (cys), a caffeine synthase (ccs) and the 60S ribosomal protein L7 (rpl7) were also tested. Their stability and suitability as reference genes were validated by geNorm, NormFinder and BestKeeper programs. The obtained results revealed significantly variable expression levels of all reference genes analyzed, with the exception of gapdh, which showed no significant changes in expression among the investigated experimental conditions. Conclusion Our data suggests that the expression of housekeeping genes is not completely stable in coffee. Based on our results, gapdh, followed by 14-3-3 and rpl7 were found to be homogeneously expressed and are therefore adequate for normalization purposes, showing equivalent transcript levels in different tissue/organ samples. Gapdh is therefore the recommended reference gene for measuring gene expression in Coffea arabica. Its use will enable more accurate and reliable normalization of tissue/organ-specific gene expression studies in this important cherry crop plant. PMID:19126214

Housekeeping gene expression during fetal brain development in the rat-validation by semi-quantitative RT-PCR.

PubMed

Al-Bader, Maie Dawoud; Al-Sarraf, Hameed Ali

2005-04-21

Mammalian gene expression is usually carried out at the level of mRNA where the amount of mRNA of interest is measured under different conditions such as growth and development. It is therefore important to use a "housekeeping gene", that does not change in relative abundance during the experimental conditions, as a standard or internal control. However, recent data suggest that expression of some housekeeping genes may vary with the extent of cell proliferation, differentiation and under various experimental conditions. In this study, the expression of various housekeeping genes (18S rRNA [18S], glyceraldehydes-3-phosphate dehydrogenase [G3PDH], beta-glucuronidase [BGLU], histone H4 [HH4], ribosomal protein L19 [RPL19] and cyclophilin [CY]) was investigated during fetal rat brain development using semi-quantitative RT-PCR at 16, 19 and 21 days gestation. It was found that all genes studied, with exception to G3PDH, did not show any change in their expression levels during development. G3PDH, on the other hand, showed increased expression with development. These results suggest that the choice of a housekeeping gene is critical to the interpretation of experimental results and should be modified according to the nature of the study.

Gene Expression Profile Analysis is Directly Affected by the Selected Reference Gene: The Case of Leaf-Cutting Atta Sexdens

PubMed Central

Máximo, Wesley P. F.; Zanetti, Ronald; Paiva, Luciano V.

2018-01-01

Although several ant species are important targets for the development of molecular control strategies, only a few studies focus on identifying and validating reference genes for quantitative reverse transcription polymerase chain reaction (RT-qPCR) data normalization. We provide here an extensive study to identify and validate suitable reference genes for gene expression analysis in the ant Atta sexdens, a threatening agricultural pest in South America. The optimal number of reference genes varies according to each sample and the result generated by RefFinder differed about which is the most suitable reference gene. Results suggest that the RPS16, NADH and SDHB genes were the best reference genes in the sample pool according to stability values. The SNF7 gene expression pattern was stable in all evaluated sample set. In contrast, when using less stable reference genes for normalization a large variability in SNF7 gene expression was recorded. There is no universal reference gene suitable for all conditions under analysis, since these genes can also participate in different cellular functions, thus requiring a systematic validation of possible reference genes for each specific condition. The choice of reference genes on SNF7 gene normalization confirmed that unstable reference genes might drastically change the expression profile analysis of target candidate genes. PMID:29419794

Histone Modifications around Individual BDNF Gene Promoters in Prefrontal Cortex Are Associated with Extinction of Conditioned Fear

ERIC Educational Resources Information Center

Bredy, Timothy W.; Wu, Hao; Crego, Cortney; Zellhoefer, Jessica; Sun, Yi E.; Barad, Mark

2007-01-01

Extinction of conditioned fear is an important model both of inhibitory learning and of behavior therapy for human anxiety disorders. Like other forms of learning, extinction learning is long-lasting and depends on regulated gene expression. Epigenetic mechanisms make an important contribution to persistent changes in gene expression; therefore,…

Selection of reference genes for expression analysis in the entomophthoralean fungus Pandora neoaphidis.

PubMed

Chen, Chun; Xie, Tingna; Ye, Sudan; Jensen, Annette Bruun; Eilenberg, Jørgen

2016-01-01

The selection of suitable reference genes is crucial for accurate quantification of gene expression and can add to our understanding of host-pathogen interactions. To identify suitable reference genes in Pandora neoaphidis, an obligate aphid pathogenic fungus, the expression of three traditional candidate genes including 18S rRNA(18S), 28S rRNA(28S) and elongation factor 1 alpha-like protein (EF1), were measured by quantitative polymerase chain reaction at different developmental stages (conidia, conidia with germ tubes, short hyphae and elongated hyphae), and under different nutritional conditions. We calculated the expression stability of candidate reference genes using four algorithms including geNorm, NormFinder, BestKeeper and Delta Ct. The analysis results revealed that the comprehensive ranking of candidate reference genes from the most stable to the least stable was 18S (1.189), 28S (1.414) and EF1 (3). The 18S was, therefore, the most suitable reference gene for real-time RT-PCR analysis of gene expression under all conditions. These results will support further studies on gene expression in P. neoaphidis. Copyright © 2015 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

Selection of suitable endogenous reference genes for qPCR in kidney and hypothalamus of rats under testosterone influence

PubMed Central

2017-01-01

Real-time quantitative PCR (qPCR) is the most reliable and accurate technique for analyses of gene expression. Endogenous reference genes are being used to normalize qPCR data even though their expression may vary under different conditions and in different tissues. Nonetheless, verification of expression of reference genes in selected studied tissue is essential in order to accurately assess the level of expression of target genes of interest. Therefore, in this study, we attempted to examine six commonly used reference genes in order to identify the gene being expressed most constantly under the influence of testosterone in the kidneys and hypothalamus. The reference genes include glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin beta (ACTB), beta-2 microglobulin (B2m), hypoxanthine phosphoribosyltransferase 1 (HPRT), peptidylprolylisomerase A (Ppia) and hydroxymethylbilane synthase (Hmbs). The cycle threshold (Ct) value for each gene was determined and data obtained were analyzed using the software programs NormFinder, geNorm, BestKeeper, and rank aggregation. Results showed that Hmbs and Ppia genes were the most stably expressed in the hypothalamus. Meanwhile, in kidneys, Hmbs and GAPDH appeared to be the most constant genes. In conclusion, variations in expression levels of reference genes occur in kidneys and hypothalamus under similar conditions; thus, it is important to verify reference gene levels in these tissues prior to commencing any studies. PMID:28591185

Existence of a photoinducible phase for ovarian development and photoperiod-related alteration of clock gene expression in a damselfish.

PubMed

Takeuchi, Yuki; Hada, Noriko; Imamura, Satoshi; Hur, Sung-Pyo; Bouchekioua, Selma; Takemura, Akihiro

2015-10-01

The sapphire devil, Chrysiptera cyanea, is a reef-associated damselfish and their ovarian development can be induced by a long photoperiod. In this study, we demonstrated the existence of a photoinducible phase for the photoperiodic ovarian development in the sapphire devil. Induction of ovarian development under night-interruption light schedules and Nanda-Hamner cycles revealed that the photoinducible phase appeared in a circadian manner between ZT12 and ZT13. To characterize the effect of photoperiod on clock gene expression in the brain of this species, we determined the expression levels of the sdPer1, sdPer2, sdCry1, and sdCry2 clock genes under constant light and dark conditions (LL and DD) and photoperiodic (short and long photoperiods). The expression of sdPer1 exhibited clear circadian oscillation under both LL and DD conditions, while sdPer2 and sdCry1 expression levels were lower under DD than under LL conditions and sdCry2 expression was lower under LL than under DD conditions. These results suggest a key role for sdPer1 in circadian clock cycling and that sdPer2, sdCry1, and sdCry2 are light-responsive clock genes in the sapphire devil. After 1 week under a long photoperiod, we observed photoperiod-related changes in sdPer1, sdPer2, and sdCry2 expression, but not in sdCry1 expression. These results suggest that the expression patterns of some clock genes exhibit seasonal variation according to seasonal changes in day length and that such seasonal alteration of clock gene expression may contribute to seasonal recognition by the sapphire devil. Copyright © 2015 Elsevier Inc. All rights reserved.

Functional genomics of commercial baker's yeasts that have different abilities for sugar utilization and high-sucrose tolerance under different sugar conditions.

PubMed

Tanaka-Tsuno, Fumiko; Mizukami-Murata, Satomi; Murata, Yoshinori; Nakamura, Toshihide; Ando, Akira; Takagi, Hiroshi; Shima, Jun

2007-10-01

In the modern baking industry, high-sucrose-tolerant (HS) and maltose-utilizing (LS) yeast were developed using breeding techniques and are now used commercially. Sugar utilization and high-sucrose tolerance differ significantly between HS and LS yeasts. We analysed the gene expression profiles of HS and LS yeasts under different sucrose conditions in order to determine their basic physiology. Two-way hierarchical clustering was performed to obtain the overall patterns of gene expression. The clustering clearly showed that the gene expression patterns of LS yeast differed from those of HS yeast. Quality threshold clustering was used to identify the gene clusters containing upregulated genes (cluster 1) and downregulated genes (cluster 2) under high-sucrose conditions. Clusters 1 and 2 contained numerous genes involved in carbon and nitrogen metabolism, respectively. The expression level of the genes involved in the metabolism of glycerol and trehalose, which are known to be osmoprotectants, in LS yeast was higher than that in HS yeast under sucrose concentrations of 5-40%. No clear correlation was found between the expression level of the genes involved in the biosynthesis of the osmoprotectants and the intracellular contents of the osmoprotectants. The present gene expression data were compared with data previously reported in a comprehensive analysis of a gene deletion strain collection. Welch's t-test for this comparison showed that the relative growth rates of the deletion strains whose deletion occurred in genes belonging to cluster 1 were significantly higher than the average growth rates of all deletion strains. Copyright 2007 John Wiley & Sons, Ltd.

Selection of reference genes for gene expression studies related to intramuscular fat deposition in Capra hircus skeletal muscle.

PubMed

Zhu, Wuzheng; Lin, Yaqiu; Liao, Honghai; Wang, Yong

2015-01-01

The identification of suitable reference genes is critical for obtaining reliable results from gene expression studies using quantitative real-time PCR (qPCR) because the expression of reference genes may vary considerably under different experimental conditions. In most cases, however, commonly used reference genes are employed in data normalization without proper validation, which may lead to incorrect data interpretation. Here, we aim to select a set of optimal reference genes for the accurate normalization of gene expression associated with intramuscular fat (IMF) deposition during development. In the present study, eight reference genes (PPIB, HMBS, RPLP0, B2M, YWHAZ, 18S, GAPDH and ACTB) were evaluated by three different algorithms (geNorm, NormFinder and BestKeeper) in two types of muscle tissues (longissimus dorsi muscle and biceps femoris muscle) across different developmental stages. All three algorithms gave similar results. PPIB and HMBS were identified as the most stable reference genes, while the commonly used reference genes 18S and GAPDH were the most variably expressed, with expression varying dramatically across different developmental stages. Furthermore, to reveal the crucial role of appropriate reference genes in obtaining a reliable result, analysis of PPARG expression was performed by normalization to the most and the least stable reference genes. The relative expression levels of PPARG normalized to the most stable reference genes greatly differed from those normalized to the least stable one. Therefore, evaluation of reference genes must be performed for a given experimental condition before the reference genes are used. PPIB and HMBS are the optimal reference genes for analysis of gene expression associated with IMF deposition in skeletal muscle during development.

Evaluation of New Reference Genes in Papaya for Accurate Transcript Normalization under Different Experimental Conditions

PubMed Central

Chen, Weixin; Chen, Jianye; Lu, Wangjin; Chen, Lei; Fu, Danwen

2012-01-01

Real-time reverse transcription PCR (RT-qPCR) is a preferred method for rapid and accurate quantification of gene expression studies. Appropriate application of RT-qPCR requires accurate normalization though the use of reference genes. As no single reference gene is universally suitable for all experiments, thus reference gene(s) validation under different experimental conditions is crucial for RT-qPCR analysis. To date, only a few studies on reference genes have been done in other plants but none in papaya. In the present work, we selected 21 candidate reference genes, and evaluated their expression stability in 246 papaya fruit samples using three algorithms, geNorm, NormFinder and RefFinder. The samples consisted of 13 sets collected under different experimental conditions, including various tissues, different storage temperatures, different cultivars, developmental stages, postharvest ripening, modified atmosphere packaging, 1-methylcyclopropene (1-MCP) treatment, hot water treatment, biotic stress and hormone treatment. Our results demonstrated that expression stability varied greatly between reference genes and that different suitable reference gene(s) or combination of reference genes for normalization should be validated according to the experimental conditions. In general, the internal reference genes EIF (Eukaryotic initiation factor 4A), TBP1 (TATA binding protein 1) and TBP2 (TATA binding protein 2) genes had a good performance under most experimental conditions, whereas the most widely present used reference genes, ACTIN (Actin 2), 18S rRNA (18S ribosomal RNA) and GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) were not suitable in many experimental conditions. In addition, two commonly used programs, geNorm and Normfinder, were proved sufficient for the validation. This work provides the first systematic analysis for the selection of superior reference genes for accurate transcript normalization in papaya under different experimental conditions. PMID:22952972

CrMAPK3 regulates the expression of iron-deficiency-responsive genes in Chlamydomonas reinhardtii.

PubMed

Fei, Xiaowen; Yu, Junmei; Li, Yajun; Deng, Xiaodong

2017-05-16

Under iron-deficient conditions, Chlamydomonas exhibits high affinity for iron absorption. Nevertheless, the response, transmission, and regulation of downstream gene expression in algae cells have not to be investigated. Considering that the MAPK pathway is essential for abiotic stress responses, we determined whether this pathway is involved in iron deficiency signal transduction in Chlamydomonas. Arabidopsis MAPK gene sequences were used as entry data to search for homologous genes in Chlamydomonas reinhardtii genome database to investigate the functions of mitogen-activated protein kinase (MAPK) gene family in C. reinhardtii under iron-free conditions. Results revealed 16 C. reinhardtii MAPK genes labeled CrMAPK2-CrMAPK17 with TXY conserved domains and low homology to MAPK in yeast, Arabidopsis, and humans. The expression levels of these genes were then analyzed through qRT-PCR and exposure to high salt (150 mM NaCl), low nitrogen, or iron-free conditions. The expression levels of these genes were also subjected to adverse stress conditions. The mRNA levels of CrMAPK2, CrMAPK3, CrMAPK4, CrMAPK5, CrMAPK6, CrMAPK8, CrMAPK9, and CrMAPK11 were remarkably upregulated under iron-deficient stress. The increase in CrMAPK3 expression was 43-fold greater than that in the control. An RNA interference vector was constructed and transformed into C. reinhardtii 2A38, an algal strain with an exogenous FOX1:ARS chimeric gene, to silence CrMAPK3. After this gene was silenced, the mRNA levels and ARS activities of FOX1:ARS chimeric gene and endogenous CrFOX1 were decreased. The mRNA levels of iron-responsive genes, such as CrNRAMP2, CrATX1, CrFTR1, and CrFEA1, were also remarkably reduced. CrMAPK3 regulates the expression of iron-deficiency-responsive genes in C. reinhardtii.

Modulation of Gene Expression in Contextual Fear Conditioning in the Rat

PubMed Central

Macchi, Monica; Ciampini, Cristina; Bernardi, Rodolfo; Baldi, Elisabetta; Bucherelli, Corrado; Brunelli, Marcello; Scuri, Rossana

2013-01-01

In contextual fear conditioning (CFC) a single training leads to long-term memory of context-aversive electrical foot-shocks association. Mid-temporal regions of the brain of trained and naive rats were obtained 2 days after conditioning and screened by two-directional suppression subtractive hybridization. A pool of differentially expressed genes was identified and some of them were randomly selected and confirmed with qRT-PCR assay. These transcripts showed high homology for rat gene sequences coding for proteins involved in different cellular processes. The expression of the selected transcripts was also tested in rats which had freely explored the experimental apparatus (exploration) and in rats to which the same number of aversive shocks had been administered in the same apparatus, but temporally compressed so as to make the association between painful stimuli and the apparatus difficult (shock-only). Some genes resulted differentially expressed only in the rats subjected to CFC, others only in exploration or shock-only rats, whereas the gene coding for translocase of outer mitochondrial membrane 20 protein and nardilysin were differentially expressed in both CFC and exploration rats. For example, the expression of stathmin 1 whose transcripts resulted up regulated was also tested to evaluate the transduction and protein localization after conditioning. PMID:24278235

Gene Expression of Type VI Secretion System Associated with Environmental Survival in Acidovorax avenae subsp. avenae by Principle Component Analysis

PubMed Central

Cui, Zhouqi; Jin, Guoqiang; Li, Bin; Kakar, Kaleem Ullah; Ojaghian, Mohammad Reza; Wang, Yangli; Xie, Guanlin; Sun, Guochang

2015-01-01

Valine glycine repeat G (VgrG) proteins are regarded as one of two effectors of Type VI secretion system (T6SS) which is a complex multi-component secretion system. In this study, potential biological roles of T6SS structural and VgrG genes in a rice bacterial pathogen, Acidovorax avenae subsp. avenae (Aaa) RS-1, were evaluated under seven stress conditions using principle component analysis of gene expression. The results showed that growth of the pathogen was reduced by H2O2 and paraquat-induced oxidative stress, high salt, low temperature, and vgrG mutation, compared to the control. However, pathogen growth was unaffected by co-culture with a rice rhizobacterium Burkholderia seminalis R456. In addition, expression of 14 T6SS structural and eight vgrG genes was significantly changed under seven conditions. Among different stress conditions, high salt, and low temperature showed a higher effect on the expression of T6SS gene compared with host infection and other environmental conditions. As a first report, this study revealed an association of T6SS gene expression of the pathogen with the host infection, gene mutation, and some common environmental stresses. The results of this research can increase understanding of the biological function of T6SS in this economically-important pathogen of rice. PMID:26378528

Use of transcriptomic data for extending a model of the AppA/PpsR system in Rhodobacter sphaeroides.

PubMed

Pandey, Rakesh; Armitage, Judith P; Wadhams, George H

2017-12-28

Photosynthetic (PS) gene expression in Rhodobacter sphaeroides is regulated in response to changes in light and redox conditions mainly by PrrB/A, FnrL and AppA/PpsR systems. The PrrB/A and FnrL systems activate the expression of them under anaerobic conditions while the AppA/PpsR system represses them under aerobic conditions. Recently, two mathematical models have been developed for the AppA/PpsR system and demonstrated how the interaction between AppA and PpsR could lead to a phenotype in which PS genes are repressed under semi-aerobic conditions. These models have also predicted that the transition from aerobic to anaerobic growth mode could occur via a bistable regime. However, they lack experimentally quantifiable inputs and outputs. Here, we extend one of them to include such quantities and combine all relevant micro-array data publically available for a PS gene of this bacterium and use that to parameterise the model. In addition, we hypothesise that the AppA/PpsR system alone might account for the observed trend of PS gene expression under semi-aerobic conditions. Our extended model of the AppA/PpsR system includes the biological input of atmospheric oxygen concentration and an output of photosynthetic gene expression. Following our hypothesis that the AppA/PpsR system alone is sufficient to describe the overall trend of PS gene expression we parameterise the model and suggest that the rate of AppA reduction in vivo should be faster than its oxidation. Also, we show that despite both the reduced and oxidised forms of PpsR binding to the PS gene promoters in vitro, binding of the oxidised form as a repressor alone is sufficient to reproduce the observed PS gene expression pattern. Finally, the combination of model parameters which fit the biological data well are broadly consistent with those which were previously determined to be required for the system to show (i) the repression of PS genes under semi-aerobic conditions, and (ii) bistability. We found that despite at least three pathways being involved in the regulation of photosynthetic genes, the AppA/PpsR system alone is capable of accounting for the observed trends in photosynthetic gene expression seen at different oxygen levels.

Expression Atlas: gene and protein expression across multiple studies and organisms

PubMed Central

Tang, Y Amy; Bazant, Wojciech; Burke, Melissa; Fuentes, Alfonso Muñoz-Pomer; George, Nancy; Koskinen, Satu; Mohammed, Suhaib; Geniza, Matthew; Preece, Justin; Jarnuczak, Andrew F; Huber, Wolfgang; Stegle, Oliver; Brazma, Alvis; Petryszak, Robert

2018-01-01

Abstract Expression Atlas (http://www.ebi.ac.uk/gxa) is an added value database that provides information about gene and protein expression in different species and contexts, such as tissue, developmental stage, disease or cell type. The available public and controlled access data sets from different sources are curated and re-analysed using standardized, open source pipelines and made available for queries, download and visualization. As of August 2017, Expression Atlas holds data from 3,126 studies across 33 different species, including 731 from plants. Data from large-scale RNA sequencing studies including Blueprint, PCAWG, ENCODE, GTEx and HipSci can be visualized next to each other. In Expression Atlas, users can query genes or gene-sets of interest and explore their expression across or within species, tissues, developmental stages in a constitutive or differential context, representing the effects of diseases, conditions or experimental interventions. All processed data matrices are available for direct download in tab-delimited format or as R-data. In addition to the web interface, data sets can now be searched and downloaded through the Expression Atlas R package. Novel features and visualizations include the on-the-fly analysis of gene set overlaps and the option to view gene co-expression in experiments investigating constitutive gene expression across tissues or other conditions. PMID:29165655

The spatial expression and regulation of transcription factors IDEF1 and IDEF2

PubMed Central

Kobayashi, Takanori; Ogo, Yuko; Aung, May Sann; Nozoye, Tomoko; Itai, Reiko Nakanishi; Nakanishi, Hiromi; Yamakawa, Takashi; Nishizawa, Naoko K.

2010-01-01

Background and Aims Under conditions of low iron availability, rice plants induce genes involved in iron uptake and utilization. The iron deficiency-responsive cis-acting element binding factors 1 and 2 (IDEF1 and IDEF2) regulate transcriptional response to iron deficiency in rice roots. Clarification of the functions of IDEF1 and IDEF2 could uncover the gene regulation mechanism. Methods Spatial patterns of IDEF1 and IDEF2 expression were analysed by histochemical staining of IDEF1 and IDEF2 promoter-GUS transgenic rice lines. Expression patterns of the target genes of IDEF1 and IDEF2 were analysed using transformants with induced or repressed expression of IDEF1 or IDEF2 grown in iron-rich or in iron-deficient solutions for 1 d. Key Results IDEF1 and IDEF2 were highly expressed in the basal parts of the lateral roots and vascular bundles. IDEF1 and IDEF2 expression was dominant in leaf mesophyll and vascular cells, respectively. These expression patterns were similar under both iron-deficient and iron-sufficient conditions. IDEF1 was strongly expressed in pollen, ovaries, the aleurone layer and embryo. IDEF2 was expressed in pollen, ovaries and the dorsal vascular region of the endosperm. During seed germination, IDEF1 and IDEF2 were expressed in the endosperm and embryo. Expression of IDEF1 target genes was regulated in iron-rich roots similar to early iron-deficiency stages. In addition, the expression patterns of IDEF2 target genes were similar between iron-rich conditions and early or subsequent iron deficiency. Conclusions IDEF1 and IDEF2 are constitutively expressed during both vegetative and reproductive stages. The spatial expression patterns of IDEF1 and IDEF2 overlap with their target genes in restricted cell types, but not in all cells. The spatial expression patterns and gene regulation of IDEF1 and IDEF2 in roots are generally conserved under conditions of iron sufficiency and deficiency, suggesting complicated interactions with unknown factors for sensing and transmitting iron-deficiency signals. PMID:20197292

Novel regulatory loci controlling oxygen- and pH-regulated gene expression in Salmonella typhimurium.

PubMed Central

Aliabadi, Z; Park, Y K; Slonczewski, J L; Foster, J W

1988-01-01

Three new loci were discovered, each of which participates in the regulation of anaerobic gene expression. The regulatory gene earA negatively regulates the expression of the anaerobiosis-inducible gene aniG as well as that of at least three other genes, as determined by two-dimensional polyacrylamide gel electrophoresis. The earA locus maps at 86 min. The expression of aniG was also shown to be controlled by changes in external pH under aerobic and anaerobic conditions. Maximal expression was observed under anaerobic conditions at an external pH of 6.0. Significant transcriptional activity was also observed under aerobic conditions at pH 6.0. This was in contrast to hyd, whose expression was dependent upon anaerobiosis and varied with external pH. The pH dependence disappeared under fully aerobic conditions. Mutations in earA had no effect upon hyd expression. The two other regulators identified were oxrF, which controls aniH, and oxrG, which, in concert with oxrA and oxrB, controls aniC and aniI. The oxrG locus was mapped to 88 min and appears to code for a positive regulator. Various oxr mutants were subjected to two-dimensional polyacrylamide electrophoretic analysis of anaerobiosis-inducible proteins. Several pathways of anaerobic control were observed by means of these techniques. Images PMID:3276666

Cell culture density affects the stemness gene expression of adipose tissue-derived mesenchymal stem cells.

PubMed

Kim, Dae Seong; Lee, Myoung Woo; Lee, Tae-Hee; Sung, Ki Woong; Koo, Hong Hoe; Yoo, Keon Hee

2017-03-01

The results of clinical trials using mesenchymal stem cells (MSCs) are controversial due to the heterogeneity of human MSCs and differences in culture conditions. In this regard, it is important to identify gene expression patterns according to culture conditions, and to determine how the cells are expanded and when they should be clinically used. In the current study, stemness gene expression was investigated in adipose tissue-derived MSCs (AT-MSCs) harvested following culture at different densities. AT-MSCs were plated at a density of 200 or 5,000 cells/cm 2 . After 7 days of culture, stemness gene expression was examined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis. The proliferation rate of AT-MSCs harvested at a low density (~50% confluent) was higher than that of AT-MSCs harvested at a high density (~90% confluent). Although there were differences in the expression levels of stemness gene, such as octamer-binding transcription factor 4, nanog homeobox ( Nanog ), SRY-box 2, Kruppel like factor 4, v-myc avian myelocytomatosis viral oncogene homolog ( c-Myc ), and lin-28 homolog A, in the AT-MSCs obtained from different donors, RT-qPCR analysis demonstrated differential gene expression patterns according to the cell culture density. Expression levels of stemness genes, particularly Nanog and c-Myc , were upregulated in AT-MSCs harvested at a low density (~50% confluent) in comparison to AT-MSCs from the same donor harvested at a high density (~90% confluent). These results imply that culture conditions, such as the cell density at harvesting, modulate the stemness gene expression and proliferation of MSCs.

Determination of internal controls for quantitative gene expression of Isochrysis zhangjiangensis at nitrogen stress condition

NASA Astrophysics Data System (ADS)

Wu, Shuang; Zhou, Jiannan; Cao, Xupeng; Xue, Song

2016-02-01

Isochrysis zhangjiangensis is a potential marine microalga for biodiesel production, which accumulates lipid under nitrogen limitation conditions, but the mechanism on molecular level is veiled. Quantitative real-time polymerase chain reaction (qPCR) provides the possibility to investigate the gene expression levels, and a valid reference for data normalization is an essential prerequisite for firing up the analysis. In this study, five housekeeping genes, actin (ACT), α-tubulin (TUA), ß-tubulin (TUB), ubiquitin (UBI), 18S rRNA (18S) and one target gene, diacylglycerol acyltransferase (DGAT), were used for determining the reference. By analyzing the stabilities based on calculation of the stability index and on operating the two types of software, geNorm and bestkeeper, it showed that the reference genes widely used in higher plant and microalgae, such as UBI, TUA and 18S, were not the most stable ones in nitrogen-stressed I. zhangjiangensis, and thus are not suitable for exploring the mRNA expression levels under these experimental conditions. Our results show that ACT together with TUB is the most feasible internal control for investigating gene expression under nitrogen-stressed conditions. Our findings will contribute not only to future qPCR studies of I. zhangjiangensis, but also to verification of comparative transcriptomics studies of the microalgae under similar conditions.

Gene expression profiling data of Schizosaccharomyces pombe under nitrosative stress using differential display.

PubMed

Biswas, Pranjal; Majumdar, Uddalak; Ghosh, Sanjay

2016-03-01

Excess production of nitric oxide (NO) and reactive nitrogen intermediates (RNIs) causes nitrosative stress on cells. Schizosaccharomyces pombe was used as a model to study nitrosative stress response. In the present data article, we have used differential display to identify the differentially expressed genes in the fission yeast under nitrosative stress conditions. We have used pure NO donor compound detaNONOate at final concentrations of 0.1 mM and 1 mM to treat the cells for 15 min alongside control before studying their gene expression profiles. At both the treated conditions, we identified genes which were commonly repressed while several genes were induced upon both 0.1 mM and 1 mM treatments. The differentially expressed genes were further analyzed in DAVID and categorized into several different pathways.

Genome-Wide Survey on Genomic Variation, Expression Divergence, and Evolution in Two Contrasting Rice Genotypes under High Salinity Stress

PubMed Central

Jiang, Shu-Ye; Ma, Ali; Ramamoorthy, Rengasamy; Ramachandran, Srinivasan

2013-01-01

Expression profiling is one of the most important tools for dissecting biological functions of genes and the upregulation or downregulation of gene expression is sufficient for recreating phenotypic differences. Expression divergence of genes significantly contributes to phenotypic variations. However, little is known on the molecular basis of expression divergence and evolution among rice genotypes with contrasting phenotypes. In this study, we have implemented an integrative approach using bioinformatics and experimental analyses to provide insights into genomic variation, expression divergence, and evolution between salinity-sensitive rice variety Nipponbare and tolerant rice line Pokkali under normal and high salinity stress conditions. We have detected thousands of differentially expressed genes between these two genotypes and thousands of up- or downregulated genes under high salinity stress. Many genes were first detected with expression evidence using custom microarray analysis. Some gene families were preferentially regulated by high salinity stress and might play key roles in stress-responsive biological processes. Genomic variations in promoter regions resulted from single nucleotide polymorphisms, indels (1–10 bp of insertion/deletion), and structural variations significantly contributed to the expression divergence and regulation. Our data also showed that tandem and segmental duplication, CACTA and hAT elements played roles in the evolution of gene expression divergence and regulation between these two contrasting genotypes under normal or high salinity stress conditions. PMID:24121498

The complexity of gene expression dynamics revealed by permutation entropy

PubMed Central

2010-01-01

Background High complexity is considered a hallmark of living systems. Here we investigate the complexity of temporal gene expression patterns using the concept of Permutation Entropy (PE) first introduced in dynamical systems theory. The analysis of gene expression data has so far focused primarily on the identification of differentially expressed genes, or on the elucidation of pathway and regulatory relationships. We aim to study gene expression time series data from the viewpoint of complexity. Results Applying the PE complexity metric to abiotic stress response time series data in Arabidopsis thaliana, genes involved in stress response and signaling were found to be associated with the highest complexity not only under stress, but surprisingly, also under reference, non-stress conditions. Genes with house-keeping functions exhibited lower PE complexity. Compared to reference conditions, the PE of temporal gene expression patterns generally increased upon stress exposure. High-complexity genes were found to have longer upstream intergenic regions and more cis-regulatory motifs in their promoter regions indicative of a more complex regulatory apparatus needed to orchestrate their expression, and to be associated with higher correlation network connectivity degree. Arabidopsis genes also present in other plant species were observed to exhibit decreased PE complexity compared to Arabidopsis specific genes. Conclusions We show that Permutation Entropy is a simple yet robust and powerful approach to identify temporal gene expression profiles of varying complexity that is equally applicable to other types of molecular profile data. PMID:21176199

Changes in growth conditions alter the male strobilus gene expression pattern in Cryptomeria japonica.

PubMed

Fukui, Mitsue

2003-11-01

Two-year old saplings grown from cuttings of Cryptomeria japonica D. Don initiate strobilus development following treatment with gibberellic acid under long-day photoperiods. At 25 degrees C with a 14-h photoperiod in a phytotron, male strobili initiated normally; however, they remained green and fell from the saplings prematurely. To examine the change in male strobilus development at the molecular level, three genes expressed specifically in male strobili were analyzed. Two were MADS box genes homologous to the B-function genes in angiosperms, CjMADS1 and CjMADS2, and the third was Cry j I, which encodes an allergen protein, and this gene is expressed mainly in microspores. Under phytotron growing conditions, the homeotic genes were expressed constantly, which reflected the extended early developmental stage of male strobili. On the other hand, Cry j I expression was detected after a long delay just before strobilus development ceased. These results indicate that the expression of the genes related to male reproductive development in C. japonica is regulated by a factor(s) that is sensitive to environmental signals.

Probe-level linear model fitting and mixture modeling results in high accuracy detection of differential gene expression.

PubMed

Lemieux, Sébastien

2006-08-25

The identification of differentially expressed genes (DEGs) from Affymetrix GeneChips arrays is currently done by first computing expression levels from the low-level probe intensities, then deriving significance by comparing these expression levels between conditions. The proposed PL-LM (Probe-Level Linear Model) method implements a linear model applied on the probe-level data to directly estimate the treatment effect. A finite mixture of Gaussian components is then used to identify DEGs using the coefficients estimated by the linear model. This approach can readily be applied to experimental design with or without replication. On a wholly defined dataset, the PL-LM method was able to identify 75% of the differentially expressed genes within 10% of false positives. This accuracy was achieved both using the three replicates per conditions available in the dataset and using only one replicate per condition. The method achieves, on this dataset, a higher accuracy than the best set of tools identified by the authors of the dataset, and does so using only one replicate per condition.

Landscape of Conditional eQTL in Dorsolateral Prefrontal Cortex and Co-localization with Schizophrenia GWAS.

PubMed

Dobbyn, Amanda; Huckins, Laura M; Boocock, James; Sloofman, Laura G; Glicksberg, Benjamin S; Giambartolomei, Claudia; Hoffman, Gabriel E; Perumal, Thanneer M; Girdhar, Kiran; Jiang, Yan; Raj, Towfique; Ruderfer, Douglas M; Kramer, Robin S; Pinto, Dalila; Akbarian, Schahram; Roussos, Panos; Domenici, Enrico; Devlin, Bernie; Sklar, Pamela; Stahl, Eli A; Sieberts, Solveig K

2018-06-07

Causal genes and variants within genome-wide association study (GWAS) loci can be identified by integrating GWAS statistics with expression quantitative trait loci (eQTL) and determining which variants underlie both GWAS and eQTL signals. Most analyses, however, consider only the marginal eQTL signal, rather than dissect this signal into multiple conditionally independent signals for each gene. Here we show that analyzing conditional eQTL signatures, which could be important under specific cellular or temporal contexts, leads to improved fine mapping of GWAS associations. Using genotypes and gene expression levels from post-mortem human brain samples (n = 467) reported by the CommonMind Consortium (CMC), we find that conditional eQTL are widespread; 63% of genes with primary eQTL also have conditional eQTL. In addition, genomic features associated with conditional eQTL are consistent with context-specific (e.g., tissue-, cell type-, or developmental time point-specific) regulation of gene expression. Integrating the 2014 Psychiatric Genomics Consortium schizophrenia (SCZ) GWAS and CMC primary and conditional eQTL data reveals 40 loci with strong evidence for co-localization (posterior probability > 0.8), including six loci with co-localization of conditional eQTL. Our co-localization analyses support previously reported genes, identify novel genes associated with schizophrenia risk, and provide specific hypotheses for their functional follow-up. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Identification of candidate genes for drought tolerance in coffee by high-throughput sequencing in the shoot apex of different Coffea arabica cultivars.

PubMed

Mofatto, Luciana Souto; Carneiro, Fernanda de Araújo; Vieira, Natalia Gomes; Duarte, Karoline Estefani; Vidal, Ramon Oliveira; Alekcevetch, Jean Carlos; Cotta, Michelle Guitton; Verdeil, Jean-Luc; Lapeyre-Montes, Fabienne; Lartaud, Marc; Leroy, Thierry; De Bellis, Fabien; Pot, David; Rodrigues, Gustavo Costa; Carazzolle, Marcelo Falsarella; Pereira, Gonçalo Amarante Guimarães; Andrade, Alan Carvalho; Marraccini, Pierre

2016-04-19

Drought is a widespread limiting factor in coffee plants. It affects plant development, fruit production, bean development and consequently beverage quality. Genetic diversity for drought tolerance exists within the coffee genus. However, the molecular mechanisms underlying the adaptation of coffee plants to drought are largely unknown. In this study, we compared the molecular responses to drought in two commercial cultivars (IAPAR59, drought-tolerant and Rubi, drought-susceptible) of Coffea arabica grown in the field under control (irrigation) and drought conditions using the pyrosequencing of RNA extracted from shoot apices and analysing the expression of 38 candidate genes. Pyrosequencing from shoot apices generated a total of 34.7 Mbp and 535,544 reads enabling the identification of 43,087 clusters (41,512 contigs and 1,575 singletons). These data included 17,719 clusters (16,238 contigs and 1,575 singletons) exclusively from 454 sequencing reads, along with 25,368 hybrid clusters assembled with 454 sequences. The comparison of DNA libraries identified new candidate genes (n = 20) presenting differential expression between IAPAR59 and Rubi and/or drought conditions. Their expression was monitored in plagiotropic buds, together with those of other (n = 18) candidates genes. Under drought conditions, up-regulated expression was observed in IAPAR59 but not in Rubi for CaSTK1 (protein kinase), CaSAMT1 (SAM-dependent methyltransferase), CaSLP1 (plant development) and CaMAS1 (ABA biosynthesis). Interestingly, the expression of lipid-transfer protein (nsLTP) genes was also highly up-regulated under drought conditions in IAPAR59. This may have been related to the thicker cuticle observed on the abaxial leaf surface in IAPAR59 compared to Rubi. The full transcriptome assembly of C. arabica, followed by functional annotation, enabled us to identify differentially expressed genes related to drought conditions. Using these data, candidate genes were selected and their differential expression profiles were confirmed by qPCR experiments in plagiotropic buds of IAPAR59 and Rubi under drought conditions. As regards the genes up-regulated under drought conditions, specifically in the drought-tolerant IAPAR59, several corresponded to orphan genes but also to genes coding proteins involved in signal transduction pathways, as well as ABA and lipid metabolism, for example. The identification of these genes should help advance our understanding of the genetic determinism of drought tolerance in coffee.

Reference genes for gene expression studies in wheat flag leaves grown under different farming conditions

PubMed Central

2011-01-01

Background Internal control genes with highly uniform expression throughout the experimental conditions are required for accurate gene expression analysis as no universal reference genes exists. In this study, the expression stability of 24 candidate genes from Triticum aestivum cv. Cubus flag leaves grown under organic and conventional farming systems was evaluated in two locations in order to select suitable genes that can be used for normalization of real-time quantitative reverse-transcription PCR (RT-qPCR) reactions. The genes were selected among the most common used reference genes as well as genes encoding proteins involved in several metabolic pathways. Findings Individual genes displayed different expression rates across all samples assayed. Applying geNorm, a set of three potential reference genes were suitable for normalization of RT-qPCR reactions in winter wheat flag leaves cv. Cubus: TaFNRII (ferredoxin-NADP(H) oxidoreductase; AJ457980.1), ACT2 (actin 2; TC234027), and rrn26 (a putative homologue to RNA 26S gene; AL827977.1). In addition of these three genes that were also top-ranked by NormFinder, two extra genes: CYP18-2 (Cyclophilin A, AY456122.1) and TaWIN1 (14-3-3 like protein, AB042193) were most consistently stably expressed. Furthermore, we showed that TaFNRII, ACT2, and CYP18-2 are suitable for gene expression normalization in other two winter wheat varieties (Tommi and Centenaire) grown under three treatments (organic, conventional and no nitrogen) and a different environment than the one tested with cv. Cubus. Conclusions This study provides a new set of reference genes which should improve the accuracy of gene expression analyses when using wheat flag leaves as those related to the improvement of nitrogen use efficiency for cereal production. PMID:21951810

Pervasive Effects of Aging on Gene Expression in Wild Wolves

PubMed Central

Charruau, Pauline; Johnston, Rachel A.; Stahler, Daniel R.; Lea, Amanda; Snyder-Mackler, Noah; Smith, Douglas W.; vonHoldt, Bridgett M.; Cole, Steven W.; Tung, Jenny; Wayne, Robert K.

2016-01-01

Abstract Gene expression levels change as an individual ages and responds to environmental conditions. With the exception of humans, such patterns have principally been studied under controlled conditions, overlooking the array of developmental and environmental influences that organisms encounter under conditions in which natural selection operates. We used high-throughput RNA sequencing (RNA-Seq) of whole blood to assess the relative impacts of social status, age, disease, and sex on gene expression levels in a natural population of gray wolves (Canis lupus). Our findings suggest that age is broadly associated with gene expression levels, whereas other examined factors have minimal effects on gene expression patterns. Further, our results reveal evolutionarily conserved signatures of senescence, such as immunosenescence and metabolic aging, between wolves and humans despite major differences in life history and environment. The effects of aging on gene expression levels in wolves exhibit conservation with humans, but the more rapid expression differences observed in aging wolves is evolutionarily appropriate given the species’ high level of extrinsic mortality due to intraspecific aggression. Some expression changes that occur with age can facilitate physical age-related changes that may enhance fitness in older wolves. However, the expression of these ancestral patterns of aging in descendant modern dogs living in highly modified domestic environments may be maladaptive and cause disease. This work provides evolutionary insight into aging patterns observed in domestic dogs and demonstrates the applicability of studying natural populations to investigate the mechanisms of aging. PMID:27189566

Combining Shapley value and statistics to the analysis of gene expression data in children exposed to air pollution

PubMed Central

Moretti, Stefano; van Leeuwen, Danitsja; Gmuender, Hans; Bonassi, Stefano; van Delft, Joost; Kleinjans, Jos; Patrone, Fioravante; Merlo, Domenico Franco

2008-01-01

Background In gene expression analysis, statistical tests for differential gene expression provide lists of candidate genes having, individually, a sufficiently low p-value. However, the interpretation of each single p-value within complex systems involving several interacting genes is problematic. In parallel, in the last sixty years, game theory has been applied to political and social problems to assess the power of interacting agents in forcing a decision and, more recently, to represent the relevance of genes in response to certain conditions. Results In this paper we introduce a Bootstrap procedure to test the null hypothesis that each gene has the same relevance between two conditions, where the relevance is represented by the Shapley value of a particular coalitional game defined on a microarray data-set. This method, which is called Comparative Analysis of Shapley value (shortly, CASh), is applied to data concerning the gene expression in children differentially exposed to air pollution. The results provided by CASh are compared with the results from a parametric statistical test for testing differential gene expression. Both lists of genes provided by CASh and t-test are informative enough to discriminate exposed subjects on the basis of their gene expression profiles. While many genes are selected in common by CASh and the parametric test, it turns out that the biological interpretation of the differences between these two selections is more interesting, suggesting a different interpretation of the main biological pathways in gene expression regulation for exposed individuals. A simulation study suggests that CASh offers more power than t-test for the detection of differential gene expression variability. Conclusion CASh is successfully applied to gene expression analysis of a data-set where the joint expression behavior of genes may be critical to characterize the expression response to air pollution. We demonstrate a synergistic effect between coalitional games and statistics that resulted in a selection of genes with a potential impact in the regulation of complex pathways. PMID:18764936

TIPMaP: a web server to establish transcript isoform profiles from reliable microarray probes.

PubMed

Chitturi, Neelima; Balagannavar, Govindkumar; Chandrashekar, Darshan S; Abinaya, Sadashivam; Srini, Vasan S; Acharya, Kshitish K

2013-12-27

Standard 3' Affymetrix gene expression arrays have contributed a significantly higher volume of existing gene expression data than other microarray platforms. These arrays were designed to identify differentially expressed genes, but not their alternatively spliced transcript forms. No resource can currently identify expression pattern of specific mRNA forms using these microarray data, even though it is possible to do this. We report a web server for expression profiling of alternatively spliced transcripts using microarray data sets from 31 standard 3' Affymetrix arrays for human, mouse and rat species. The tool has been experimentally validated for mRNAs transcribed or not-detected in a human disease condition (non-obstructive azoospermia, a male infertility condition). About 4000 gene expression datasets were downloaded from a public repository. 'Good probes' with complete coverage and identity to latest reference transcript sequences were first identified. Using them, 'Transcript specific probe-clusters' were derived for each platform and used to identify expression status of possible transcripts. The web server can lead the user to datasets corresponding to specific tissues, conditions via identifiers of the microarray studies or hybridizations, keywords, official gene symbols or reference transcript identifiers. It can identify, in the tissues and conditions of interest, about 40% of known transcripts as 'transcribed', 'not-detected' or 'differentially regulated'. Corresponding additional information for probes, genes, transcripts and proteins can be viewed too. We identified the expression of transcripts in a specific clinical condition and validated a few of these transcripts by experiments (using reverse transcription followed by polymerase chain reaction). The experimental observations indicated higher agreements with the web server results, than contradictions. The tool is accessible at http://resource.ibab.ac.in/TIPMaP. The newly developed online tool forms a reliable means for identification of alternatively spliced transcript-isoforms that may be differentially expressed in various tissues, cell types or physiological conditions. Thus, by making better use of existing data, TIPMaP avoids the dependence on precious tissue-samples, in experiments with a goal to establish expression profiles of alternative splice forms--at least in some cases.

Changes in expression of genes involved in apoptosis in activated human T-cells in response to modeled microgravity

NASA Astrophysics Data System (ADS)

Ward, Nancy E.; Pellis, Neal R.; Risin, Diana; Risin, Semyon A.; Liu, Wenbin

2006-09-01

Space flights result in remarkable effects on various physiological systems, including a decline in cellular immune functions. Previous studies have shown that exposure to microgravity, both true and modeled, can cause significant changes in numerous lymphocyte functions. The purpose of this study was to search for microgravity-sensitive genes, and specifically for apoptotic genes influenced by the microgravity environment and other genes related to immune response. The experiments were performed on anti-CD3 and IL-2 activated human T cells. To model microgravity conditions we have utilized the NASA rotating wall vessel bioreactor. Control lymphocytes were cultured in static 1g conditions. To assess gene expression we used DNA microarray chip technology. We had shown that multiple genes (approximately 3-8% of tested genes) respond to microgravity conditions by 1.5 and more fold change in expression. There is a significant variability in the response. However, a certain reproducible pattern in gene response could be identified. Among the genes showing reproducible changes in expression in modeled microgravity, several genes involved in apoptosis as well as in immune response were identified. These are IL-7 receptor, Granzyme B, Beta-3-endonexin, Apo2 ligand and STAT1. Possible functional consequences of these changes are discussed.

STUDIES OF NORMAL GENE EXPRESSION IN THE RAT NASAL EPITHELIUM USNG CDNA ARRAY TECHNOLOGY

EPA Science Inventory

Studies of Normal Gene Expression in the Rat Nasal Epithelium Using cDNA Array

The nasal epithelium is an important target site for chemically-induced toxicity and carcinogenicity .Gene expression data are being used increasingly for studies of such conditions. In or...

Environmental history impacts gene expression during diapause development in the alfalfa leafcutting bee, Megachile rotundata

USDA-ARS?s Scientific Manuscript database

Our understanding of the mechanisms controlling insect diapause has increased dramatically with the introduction of global gene expression techniques, such as RNAseq. However, little attention has been given to how ecologically relevant field conditions may affect gene expression during diapause dev...

Changes of gene expression of iron regulatory proteins during turpentine oil-induced acute-phase response in the rat.

PubMed

Sheikh, Nadeem; Dudas, Jozsef; Ramadori, Giuliano

2007-07-01

In the present study, turpentine oil was injected in the hind limb muscle of the rat to stimulate an acute-phase response (APR). The changes in the gene expression of cytokines and proteins known to be involved in the iron regulatory pathway were then studied in the liver and in extra-hepatic tissue. In addition to the strong upregulation of interleukin-6 (IL-6) and IL-1 beta observed in the inflamed muscle, an upregulation of the genes for IL1-beta and tumor necrosis factor-alpha, but not IL-6, were detectable in the liver. Hepatic Hepc gene expression increased to a maximum at 6 h after the onset of APR. An upregulation of transferrin, transferrin receptor 1 (TfR1), TfR2, ferritin-H, iron responsive element binding protein-1 (IRP1), IRP2 and divalent metal transporter gene expression was also found. Hemojuvelin (Hjv)-, ferroportin 1-, Dcytb-, hemochromatosis-gene- and hephaestin gene expression was downregulated. Hepcidin (Hepc) gene expression was not only detectable in extra-hepatic tissues such as heart, small intestine, colon, spleen and kidney but it was also upregulated under acute-phase conditions, with the Hjv gene being regulated antagonistically. Fpn-1 gene expression was downregulated significantly in heart, colon and spleen. Most of the genes of the known proteins involved in iron metabolism are expressed not only in the liver but also in extra-hepatic tissues. Under acute-phase conditions, acute-phase cytokines (eg IL-6) may modulate the gene expression of such proteins not only in the liver but also in other organs.

Multiple abiotic stimuli are integrated in the regulation of rice gene expression under field conditions.

PubMed

Plessis, Anne; Hafemeister, Christoph; Wilkins, Olivia; Gonzaga, Zennia Jean; Meyer, Rachel Sarah; Pires, Inês; Müller, Christian; Septiningsih, Endang M; Bonneau, Richard; Purugganan, Michael

2015-11-26

Plants rely on transcriptional dynamics to respond to multiple climatic fluctuations and contexts in nature. We analyzed the genome-wide gene expression patterns of rice (Oryza sativa) growing in rainfed and irrigated fields during two distinct tropical seasons and determined simple linear models that relate transcriptomic variation to climatic fluctuations. These models combine multiple environmental parameters to account for patterns of expression in the field of co-expressed gene clusters. We examined the similarities of our environmental models between tropical and temperate field conditions, using previously published data. We found that field type and macroclimate had broad impacts on transcriptional responses to environmental fluctuations, especially for genes involved in photosynthesis and development. Nevertheless, variation in solar radiation and temperature at the timescale of hours had reproducible effects across environmental contexts. These results provide a basis for broad-based predictive modeling of plant gene expression in the field.

Stress amplifies sex differences in primate prefrontal profiles of gene expression.

PubMed

Lee, Alex G; Hagenauer, Megan; Absher, Devin; Morrison, Kathleen E; Bale, Tracy L; Myers, Richard M; Watson, Stanley J; Akil, Huda; Schatzberg, Alan F; Lyons, David M

2017-11-02

Stress is a recognized risk factor for mood and anxiety disorders that occur more often in women than men. Prefrontal brain regions mediate stress coping, cognitive control, and emotion. Here, we investigate sex differences and stress effects on prefrontal cortical profiles of gene expression in squirrel monkey adults. Dorsolateral, ventrolateral, and ventromedial prefrontal cortical regions from 18 females and 12 males were collected after stress or no-stress treatment conditions. Gene expression profiles were acquired using HumanHT-12v4.0 Expression BeadChip arrays adapted for squirrel monkeys. Extensive variation between prefrontal cortical regions was discerned in the expression of numerous autosomal and sex chromosome genes. Robust sex differences were also identified across prefrontal cortical regions in the expression of mostly autosomal genes. Genes with increased expression in females compared to males were overrepresented in mitogen-activated protein kinase and neurotrophin signaling pathways. Many fewer genes with increased expression in males compared to females were discerned, and no molecular pathways were identified. Effect sizes for sex differences were greater in stress compared to no-stress conditions for ventromedial and ventrolateral prefrontal cortical regions but not dorsolateral prefrontal cortex. Stress amplifies sex differences in gene expression profiles for prefrontal cortical regions involved in stress coping and emotion regulation. Results suggest molecular targets for new treatments of stress disorders in human mental health.

Evaluation of Candidate Reference Genes for Quantitative Gene Expression Analysis in Spodoptera exigu a after Long-time Exposure to Cadmium.

PubMed

Płachetka-Bożek, Anna; Augustyniak, Maria

2017-08-21

Studies on the transcriptional control of gene expression play an important role in many areas of biology. Reference genes, which are often referred to as housekeeping genes, such as GAPDH, G3PDH, EF2, RpL7A, RpL10, TUBα and Actin, have traditionally been assumed to be stably expressed in all conditions, and they are frequently used to normalize mRNA levels between different samples in qPCR analysis. However, it is known that the expression of these genes is influenced by numerous factors, such as experimental conditions. The difference in gene expression underlies a range of biological processes, including development, reproduction and behavior. The aim of this study was to show the problems associated with using reference genes in the qPCR technique, in a study on inbred strains of Spodoptera exigua selected toward cadmium resistance. We present and discuss our results and observations, and give some recommendations concerning the use and limitations of housekeeping genes as internal standards, especially in research on insects. Our results suggest that holometabolism and poikilothermia, as well as time since metamorphosis and the level of exposure to the selective factor (cadmium in this case), have a significant effect on the expression of reference genes.

Transcriptomic Analysis and the Expression of Disease-Resistant Genes in Oryza meyeriana under Native Condition

PubMed Central

He, Bin; Tao, Xiang; Gu, Yinghong; Wei, Changhe; Cheng, Xiaojie; Xiao, Suqin; Cheng, Zaiquan; Zhang, Yizheng

2015-01-01

Oryza meyeriana (O. meyeriana), with a GG genome type (2n = 24), accumulated plentiful excellent characteristics with respect to resistance to many diseases such as rice shade and blast, even immunity to bacterial blight. It is very important to know if the diseases-resistant genes exist and express in this wild rice under native conditions. However, limited genomic or transcriptomic data of O. meyeriana are currently available. In this study, we present the first comprehensive characterization of the O. meyeriana transcriptome using RNA-seq and obtained 185,323 contigs with an average length of 1,692 bp and an N50 of 2,391 bp. Through differential expression analysis, it was found that there were most tissue-specifically expressed genes in roots, and next to stems and leaves. By similarity search against protein databases, 146,450 had at least a significant alignment to existed gene models. Comparison with the Oryza sativa (japonica-type Nipponbare and indica-type 93–11) genomes revealed that 13% of the O. meyeriana contigs had not been detected in O. sativa. Many diseases-resistant genes, such as bacterial blight resistant, blast resistant, rust resistant, fusarium resistant, cyst nematode resistant and downy mildew gene, were mined from the transcriptomic database. There are two kinds of rice bacterial blight-resistant genes (Xa1 and Xa26) differentially or specifically expressed in O. meyeriana. The 4 Xa1 contigs were all only expressed in root, while three of Xa26 contigs have the highest expression level in leaves, two of Xa26 contigs have the highest expression profile in stems and one of Xa26 contigs was expressed dominantly in roots. The transcriptomic database of O. meyeriana has been constructed and many diseases-resistant genes were found to express under native condition, which provides a foundation for future discovery of a number of novel genes and provides a basis for studying the molecular mechanisms associated with disease resistance in O. meyeriana. PMID:26640944

Identification of optimal reference genes for RT-qPCR in the rat hypothalamus and intestine for the study of obesity.

PubMed

Li, B; Matter, E K; Hoppert, H T; Grayson, B E; Seeley, R J; Sandoval, D A

2014-02-01

Obesity has a complicated metabolic pathology, and defining the underlying mechanisms of obesity requires integrative studies with molecular end points. Real-time quantitative PCR (RT-qPCR) is a powerful tool that has been widely utilized. However, the importance of using carefully validated reference genes in RT-qPCR seems to have been overlooked in obesity-related research. The objective of this study was to select a set of reference genes with stable expressions to be used for RT-qPCR normalization in rats under fasted vs re-fed and chow vs high-fat diet (HFD) conditions. Male long-Evans rats were treated under four conditions: chow/fasted, chow/re-fed, HFD/fasted and HFD/re-fed. Expression stabilities of 13 candidate reference genes were evaluated in the rat hypothalamus, duodenum, jejunum and ileum using the ReFinder software program. The optimal number of reference genes needed for RT-qPCR analyses was determined using geNorm. Using geNorm analysis, we found that it was sufficient to use the two most stably expressed genes as references in RT-qPCR analyses for each tissue under specific experimental conditions. B2M and RPLP0 in the hypothalamus, RPS18 and HMBS in the duodenum, RPLP2 and RPLP0 in the jejunum and RPS18 and YWHAZ in the ileum were the most suitable pairs for a normalization study when the four aforementioned experimental conditions were considered. Our study demonstrates that gene expression levels of reference genes commonly used in obesity-related studies, such as ACTB or RPS18, are altered by changes in acute or chronic energy status. These findings underline the importance of using reference genes that are stable in expression across experimental conditions when studying the rat hypothalamus and intestine, because these tissues have an integral role in the regulation of energy homeostasis. It is our hope that this study will raise awareness among obesity researchers on the essential need for reference gene validation in gene expression studies.

Optimization of conditions for transient Agrobacterium-mediated gene expression assays in Arabidopsis.

PubMed

Kim, Mi Jung; Baek, Kon; Park, Chung-Mo

2009-08-01

Transient genetic transformation of plant organs is an indispensable way of studying gene function in plants. This study was aimed to develop an optimized system for transient Agrobacterium-mediated transformation of the Arabidopsis leaves. The beta-glucuronidase (GUS) reporter gene was employed to evaluate growth and biochemical parameters that influence the levels of transient expression. The effects of plant culture conditions, Agrobacterial genetic backgrounds, densities of Agrobacterial cell suspensions, and of several detergents were analyzed. We found that optimization of plant culture conditions is the most critical factor among the parameters analyzed. Higher levels of transient expression were observed in plants grown under short day conditions (SDs) than in plants grown under long day conditions (LDs). Furthermore, incubation of the plants under SDs at high relative humidity (85-90%) for 24 h after infiltration greatly improved the levels of transient expression. Under the optimized culture conditions, expression of the reporter gene reached the peak 3 days after infiltration and was rapidly decreased after the peak. Among the five Agrobacterial strains examined, LAB4404 produced the highest levels of expression. We also examined the effects of detergents, including Triton X-100, Tween-20, and Silwet L-77. Supplementation of the infiltration media either with 0.01% Triton X-100 or 0.01% Tween-20 improved the levels of expression by approximately 1.6-fold. Our observations indicate that transient transformation of the Arabidopsis leaves in the infiltration media supplemented with 0.01% Triton X-100 and incubation of the infiltrated plants under SDs at high relative humidity are necessary for maximal levels of expression.

Selection of reliable reference genes for quantitative real-time PCR gene expression analysis in Jute (Corchorus capsularis) under stress treatments

PubMed Central

Niu, Xiaoping; Qi, Jianmin; Zhang, Gaoyang; Xu, Jiantang; Tao, Aifen; Fang, Pingping; Su, Jianguang

2015-01-01

To accurately measure gene expression using quantitative reverse transcription PCR (qRT-PCR), reliable reference gene(s) are required for data normalization. Corchorus capsularis, an annual herbaceous fiber crop with predominant biodegradability and renewability, has not been investigated for the stability of reference genes with qRT-PCR. In this study, 11 candidate reference genes were selected and their expression levels were assessed using qRT-PCR. To account for the influence of experimental approach and tissue type, 22 different jute samples were selected from abiotic and biotic stress conditions as well as three different tissue types. The stability of the candidate reference genes was evaluated using geNorm, NormFinder, and BestKeeper programs, and the comprehensive rankings of gene stability were generated by aggregate analysis. For the biotic stress and NaCl stress subsets, ACT7 and RAN were suitable as stable reference genes for gene expression normalization. For the PEG stress subset, UBC, and DnaJ were sufficient for accurate normalization. For the tissues subset, four reference genes TUBβ, UBI, EF1α, and RAN were sufficient for accurate normalization. The selected genes were further validated by comparing expression profiles of WRKY15 in various samples, and two stable reference genes were recommended for accurate normalization of qRT-PCR data. Our results provide researchers with appropriate reference genes for qRT-PCR in C. capsularis, and will facilitate gene expression study under these conditions. PMID:26528312

MALDI-TOF mass spectrometry for quantitative gene expression analysis of acid responses in Staphylococcus aureus.

PubMed

Rode, Tone Mari; Berget, Ingunn; Langsrud, Solveig; Møretrø, Trond; Holck, Askild

2009-07-01

Microorganisms are constantly exposed to new and altered growth conditions, and respond by changing gene expression patterns. Several methods for studying gene expression exist. During the last decade, the analysis of microarrays has been one of the most common approaches applied for large scale gene expression studies. A relatively new method for gene expression analysis is MassARRAY, which combines real competitive-PCR and MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) mass spectrometry. In contrast to microarray methods, MassARRAY technology is suitable for analysing a larger number of samples, though for a smaller set of genes. In this study we compare the results from MassARRAY with microarrays on gene expression responses of Staphylococcus aureus exposed to acid stress at pH 4.5. RNA isolated from the same stress experiments was analysed using both the MassARRAY and the microarray methods. The MassARRAY and microarray methods showed good correlation. Both MassARRAY and microarray estimated somewhat lower fold changes compared with quantitative real-time PCR (qRT-PCR). The results confirmed the up-regulation of the urease genes in acidic environments, and also indicated the importance of metal ion regulation. This study shows that the MassARRAY technology is suitable for gene expression analysis in prokaryotes, and has advantages when a set of genes is being analysed for an organism exposed to many different environmental conditions.

Allele specific expression analysis identifies regulatory variation associated with stress-related genes in the Mexican highland maize landrace Palomero Toluqueño

PubMed Central

González-Segovia, Eric; Ross-Ibarra, Jeffrey; Simpson, June K.

2017-01-01

Background Gene regulatory variation has been proposed to play an important role in the adaptation of plants to environmental stress. In the central highlands of Mexico, farmer selection has generated a unique group of maize landraces adapted to the challenges of the highland niche. In this study, gene expression in Mexican highland maize and a reference maize breeding line were compared to identify evidence of regulatory variation in stress-related genes. It was hypothesised that local adaptation in Mexican highland maize would be associated with a transcriptional signature observable even under benign conditions. Methods Allele specific expression analysis was performed using the seedling-leaf transcriptome of an F1 individual generated from the cross between the highland adapted Mexican landrace Palomero Toluqueño and the reference line B73, grown under benign conditions. Results were compared with a published dataset describing the transcriptional response of B73 seedlings to cold, heat, salt and UV treatments. Results A total of 2,386 genes were identified to show allele specific expression. Of these, 277 showed an expression difference between Palomero Toluqueño and B73 alleles under benign conditions that anticipated the response of B73 cold, heat, salt and/or UV treatments, and, as such, were considered to display a prior stress response. Prior stress response candidates included genes associated with plant hormone signaling and a number of transcription factors. Construction of a gene co-expression network revealed further signaling and stress-related genes to be among the potential targets of the transcription factors candidates. Discussion Prior activation of responses may represent the best strategy when stresses are severe but predictable. Expression differences observed here between Palomero Toluqueño and B73 alleles indicate the presence of cis-acting regulatory variation linked to stress-related genes in Palomero Toluqueño. Considered alongside gene annotation and population data, allele specific expression analysis of plants grown under benign conditions provides an attractive strategy to identify functional variation potentially linked to local adaptation. PMID:28852597

Exploring Valid Reference Genes for Quantitative Real-time PCR Analysis in Plutella xylostella (Lepidoptera: Plutellidae)

PubMed Central

Fu, Wei; Xie, Wen; Zhang, Zhuo; Wang, Shaoli; Wu, Qingjun; Liu, Yong; Zhou, Xiaomao; Zhou, Xuguo; Zhang, Youjun

2013-01-01

Abstract: Quantitative real-time PCR (qRT-PCR), a primary tool in gene expression analysis, requires an appropriate normalization strategy to control for variation among samples. The best option is to compare the mRNA level of a target gene with that of reference gene(s) whose expression level is stable across various experimental conditions. In this study, expression profiles of eight candidate reference genes from the diamondback moth, Plutella xylostella, were evaluated under diverse experimental conditions. RefFinder, a web-based analysis tool, integrates four major computational programs including geNorm, Normfinder, BestKeeper, and the comparative ΔCt method to comprehensively rank the tested candidate genes. Elongation factor 1 (EF1) was the most suited reference gene for the biotic factors (development stage, tissue, and strain). In contrast, although appropriate reference gene(s) do exist for several abiotic factors (temperature, photoperiod, insecticide, and mechanical injury), we were not able to identify a single universal reference gene. Nevertheless, a suite of candidate reference genes were specifically recommended for selected experimental conditions. Our finding is the first step toward establishing a standardized qRT-PCR analysis of this agriculturally important insect pest. PMID:23983612

Comprehensive gene expression analysis of the NAC gene family under normal growth conditions, hormone treatment, and drought stress conditions in rice using near-isogenic lines (NILs) generated from crossing Aday Selection (drought tolerant) and IR64.

PubMed

Nuruzzaman, Mohammed; Sharoni, Akhter Most; Satoh, Kouji; Moumeni, Ali; Venuprasad, Ramiah; Serraj, Rachid; Kumar, Arvind; Leung, Hei; Attia, Kotb; Kikuchi, Shoshi

2012-05-01

The NAC (NAM, ATAF1/2 and CUC2) genes are plant-specific transcriptional factors known to play diverse roles in various plant developmental processes. We describe the rice (Oryza sativa) OsNAC genes expression profiles (GEPs) under normal and water-deficit treatments (WDTs). The GEPs of the OsNAC genes were analyzed in 25 tissues covering the entire life cycle of Minghui 63. High expression levels of 17 genes were demonstrated in certain tissues under normal conditions suggesting that these genes may play important roles in specific organs. We determined that 16 genes were differentially expressed under at least 1 phytohormone (NAA, GA3, KT, SA, ABA, and JA) treatment. To investigate the GEPs in the root, leaf, and panicle of three rice genotypes [e.g., 2 near-isogenic lines (NILs) and IR64], we used two NILs from a common genetic combination backcross developed by Aday Selection and IR64. WDTs were applied using the fraction of transpirable soil water at severe, mild, and control conditions. Transcriptomic analysis using a 44K oligoarray from Agilent was performed on all the tissue samples. We identified common and specific genes in all tissues from the two NILs under both WDTs, and the majority of the OsNAC genes that were activated were in the drought-tolerant IR77298-14-1-2-B-10 line compared with the drought-susceptible IR77298-14-1-2-B-13 or IR64. In IR77298-14-1-2-B-10, seventeen genes were very specific in their expression levels. Approximately 70 % of the genes from subgroups SNAC and NAM/CUC3 were activated in the leaf, but 37 % genes from subgroup SND were inactivated in the root compared with the control under severe stress conditions. These results provide a useful reference for the cloning of candidate genes from the specific subgroup for further functional analysis.

Selection of Reference Genes for Expression Studies of Xenobiotic Adaptation in Tetranychus urticae.

PubMed

Morales, Mariany Ashanty; Mendoza, Bianca Marie; Lavine, Laura Corley; Lavine, Mark Daniel; Walsh, Douglas Bruce; Zhu, Fang

2016-01-01

Quantitative real-time PCR (qRT-PCR) is an extensively used, high-throughput method to analyze transcriptional expression of genes of interest. An appropriate normalization strategy with reliable reference genes is required for calculating gene expression across diverse experimental conditions. In this study, we aim to identify the most stable reference genes for expression studies of xenobiotic adaptation in Tetranychus urticae, an extremely polyphagous herbivore causing significant yield reduction of agriculture. We chose eight commonly used housekeeping genes as candidates. The qRT-PCR expression data for these genes were evaluated from seven populations: a susceptible and three acaricide resistant populations feeding on lima beans, and three other susceptible populations which had been shifted host from lima beans to three other plant species. The stability of the candidate reference genes was then assessed using four different algorithms (comparative ΔCt method, geNorm, NormFinder, and BestKeeper). Additionally, we used an online web-based tool (RefFinder) to assign an overall final rank for each candidate gene. Our study found that CycA and Rp49 are best for investigating gene expression in acaricide susceptible and resistant populations. GAPDH, Rp49, and Rpl18 are best for host plant shift studies. And GAPDH and Rp49 were the most stable reference genes when investigating gene expression under changes in both experimental conditions. These results will facilitate research in revealing molecular mechanisms underlying the xenobiotic adaptation of this notorious agricultural pest.

Selection of Reference Genes for Expression Studies of Xenobiotic Adaptation in Tetranychus urticae

PubMed Central

Morales, Mariany Ashanty; Mendoza, Bianca Marie; Lavine, Laura Corley; Lavine, Mark Daniel; Walsh, Douglas Bruce; Zhu, Fang

2016-01-01

Quantitative real-time PCR (qRT-PCR) is an extensively used, high-throughput method to analyze transcriptional expression of genes of interest. An appropriate normalization strategy with reliable reference genes is required for calculating gene expression across diverse experimental conditions. In this study, we aim to identify the most stable reference genes for expression studies of xenobiotic adaptation in Tetranychus urticae, an extremely polyphagous herbivore causing significant yield reduction of agriculture. We chose eight commonly used housekeeping genes as candidates. The qRT-PCR expression data for these genes were evaluated from seven populations: a susceptible and three acaricide resistant populations feeding on lima beans, and three other susceptible populations which had been shifted host from lima beans to three other plant species. The stability of the candidate reference genes was then assessed using four different algorithms (comparative ΔCt method, geNorm, NormFinder, and BestKeeper). Additionally, we used an online web-based tool (RefFinder) to assign an overall final rank for each candidate gene. Our study found that CycA and Rp49 are best for investigating gene expression in acaricide susceptible and resistant populations. GAPDH, Rp49, and Rpl18 are best for host plant shift studies. And GAPDH and Rp49 were the most stable reference genes when investigating gene expression under changes in both experimental conditions. These results will facilitate research in revealing molecular mechanisms underlying the xenobiotic adaptation of this notorious agricultural pest. PMID:27570487

Transcriptional Activity, Chromosomal Distribution and Expression Effects of Transposable Elements in Coffea Genomes

PubMed Central

da Silva, Carlos R. M.; Andrade, Alan C.; Marraccini, Pierre; Teixeira, João B.; Carazzolle, Marcelo F.; Pereira, Gonçalo A. G.; Pereira, Luiz Filipe P.; Vanzela, André L. L.; Wang, Lu; Jordan, I. King; Carareto, Claudia M. A.

2013-01-01

Plant genomes are massively invaded by transposable elements (TEs), many of which are located near host genes and can thus impact gene expression. In flowering plants, TE expression can be activated (de-repressed) under certain stressful conditions, both biotic and abiotic, as well as by genome stress caused by hybridization. In this study, we examined the effects of these stress agents on TE expression in two diploid species of coffee, Coffea canephora and C. eugenioides, and their allotetraploid hybrid C. arabica. We also explored the relationship of TE repression mechanisms to host gene regulation via the effects of exonized TE sequences. Similar to what has been seen for other plants, overall TE expression levels are low in Coffea plant cultivars, consistent with the existence of effective TE repression mechanisms. TE expression patterns are highly dynamic across the species and conditions assayed here are unrelated to their classification at the level of TE class or family. In contrast to previous results, cell culture conditions per se do not lead to the de-repression of TE expression in C. arabica. Results obtained here indicate that differing plant drought stress levels relate strongly to TE repression mechanisms. TEs tend to be expressed at significantly higher levels in non-irrigated samples for the drought tolerant cultivars but in drought sensitive cultivars the opposite pattern was shown with irrigated samples showing significantly higher TE expression. Thus, TE genome repression mechanisms may be finely tuned to the ideal growth and/or regulatory conditions of the specific plant cultivars in which they are active. Analysis of TE expression levels in cell culture conditions underscored the importance of nonsense-mediated mRNA decay (NMD) pathways in the repression of Coffea TEs. These same NMD mechanisms can also regulate plant host gene expression via the repression of genes that bear exonized TE sequences. PMID:24244387

Influence of in vivo growth on human glioma cell line gene expression: Convergent profiles under orthotopic conditions

PubMed Central

Camphausen, Kevin; Purow, Benjamin; Sproull, Mary; Scott, Tamalee; Ozawa, Tomoko; Deen, Dennis F.; Tofilon, Philip J.

2005-01-01

Defining the molecules that regulate tumor cell survival is an essential prerequisite for the development of targeted approaches to cancer treatment. Whereas many studies aimed at identifying such targets use human tumor cells grown in vitro or as s.c. xenografts, it is unclear whether such experimental models replicate the phenotype of the in situ tumor cell. To begin addressing this issue, we have used microarray analysis to define the gene expression profile of two human glioma cell lines (U251 and U87) when grown in vitro and in vivo as s.c. or as intracerebral (i.c.) xenografts. For each cell line, the gene expression profile generated from tissue culture was significantly different from that generated from the s.c. tumor, which was significantly different from those grown i.c. The disparity between the i.c gene expression profiles and those generated from s.c. xenografts suggests that whereas an in vivo growth environment modulates gene expression, orthotopic growth conditions induce a different set of modifications. In this study the U251 and U87 gene expression profiles generated under the three growth conditions were also compared. As expected, the profiles of the two glioma cell lines were significantly different when grown as monolayer cultures. However, the glioma cell lines had similar gene expression profiles when grown i.c. These results suggest that tumor cell gene expression, and thus phenotype, as defined in vitro is affected not only by in vivo growth but also by orthotopic growth, which may have implications regarding the identification of relevant targets for cancer therapy. PMID:15928080

β-Glucuronidase is a suitable internal control gene for mRNA quantitation in pathophysiological and non-pathological livers.

PubMed

Yamaguchi, Hiromi; Matsumoto, Sawako; Ishibashi, Mariko; Hasegawa, Kiyoshi; Sugitani, Masahiko; Takayama, Tadatoshi; Esumi, Mariko

2013-10-01

The level of expression of housekeeping genes is in general considered stable, and a representative gene such as glyceraldehyde-3-phosphate dehydrogenase is commonly used as an internal control for quantitating mRNA. However, expression of housekeeping genes is not always constant under pathological conditions. To determine which genes would be most suitable as internal controls for quantitative gene expression studies in human liver diseases, we quantified 12 representative housekeeping genes in 27 non-cancerous liver tissues (normal, chronic hepatitis C with and without liver cirrhosis). We identified β-glucuronidase as the most suitable gene for studies on liver by rigorous statistical analysis of inter- and intra-group comparisons. We conclude that it is important to determine the most appropriate control gene for the particular condition to be analyzed. © 2013 Elsevier Inc. All rights reserved.

Implementation of plaid model biclustering method on microarray of carcinoma and adenoma tumor gene expression data

NASA Astrophysics Data System (ADS)

Ardaneswari, Gianinna; Bustamam, Alhadi; Sarwinda, Devvi

2017-10-01

A Tumor is an abnormal growth of cells that serves no purpose. Carcinoma is a tumor that grows from the top of the cell membrane and the organ adenoma is a benign tumor of the gland-like cells or epithelial tissue. In the field of molecular biology, the development of microarray technology is used in the data store of disease genetic expression. For each of microarray gene, an amount of information is stored for each trait or condition. In gene expression data clustering can be done with a bicluster algorithm, thats clustering method which not only the objects to be clustered, but also the properties or condition of the object. This research proposed Plaid Model Biclustering as one of biclustering method. In this study, we discuss the implementation of Plaid Model Biclustering Method on microarray of Carcinoma and Adenoma tumor gene expression data. From the experimental results, we found three biclusters are formed by Carcinoma gene expression data and four biclusters are formed by Adenoma gene expression data.

Broad distribution spectrum from Gaussian to power law appears in stochastic variations in RNA-seq data.

PubMed

Awazu, Akinori; Tanabe, Takahiro; Kamitani, Mari; Tezuka, Ayumi; Nagano, Atsushi J

2018-05-29

Gene expression levels exhibit stochastic variations among genetically identical organisms under the same environmental conditions. In many recent transcriptome analyses based on RNA sequencing (RNA-seq), variations in gene expression levels among replicates were assumed to follow a negative binomial distribution, although the physiological basis of this assumption remains unclear. In this study, RNA-seq data were obtained from Arabidopsis thaliana under eight conditions (21-27 replicates), and the characteristics of gene-dependent empirical probability density function (ePDF) profiles of gene expression levels were analyzed. For A. thaliana and Saccharomyces cerevisiae, various types of ePDF of gene expression levels were obtained that were classified as Gaussian, power law-like containing a long tail, or intermediate. These ePDF profiles were well fitted with a Gauss-power mixing distribution function derived from a simple model of a stochastic transcriptional network containing a feedback loop. The fitting function suggested that gene expression levels with long-tailed ePDFs would be strongly influenced by feedback regulation. Furthermore, the features of gene expression levels are correlated with their functions, with the levels of essential genes tending to follow a Gaussian-like ePDF while those of genes encoding nucleic acid-binding proteins and transcription factors exhibit long-tailed ePDF.

Xylella fastidiosa gene expression analysis by DNA microarrays.

PubMed

Travensolo, Regiane F; Carareto-Alves, Lucia M; Costa, Maria V C G; Lopes, Tiago J S; Carrilho, Emanuel; Lemos, Eliana G M

2009-04-01

Xylella fastidiosa genome sequencing has generated valuable data by identifying genes acting either on metabolic pathways or in associated pathogenicity and virulence. Based on available information on these genes, new strategies for studying their expression patterns, such as microarray technology, were employed. A total of 2,600 primer pairs were synthesized and then used to generate fragments using the PCR technique. The arrays were hybridized against cDNAs labeled during reverse transcription reactions and which were obtained from bacteria grown under two different conditions (liquid XDM(2) and liquid BCYE). All data were statistically analyzed to verify which genes were differentially expressed. In addition to exploring conditions for X. fastidiosa genome-wide transcriptome analysis, the present work observed the differential expression of several classes of genes (energy, protein, amino acid and nucleotide metabolism, transport, degradation of substances, toxins and hypothetical proteins, among others). The understanding of expressed genes in these two different media will be useful in comprehending the metabolic characteristics of X. fastidiosa, and in evaluating how important certain genes are for the functioning and survival of these bacteria in plants.

Cyclophosphamide Alters the Gene Expression Profile in Patients Treated with High Doses Prior to Stem Cell Transplantation

PubMed Central

El-Serafi, Ibrahim; Abedi-Valugerdi, Manuchehr; Potácová, Zuzana; Afsharian, Parvaneh; Mattsson, Jonas; Moshfegh, Ali; Hassan, Moustapha

2014-01-01

Background Hematopoietic stem cell transplantation is a curative treatment for several haematological malignancies. However, treatment related morbidity and mortality still is a limiting factor. Cyclophosphamide is widely used in condition regimens either in combination with other chemotherapy or with total body irradiation. Methods We present the gene expression profile during cyclophosphamide treatment in 11 patients conditioned with cyclophosphamide for 2 days followed by total body irradiation prior to hematopoietic stem cell transplantation. 299 genes were identified as specific for cyclophosphamide treatment and were arranged into 4 clusters highly down-regulated genes, highly up-regulated genes, early up-regulated but later normalized genes and moderately up-regulated genes. Results Cyclophosphamide treatment down-regulated expression of several genes mapped to immune/autoimmune activation and graft rejection including CD3, CD28, CTLA4, MHC II, PRF1, GZMB and IL-2R, and up-regulated immune-related receptor genes, e.g. IL1R2, IL18R1, and FLT3. Moreover, a high and significant expression of ANGPTL1 and c-JUN genes was observed independent of cyclophosphamide treatment. Conclusion This is the first investigation to provide significant information about alterations in gene expression following cyclophosphamide treatment that may increase our understanding of the cyclophosphamide mechanism of action and hence, in part, avoid its toxicity. Furthermore, ANGPTL1 remained highly expressed throughout the treatment and, in contrast to several other alkylating agents, cyclophosphamide did not influence c-JUN expression. PMID:24466173

Cyclophosphamide alters the gene expression profile in patients treated with high doses prior to stem cell transplantation.

PubMed

El-Serafi, Ibrahim; Abedi-Valugerdi, Manuchehr; Potácová, Zuzana; Afsharian, Parvaneh; Mattsson, Jonas; Moshfegh, Ali; Hassan, Moustapha

2014-01-01

Hematopoietic stem cell transplantation is a curative treatment for several haematological malignancies. However, treatment related morbidity and mortality still is a limiting factor. Cyclophosphamide is widely used in condition regimens either in combination with other chemotherapy or with total body irradiation. We present the gene expression profile during cyclophosphamide treatment in 11 patients conditioned with cyclophosphamide for 2 days followed by total body irradiation prior to hematopoietic stem cell transplantation. 299 genes were identified as specific for cyclophosphamide treatment and were arranged into 4 clusters highly down-regulated genes, highly up-regulated genes, early up-regulated but later normalized genes and moderately up-regulated genes. Cyclophosphamide treatment down-regulated expression of several genes mapped to immune/autoimmune activation and graft rejection including CD3, CD28, CTLA4, MHC II, PRF1, GZMB and IL-2R, and up-regulated immune-related receptor genes, e.g. IL1R2, IL18R1, and FLT3. Moreover, a high and significant expression of ANGPTL1 and c-JUN genes was observed independent of cyclophosphamide treatment. This is the first investigation to provide significant information about alterations in gene expression following cyclophosphamide treatment that may increase our understanding of the cyclophosphamide mechanism of action and hence, in part, avoid its toxicity. Furthermore, ANGPTL1 remained highly expressed throughout the treatment and, in contrast to several other alkylating agents, cyclophosphamide did not influence c-JUN expression.

Environmental Conditions Influence Induction of Key ABC-Transporter Genes Affecting Glyphosate Resistance Mechanism in Conyza canadensis.

PubMed

Tani, Eleni; Chachalis, Demosthenis; Travlos, Ilias S; Bilalis, Dimitrios

2016-04-20

Conyza canadensis has been reported to be the most frequent weed species that evolved resistance to glyphosate in various parts of the world. The objective of the present study was to investigate the effect of environmental conditions (temperature and light) on the expression levels of the EPSPS gene and two major ABC-transporter genes (M10 and M11) on glyphosate susceptible (GS) and glyphosate resistant (GR) horseweed populations, collected from several regions across Greece. Real-time PCR was conducted to determine the expression level of the aforementioned genes when glyphosate was applied at normal (1×; 533 g·a.e.·ha(-1)) and high rates (4×, 8×), measured at an early one day after treatment (DAT) and a later stage (four DAT) of expression. Plants were exposed to light or dark conditions, at three temperature regimes (8, 25, 35 °C). GR plants were made sensitive when exposed to 8 °C with light; those sensitized plants behaved biochemically (shikimate accumulation) and molecularly (expression of EPSPS and ABC-genes) like the GS plants. Results from the current study show the direct link between the environmental conditions and the induction level of the above key genes that likely affect the efficiency of the proposed mechanism of glyphosate resistance.

Environmental Conditions Influence Induction of Key ABC-Transporter Genes Affecting Glyphosate Resistance Mechanism in Conyza canadensis

PubMed Central

Tani, Eleni; Chachalis, Demosthenis; Travlos, Ilias S.; Bilalis, Dimitrios

2016-01-01

Conyza canadensis has been reported to be the most frequent weed species that evolved resistance to glyphosate in various parts of the world. The objective of the present study was to investigate the effect of environmental conditions (temperature and light) on the expression levels of the EPSPS gene and two major ABC-transporter genes (M10 and M11) on glyphosate susceptible (GS) and glyphosate resistant (GR) horseweed populations, collected from several regions across Greece. Real-time PCR was conducted to determine the expression level of the aforementioned genes when glyphosate was applied at normal (1×; 533 g·a.e.·ha−1) and high rates (4×, 8×), measured at an early one day after treatment (DAT) and a later stage (four DAT) of expression. Plants were exposed to light or dark conditions, at three temperature regimes (8, 25, 35 °C). GR plants were made sensitive when exposed to 8 °C with light; those sensitized plants behaved biochemically (shikimate accumulation) and molecularly (expression of EPSPS and ABC-genes) like the GS plants. Results from the current study show the direct link between the environmental conditions and the induction level of the above key genes that likely affect the efficiency of the proposed mechanism of glyphosate resistance. PMID:27104532

Mimosa: Mixture Model of Co-expression to Detect Modulators of Regulatory Interaction

NASA Astrophysics Data System (ADS)

Hansen, Matthew; Everett, Logan; Singh, Larry; Hannenhalli, Sridhar

Functionally related genes tend to be correlated in their expression patterns across multiple conditions and/or tissue-types. Thus co-expression networks are often used to investigate functional groups of genes. In particular, when one of the genes is a transcription factor (TF), the co-expression-based interaction is interpreted, with caution, as a direct regulatory interaction. However, any particular TF, and more importantly, any particular regulatory interaction, is likely to be active only in a subset of experimental conditions. Moreover, the subset of expression samples where the regulatory interaction holds may be marked by presence or absence of a modifier gene, such as an enzyme that post-translationally modifies the TF. Such subtlety of regulatory interactions is overlooked when one computes an overall expression correlation. Here we present a novel mixture modeling approach where a TF-Gene pair is presumed to be significantly correlated (with unknown coefficient) in a (unknown) subset of expression samples. The parameters of the model are estimated using a Maximum Likelihood approach. The estimated mixture of expression samples is then mined to identify genes potentially modulating the TF-Gene interaction. We have validated our approach using synthetic data and on three biological cases in cow and in yeast. While limited in some ways, as discussed, the work represents a novel approach to mine expression data and detect potential modulators of regulatory interactions.

Effects of Leucine Supplementation and Serum Withdrawal on Branched-Chain Amino Acid Pathway Gene and Protein Expression in Mouse Adipocytes

PubMed Central

Vivar, Juan C.; Knight, Megan S.; Pointer, Mildred A.; Gwathmey, Judith K.; Ghosh, Sujoy

2014-01-01

The essential branched-chain amino acids (BCAA), leucine, valine and isoleucine, are traditionally associated with skeletal muscle growth and maintenance, energy production, and generation of neurotransmitter and gluconeogenic precursors. Recent evidence from human and animal model studies has established an additional link between BCAA levels and obesity. However, details of the mechanism of regulation of BCAA metabolism during adipogenesis are largely unknown. We interrogated whether the expression of genes and proteins involved in BCAA metabolism are sensitive to the adipocyte differentiation process, and responsive to nutrient stress from starvation or BCAA excess. Murine 3T3-L1 preadipocytes were differentiated to adipocytes under control conditions and under conditions of L-leucine supplementation or serum withdrawal. RNA and proteins were isolated at days 0, 4 and 10 of differentiation to represent pre-differentiation, early differentiation and late differentiation stages. Expression of 16 BCAA metabolism genes was quantified by quantitative real-time PCR. Expression of the protein levels of branched-chain amino acid transaminase 2 (Bcat2) and branched-chain alpha keto acid dehydrogenase (Bckdha) was quantified by immunoblotting. Under control conditions, all genes displayed induction of gene expression during early adipogenesis (Day 4) compared to Day 0. Leucine supplementation resulted in an induction of Bcat2 and Bckdha genes during early and late differentiation. Western blot analysis demonstrated condition-specific concordance between gene and protein expression. Serum withdrawal resulted in undetectable Bcat2 and Bckdha protein levels at all timepoints. These results demonstrate that the expression of genes related to BCAA metabolism are regulated during adipocyte differentiation and influenced by nutrient levels. These results provide additional insights on how BCAA metabolism is associated with adipose tissue function and extends our understanding of the transcriptomic response of this pathway to variations in nutrient availability. PMID:25050624

[Differential expression genes of bone tissues surrounding implants in diabetic rats by gene chip].

PubMed

Wang, Xin-xin; Ma, Yue; Li, Qing; Jiang, Bao-qi; Lan, Jing

2012-10-01

To compare mRNA expression profiles of bone tissues surrounding implants between normal rats and rats with diabetes using microarray technology. Six Wistar rats were randomly selected and divided into normal model group and diabetic group. Diabetic model condition was established by injecting Streptozotocin into peritoneal space. Titanium implants were implanted into the epiphyseal end of the rats' tibia. Bone tissues surrounding implant were harvested and sampled after 3 months to perform comprehensive RNA gene expression profiling, including 17983 for genome-wide association study.GO analysis was used to compare different gene expression and real-time PCR was used to confirm the results on core samples. The results indicated that there were 1084 differential gene expression. In the diabetic model, there were 352 enhanced expression genes, 732 suppressed expression genes. GO analysis involved 1154 different functional type. Osteoblast related gene expressions in bone tissue samples of diabetic rats were decreased, and lipid metabolism pathway related gene expression was increased.

Changes in DnaA-dependent gene expression contribute to the transcriptional and developmental response of Bacillus subtilis to manganese limitation in Luria-Bertani medium.

PubMed

Hoover, Sharon E; Xu, Weihong; Xiao, Wenzhong; Burkholder, William F

2010-08-01

The SOS response to DNA damage in bacteria is a well-known component of the complex transcriptional responses to genotoxic environmental stresses such as exposure to reactive oxygen species, alkylating agents, and many of the antibiotics targeting DNA replication. However, bacteria such as Bacillus subtilis also respond to conditions that perturb DNA replication via a transcriptional response mediated by the replication initiation protein DnaA. In addition to regulating the initiation of DNA replication, DnaA directly regulates the transcription of specific genes. Conditions that perturb DNA replication can trigger the accumulation of active DnaA, activating or repressing the transcription of genes in the DnaA regulon. We report here that simply growing B. subtilis in LB medium altered DnaA-dependent gene expression in a manner consistent with the accumulation of active DnaA and that this was part of a general transcriptional response to manganese limitation. The SOS response to DNA damage was not induced under these conditions. One of the genes positively regulated by DnaA in Bacillus subtilis encodes a protein that inhibits the initiation of sporulation, Sda. Sda expression was induced as cells entered stationary phase in LB medium but not in LB medium supplemented with manganese, and the induction of Sda inhibited sporulation-specific gene expression and the onset of spore morphogenesis. In the absence of Sda, manganese-limited cells initiated spore development but failed to form mature spores. These data highlight that DnaA-dependent gene expression may influence the response of bacteria to a range of environmental conditions, including conditions that are not obviously associated with genotoxic stress.

Changes in DnaA-Dependent Gene Expression Contribute to the Transcriptional and Developmental Response of Bacillus subtilis to Manganese Limitation in Luria-Bertani Medium▿ †

PubMed Central

Hoover, Sharon E.; Xu, Weihong; Xiao, Wenzhong; Burkholder, William F.

2010-01-01

The SOS response to DNA damage in bacteria is a well-known component of the complex transcriptional responses to genotoxic environmental stresses such as exposure to reactive oxygen species, alkylating agents, and many of the antibiotics targeting DNA replication. However, bacteria such as Bacillus subtilis also respond to conditions that perturb DNA replication via a transcriptional response mediated by the replication initiation protein DnaA. In addition to regulating the initiation of DNA replication, DnaA directly regulates the transcription of specific genes. Conditions that perturb DNA replication can trigger the accumulation of active DnaA, activating or repressing the transcription of genes in the DnaA regulon. We report here that simply growing B. subtilis in LB medium altered DnaA-dependent gene expression in a manner consistent with the accumulation of active DnaA and that this was part of a general transcriptional response to manganese limitation. The SOS response to DNA damage was not induced under these conditions. One of the genes positively regulated by DnaA in Bacillus subtilis encodes a protein that inhibits the initiation of sporulation, Sda. Sda expression was induced as cells entered stationary phase in LB medium but not in LB medium supplemented with manganese, and the induction of Sda inhibited sporulation-specific gene expression and the onset of spore morphogenesis. In the absence of Sda, manganese-limited cells initiated spore development but failed to form mature spores. These data highlight that DnaA-dependent gene expression may influence the response of bacteria to a range of environmental conditions, including conditions that are not obviously associated with genotoxic stress. PMID:20511500

Genome-wide transcriptional analysis of two soybean genotypes under dehydration and rehydration conditions

PubMed Central

2013-01-01

Background Soybean is an important crop that provides valuable proteins and oils for human use. Because soybean growth and development is extremely sensitive to water deficit, quality and crop yields are severely impacted by drought stress. In the face of limited water resources, drought-responsive genes are therefore of interest. Identification and analysis of dehydration- and rehydration-inducible differentially expressed genes (DEGs) would not only aid elucidation of molecular mechanisms of stress response, but also enable improvement of crop stress tolerance via gene transfer. Using Digital Gene Expression Tag profiling (DGE), a new technique based on Illumina sequencing, we analyzed expression profiles between two soybean genotypes to identify drought-responsive genes. Results Two soybean genotypes—drought-tolerant Jindou21 and drought-sensitive Zhongdou33—were subjected to dehydration and rehydration conditions. For analysis of DEGs under dehydration conditions, 20 cDNA libraries were generated from roots and leaves at two different time points under well-watered and dehydration conditions. We also generated eight libraries for analysis under rehydration conditions. Sequencing of the 28 libraries produced 25,000–33,000 unambiguous tags, which were mapped to reference sequences for annotation of expressed genes. Many genes exhibited significant expression differences among the libraries. DEGs in the drought-tolerant genotype were identified by comparison of DEGs among treatments and genotypes. In Jindou21, 518 and 614 genes were differentially expressed under dehydration in leaves and roots, respectively, with 24 identified both in leaves and roots. The main functional categories enriched in these DEGs were metabolic process, response to stresses, plant hormone signal transduction, protein processing, and plant-pathogen interaction pathway; the associated genes primarily encoded transcription factors, protein kinases, and other regulatory proteins. The seven most significantly expressed (|log2 ratio| ≥ 8) genes— Glyma15g03920, Glyma05g02470, Glyma15g15010, Glyma05g09070, Glyma06g35630, Glyma08g12590, and Glyma11g16000—are more likely to determine drought stress tolerance. The expression patterns of eight randomly-selected genes were confirmed by quantitative RT-PCR; the results of QRT-PCR analysis agreed with transcriptional profile data for 96 out of 128 (75%) data points. Conclusions Many soybean genes were differentially expressed between drought-tolerant and drought-sensitive genotypes. Based on GO functional annotation and pathway enrichment analysis, some of these genes encoded transcription factors, protein kinases, and other regulatory proteins. The seven most significant DEGs are candidates for improving soybean drought tolerance. These findings will be helpful for analysis and elucidation of molecular mechanisms of drought tolerance; they also provide a basis for cultivating new varieties of drought-tolerant soybean. PMID:24093224

Identification and Validation of Reference Genes and Their Impact on Normalized Gene Expression Studies across Cultivated and Wild Cicer Species

PubMed Central

Reddy, Palakolanu Sudhakar; Sri Cindhuri, Katamreddy; Sivaji Ganesh, Adusumalli; Sharma, Kiran Kumar

2016-01-01

Quantitative Real-Time PCR (qPCR) is a preferred and reliable method for accurate quantification of gene expression to understand precise gene functions. A total of 25 candidate reference genes including traditional and new generation reference genes were selected and evaluated in a diverse set of chickpea samples. The samples used in this study included nine chickpea genotypes (Cicer spp.) comprising of cultivated and wild species, six abiotic stress treatments (drought, salinity, high vapor pressure deficit, abscisic acid, cold and heat shock), and five diverse tissues (leaf, root, flower, seedlings and seed). The geNorm, NormFinder and RefFinder algorithms used to identify stably expressed genes in four sample sets revealed stable expression of UCP and G6PD genes across genotypes, while TIP41 and CAC were highly stable under abiotic stress conditions. While PP2A and ABCT genes were ranked as best for different tissues, ABCT, UCP and CAC were most stable across all samples. This study demonstrated the usefulness of new generation reference genes for more accurate qPCR based gene expression quantification in cultivated as well as wild chickpea species. Validation of the best reference genes was carried out by studying their impact on normalization of aquaporin genes PIP1;4 and TIP3;1, in three contrasting chickpea genotypes under high vapor pressure deficit (VPD) treatment. The chickpea TIP3;1 gene got significantly up regulated under high VPD conditions with higher relative expression in the drought susceptible genotype, confirming the suitability of the selected reference genes for expression analysis. This is the first comprehensive study on the stability of the new generation reference genes for qPCR studies in chickpea across species, different tissues and abiotic stresses. PMID:26863232

Identification and Validation of Reference Genes and Their Impact on Normalized Gene Expression Studies across Cultivated and Wild Cicer Species.

PubMed

Reddy, Dumbala Srinivas; Bhatnagar-Mathur, Pooja; Reddy, Palakolanu Sudhakar; Sri Cindhuri, Katamreddy; Sivaji Ganesh, Adusumalli; Sharma, Kiran Kumar

2016-01-01

Quantitative Real-Time PCR (qPCR) is a preferred and reliable method for accurate quantification of gene expression to understand precise gene functions. A total of 25 candidate reference genes including traditional and new generation reference genes were selected and evaluated in a diverse set of chickpea samples. The samples used in this study included nine chickpea genotypes (Cicer spp.) comprising of cultivated and wild species, six abiotic stress treatments (drought, salinity, high vapor pressure deficit, abscisic acid, cold and heat shock), and five diverse tissues (leaf, root, flower, seedlings and seed). The geNorm, NormFinder and RefFinder algorithms used to identify stably expressed genes in four sample sets revealed stable expression of UCP and G6PD genes across genotypes, while TIP41 and CAC were highly stable under abiotic stress conditions. While PP2A and ABCT genes were ranked as best for different tissues, ABCT, UCP and CAC were most stable across all samples. This study demonstrated the usefulness of new generation reference genes for more accurate qPCR based gene expression quantification in cultivated as well as wild chickpea species. Validation of the best reference genes was carried out by studying their impact on normalization of aquaporin genes PIP1;4 and TIP3;1, in three contrasting chickpea genotypes under high vapor pressure deficit (VPD) treatment. The chickpea TIP3;1 gene got significantly up regulated under high VPD conditions with higher relative expression in the drought susceptible genotype, confirming the suitability of the selected reference genes for expression analysis. This is the first comprehensive study on the stability of the new generation reference genes for qPCR studies in chickpea across species, different tissues and abiotic stresses.

Gene Expression Analysis to Assess the Relevance of Rodent Models to Human Lung Injury.

PubMed

Sweeney, Timothy E; Lofgren, Shane; Khatri, Purvesh; Rogers, Angela J

2017-08-01

The relevance of animal models to human diseases is an area of intense scientific debate. The degree to which mouse models of lung injury recapitulate human lung injury has never been assessed. Integrating data from both human and animal expression studies allows for increased statistical power and identification of conserved differential gene expression across organisms and conditions. We sought comprehensive integration of gene expression data in experimental acute lung injury (ALI) in rodents compared with humans. We performed two separate gene expression multicohort analyses to determine differential gene expression in experimental animal and human lung injury. We used correlational and pathway analyses combined with external in vitro gene expression data to identify both potential drivers of underlying inflammation and therapeutic drug candidates. We identified 21 animal lung tissue datasets and three human lung injury bronchoalveolar lavage datasets. We show that the metasignatures of animal and human experimental ALI are significantly correlated despite these widely varying experimental conditions. The gene expression changes among mice and rats across diverse injury models (ozone, ventilator-induced lung injury, LPS) are significantly correlated with human models of lung injury (Pearson r = 0.33-0.45, P < 1E -16 ). Neutrophil signatures are enriched in both animal and human lung injury. Predicted therapeutic targets, peptide ligand signatures, and pathway analyses are also all highly overlapping. Gene expression changes are similar in animal and human experimental ALI, and provide several physiologic and therapeutic insights to the disease.

Gene expression profile of activated microglia under conditions associated with dopamine neuronal damage.

PubMed

Thomas, David M; Francescutti-Verbeem, Dina M; Kuhn, Donald M

2006-03-01

Microglia are the resident antigen-presenting cells within the central nervous system (CNS), and they serve immune-like functions in protecting the brain against injury and invading pathogens. By contrast, activated microglia can secrete numerous reactants that damage neurons. The pathogenesis of various neurodegenerative diseases has been associated with microglial activation, but the signaling pathways that program a neuronally protective or destructive phenotype in microglia are not known. To increase the understanding of microglial activation, microarray analysis was used to profile the transcriptome of BV-2 microglial cells after activation. Microglia were activated by lipopolysaccharide, the HIV neurotoxic protein TAT, and dopamine quinone, each of which has been linked to dopamine neuronal damage. We identified 210 of 9882 genes whose expression was differentially regulated by all activators (116 increased and 94 decreased in expression). Gene ontology analysis assigned up-regulated genes to a number of specific biological processes and molecular functions, including immune response, inflammation, and cytokine/chemokine activity. Genes down-regulated in expression contribute to conditions that are permissive of microglial migration, lowered adhesion to matrix, lessened phagocytosis, and reduction in receptors that oppose chemotaxis and inflammation. These results elaborate a broad profile of microglial genes whose expression is altered by conditions associated with both neurodegenerative diseases and microglial activation.

Expression profiles of sugarcane under drought conditions: Variation in gene regulation.

PubMed

Andrade, Júlio César Farias de; Terto, Jackeline; Silva, José Vieira; Almeida, Cícero

2015-12-01

Drought is a major factor in decreased sugarcane productivity because of the resulting morphophysiological effects that it causes. Gene expression studies that have examined the influence of water stress in sugarcane have yielded divergent results, indicating the absence of a fixed pattern of changes in gene expression. In this work, we investigated the expression profiles of 12 genes in the leaves of a drought-tolerant genotype (RB72910) of sugarcane and compared the results with those of other studies. The genotype was subjected to 80-100% water availability (control condition) and 0-20% water availability (simulated drought). To analyze the physiological status, the SPAD index, Fv/Fm ratio, net photosynthesis (A), stomatal conductance (gs) and stomatal transpiration (E) were measured. Total RNA was extracted from leaves and the expression of SAMDC, ZmPIP2-1 protein, ZmTIP4-2 protein, WIP protein, LTP protein, histone H3, DNAj, ferredoxin I, β-tubulin, photosystem I, gene 1 and gene 2 was analyzed by quantitative real-time PCR (RT-PCR). Important differences in the expression profiles of these genes were observed when compared with other genotypes, suggesting that complex defense mechanisms are activated in response to water stress. However, there was no recognizable pattern for the changes in expression of the different proteins associated with tolerance to drought stress.

Stress-Related Gene Expression Reflects Morphophysiological Responses to Water Deficit1[OPEN

PubMed Central

Vile, Denis; Bediee, Alexis; Dauzat, Myriam; Luchaire, Nathalie; Kamrowska, Dominika; Granier, Christine

2017-01-01

Acclimation to water deficit (WD) enables plants to maintain growth under unfavorable environmental conditions, although the mechanisms are not completely understood. In this study, the natural variation of long-term acclimation to moderate and severe soil WD was investigated in 18 Arabidopsis (Arabidopsis thaliana) accessions using PHENOPSIS, an automated phenotyping platform. Soil water content was adjusted at an early stage of plant development and maintained at a constant level until reproductive age was achieved. The accessions were selected based on the expression levels of ANNEXIN1, a drought-related marker. Severe WD conditions had a greater effect on most of the measured morphophysiological traits than moderate WD conditions. Multivariate analyses indicated that trait responses associated with plant size and water management drove most of the variation. Accessions with similar responses at these two levels were grouped in clusters that displayed different response strategies to WD. The expression levels of selected stress-response genes revealed large natural variation under WD conditions. Responses of morphophysiological traits, such as projected rosette area, transpiration rate, and rosette water content, were correlated with changes in the expression of stress-related genes, such as NINE-CIS-EPOXYCAROTENOID DIOXYGENASE3 and N-MYC DOWNREGULATED-LIKE1 (NDL1), in response to WD. Interestingly, the morphophysiological acclimation response to WD also was reflected in the gene expression levels (most notably those of NDL1, CHALCONE SYNTHASE, and MYB DOMAIN PROTEIN44) in plants cultivated under well-watered conditions. Our results may lead to the development of biomarkers and predictors of plant morphophysiological responses based on gene expression patterns. PMID:28522456

Effect of hypoxia on the expression of nuclear genes encoding mitochondrial proteins in U87 glioma cells.

PubMed

Minchenko, O H; Riabovol, O O; Tsymbal, D O; Minchenko, D O; Ratushna, O O

2016-01-01

We have studied the effect of hypoxia on the expression of nuclear genes encoding mitochondrial proteins in U87 glioma cells under the inhibition of IRE1 (inositol requiring enzyme-1), which controls cell proliferation and tumor growth as a central mediator of endoplasmic reticulum stress. It was shown that hypoxia down-regulated gene expression of malate dehydrogenase 2 (MDH2), malic enzyme 2 (ME2), mitochondrial aspartate aminotransferase (GOT2), and subunit B of succinate dehydrogenase (SDHB) in control (transfected by empty vector) glioma cells in a gene specific manner. At the same time, the expression level of mitochondrial NADP+-dependent isocitrate dehydrogenase 2 (IDH2) and subunit D of succinate dehydrogenase (SDHD) genes in these cells does not significantly change in hypoxic conditions. It was also shown that the inhibition of ІRE1 signaling enzyme function in U87 glioma cells decreases the effect of hypoxia on the expression of ME2, GOT2, and SDHB genes and introduces the sensitivity of IDH2 gene to hypoxia. Furthermore, the expression of all studied genes depends on IRE1-mediated endoplasmic reticulum stress signaling in gene specific manner, because ІRE1 knockdown significantly decreases their expression in normoxic conditions, except for IDH2 gene, which expression level is strongly up-regulated. Therefore, changes in the expression level of nuclear genes encoding ME2, MDH2, IDH2, SDHB, SDHD, and GOT2 proteins possibly reflect metabolic reprogramming of mitochondria by hypoxia and IRE1-mediated endoplasmic reticulum stress signaling and correlate with suppression of glioma cell proliferation under inhibition of the IRE1 enzyme function.

Now You See It, Now You Don't! Making Regulation of Gene Expression Come Alive for All Students.

ERIC Educational Resources Information Center

Brock, David L.

1998-01-01

Presents a protocol that can be used to discover that S. marcescens produces a bright red pigment when exposed to one temperature condition and does not produce this color under other conditions. Enables students to connect gene expression and environmental changes. (DDR)

The Transcriptome of the Reference Potato Genome Solanum tuberosum Group Phureja Clone DM1-3 516R44

PubMed Central

Massa, Alicia N.; Childs, Kevin L.; Lin, Haining; Bryan, Glenn J.; Giuliano, Giovanni; Buell, C. Robin

2011-01-01

Advances in molecular breeding in potato have been limited by its complex biological system, which includes vegetative propagation, autotetraploidy, and extreme heterozygosity. The availability of the potato genome and accompanying gene complement with corresponding gene structure, location, and functional annotation are powerful resources for understanding this complex plant and advancing molecular breeding efforts. Here, we report a reference for the potato transcriptome using 32 tissues and growth conditions from the doubled monoploid Solanum tuberosum Group Phureja clone DM1-3 516R44 for which a genome sequence is available. Analysis of greater than 550 million RNA-Seq reads permitted the detection and quantification of expression levels of over 22,000 genes. Hierarchical clustering and principal component analyses captured the biological variability that accounts for gene expression differences among tissues suggesting tissue-specific gene expression, and genes with tissue or condition restricted expression. Using gene co-expression network analysis, we identified 18 gene modules that represent tissue-specific transcriptional networks of major potato organs and developmental stages. This information provides a powerful resource for potato research as well as studies on other members of the Solanaceae family. PMID:22046362

Rapid and tunable method to temporally control gene editing based on conditional Cas9 stabilization. | Office of Cancer Genomics

Cancer.gov

The CRISPR/Cas9 system is a powerful tool for studying gene function. Here, we describe a method that allows temporal control of CRISPR/Cas9 activity based on conditional Cas9 destabilization. We demonstrate that fusing an FKBP12-derived destabilizing domain to Cas9 (DD-Cas9) enables conditional Cas9 expression and temporal control of gene editing in the presence of an FKBP12 synthetic ligand. This system can be easily adapted to co-express, from the same promoter, DD-Cas9 with any other gene of interest without co-modulation of the latter.

Transcriptome responses to temperature, water availability and photoperiod are conserved among mature trees of two divergent Douglas-fir provenances from a coastal and an interior habitat.

PubMed

Hess, Moritz; Wildhagen, Henning; Junker, Laura Verena; Ensminger, Ingo

2016-08-26

Local adaptation and phenotypic plasticity are important components of plant responses to variations in environmental conditions. While local adaptation has been widely studied in trees, little is known about plasticity of gene expression in adult trees in response to ever changing environmental conditions in natural habitats. Here we investigate plasticity of gene expression in needle tissue between two Douglas-fir provenances represented by 25 adult trees using deep RNA sequencing (RNA-Seq). Using linear mixed models we investigated the effect of temperature, soil water availability and photoperiod on the abundance of 59189 detected transcripts. Expression of more than 80 % of all identified transcripts revealed a response to variations in environmental conditions in the field. GO term overrepresentation analysis revealed gene expression responses to temperature, soil water availability and photoperiod that are highly conserved among many plant taxa. However, expression differences between the two Douglas-fir provenances were rather small compared to the expression differences observed between individual trees. Although the effect of environment on global transcript expression was high, the observed genotype by environment (GxE) interaction of gene expression was surprisingly low, since only 21 of all detected transcripts showed a GxE interaction. The majority of the transcriptome responses in plant leaf tissue is driven by variations in environmental conditions. The small variation between individuals and populations suggests strong conservation of this response within Douglas-fir. Therefore we conclude that plastic transcriptome responses to variations in environmental conditions are only weakly affected by local adaptation in Douglas-fir.

[Effect of human oviductal embryotrophic factors on gene expression of mouse preimplantation embryos].

PubMed

Yao, Yuan-Qing; Lee, Kai-Fai; Xu, Jia-Seng; Ho, Pak-Chung; Yeung, Shu-Biu

2007-09-01

To investigate the effect of embryotrophic factors (ETF) from human oviductal cells on gene expression of mouse early developmental embryos and discuss the role of fallopian tube in early development of embryos. ETF was isolated from conditioned medium of human oviductal cell line by sequential liquid chromatographic systems. Mouse embryos were treated by ETF in vitro. Using differential display RT-PCR, the gene expression of embryos treated by ETF was compared with embryos without ETF treatment. The differentially expressed genes were separated, re-amplified, cloned and sequenced. Gene expression profiles of embryos with ETF treatment was different from embryos without this treatment. Eight differentially expressed genes were cloned and sequenced. These genes functioned in RNA degradation, synthesis, splicing, protein trafficking, cellular differentiation and embryo development. Embryotrophic factors from human oviductal cells affect gene expression of early developmental embryos. The human oviductal cells play wide roles in early developmental stages of embryos.

Reference Gene Selection for qPCR Normalization of Kosteletzkya virginica under Salt Stress

PubMed Central

Tang, Xiaoli; Wang, Hongyan; Shao, Chuyang; Shao, Hongbo

2015-01-01

Kosteletzkya virginica (L.) is a newly introduced perennial halophytic plant. Presently, reverse transcription quantitative real-time PCR (qPCR) is regarded as the best choice for analyzing gene expression and its accuracy mainly depends on the reference genes which are used for gene expression normalization. In this study, we employed qPCR to select the most stable reference gene in K. virginica which showed stable expression profiles under our experimental conditions. The candidate reference genes were 18S ribosomal RNA (18SrRNA), β-actin (ACT), α-tubulin (TUA), and elongation factor (EF). We tracked the gene expression profiles of the candidate genes and analyzed their stabilities through BestKeeper, geNorm, and NormFinder software programs. The results of the three programs were identical and 18SrRNA was assessed to be the most stable reference gene in this study. However, TUA was identified to be the most unstable. Our study proved again that the traditional reference genes indeed displayed a certain degree of variations under given experimental conditions. Importantly, our research also provides guidance for selecting most suitable reference genes and lays the foundation for further studies in K. virginica. PMID:26581422

Bayesian median regression for temporal gene expression data

NASA Astrophysics Data System (ADS)

Yu, Keming; Vinciotti, Veronica; Liu, Xiaohui; 't Hoen, Peter A. C.

2007-09-01

Most of the existing methods for the identification of biologically interesting genes in a temporal expression profiling dataset do not fully exploit the temporal ordering in the dataset and are based on normality assumptions for the gene expression. In this paper, we introduce a Bayesian median regression model to detect genes whose temporal profile is significantly different across a number of biological conditions. The regression model is defined by a polynomial function where both time and condition effects as well as interactions between the two are included. MCMC-based inference returns the posterior distribution of the polynomial coefficients. From this a simple Bayes factor test is proposed to test for significance. The estimation of the median rather than the mean, and within a Bayesian framework, increases the robustness of the method compared to a Hotelling T2-test previously suggested. This is shown on simulated data and on muscular dystrophy gene expression data.

Effect of Ppd-1 on the expression of flowering-time genes in vegetative and reproductive growth stages of wheat.

PubMed

Kitagawa, Satoshi; Shimada, Sanae; Murai, Koji

2012-01-01

The photoperiod sensitivity gene Ppd-1 influences the timing of flowering in temperate cereals such as wheat and barley. The effect of Ppd-1 on the expression of flowering-time genes was assessed by examining the expression levels of the vernalization genes VRN1 and VRN3/WFT and of two CONSTANS-like genes, WCO1 and TaHd1, during vegetative and reproductive growth stages. Two near-isogenic lines (NILs) were used: the first carried a photoperiod-insensitive allele of Ppd-1 (Ppd-1a-NIL), the other, a photoperiod-sensitive allele (Ppd-1b-NIL). We found that the expression pattern of VRN1 was similar in Ppd-1a-NIL and Ppd-1b-NIL plants, suggesting that VRN1 is not regulated by Ppd-1. Under long day conditions, VRN3/WFT showed similar expression patterns in Ppd-1a-NIL and Ppd-1b-NIL plants. However, expression differed greatly under short day conditions: VRN3/WFT expression was detected in Ppd-1a-NIL plants at the 5-leaf stage when they transited from vegetative to reproductive growth; very low expression was present in Ppd-1b-NIL throughout all growth stages. Thus, the Ppd-1b allele acts to down-regulate VRN3/WFT under short day conditions. WCO1 showed high levels of expression at the vegetative stage, which decreased during the phase transition and reproductive growth stages in both Ppd-1a-NIL and Ppd-1b-NIL plants under short day conditions. By contrast to WCO1, TaHd1 was up-regulated during the reproductive stage. The level of TaHd1 expression was much higher in Ppd-1a-NIL than the Ppd-1b-NIL plants, suggesting that the Ppd-1b allele down-regulates TaHd1 under short day conditions. The present study indicates that down-regulation of VRN3/WFT together with TaHd1 is the cause of late flowering in the Ppd-1b-NIL plants under short day conditions.

Transcriptional response of Pasteurella multocida to defined iron sources.

PubMed

Paustian, Michael L; May, Barbara J; Cao, Dongwei; Boley, Daniel; Kapur, Vivek

2002-12-01

Pasteurella multocida was grown in iron-free chemically defined medium supplemented with hemoglobin, transferrin, ferritin, and ferric citrate as iron sources. Whole-genome DNA microarrays were used to monitor global gene expression over seven time points after the addition of the defined iron source to the medium. This resulted in a set of data containing over 338,000 gene expression observations. On average, 12% of P. multocida genes were differentially expressed under any single condition. A majority of these genes encoded P. multocida proteins that were involved in either transport and binding or were annotated as hypothetical proteins. Several trends are evident when the data from different iron sources are compared. In general, only two genes (ptsN and sapD) were expressed at elevated levels under all of the conditions tested. The results also show that genes with increased expression in the presence of hemoglobin did not respond to transferrin or ferritin as an iron source. Correspondingly, genes with increased expression in the transferrin and ferritin experiments were expressed at reduced levels when hemoglobin was supplied as the sole iron source. Finally, the data show that genes that were most responsive to the presence of ferric citrate did not follow a trend similar to that of the other iron sources, suggesting that different pathways respond to inorganic or organic sources of iron in P. multocida. Taken together, our results demonstrate that unique subsets of P. multocida genes are expressed in response to different iron sources and that many of these genes have yet to be functionally characterized.

CRISPR/Cas9 mediates efficient conditional mutagenesis in Drosophila.

PubMed

Xue, Zhaoyu; Wu, Menghua; Wen, Kejia; Ren, Menda; Long, Li; Zhang, Xuedi; Gao, Guanjun

2014-09-05

Existing transgenic RNA interference (RNAi) methods greatly facilitate functional genome studies via controlled silencing of targeted mRNA in Drosophila. Although the RNAi approach is extremely powerful, concerns still linger about its low efficiency. Here, we developed a CRISPR/Cas9-mediated conditional mutagenesis system by combining tissue-specific expression of Cas9 driven by the Gal4/upstream activating site system with various ubiquitously expressed guide RNA transgenes to effectively inactivate gene expression in a temporally and spatially controlled manner. Furthermore, by including multiple guide RNAs in a transgenic vector to target a single gene, we achieved a high degree of gene mutagenesis in specific tissues. The CRISPR/Cas9-mediated conditional mutagenesis system provides a simple and effective tool for gene function analysis, and complements the existing RNAi approach. Copyright © 2014 Xue et al.

Multiple abiotic stimuli are integrated in the regulation of rice gene expression under field conditions

PubMed Central

Plessis, Anne; Hafemeister, Christoph; Wilkins, Olivia; Gonzaga, Zennia Jean; Meyer, Rachel Sarah; Pires, Inês; Müller, Christian; Septiningsih, Endang M; Bonneau, Richard; Purugganan, Michael

2015-01-01

Plants rely on transcriptional dynamics to respond to multiple climatic fluctuations and contexts in nature. We analyzed the genome-wide gene expression patterns of rice (Oryza sativa) growing in rainfed and irrigated fields during two distinct tropical seasons and determined simple linear models that relate transcriptomic variation to climatic fluctuations. These models combine multiple environmental parameters to account for patterns of expression in the field of co-expressed gene clusters. We examined the similarities of our environmental models between tropical and temperate field conditions, using previously published data. We found that field type and macroclimate had broad impacts on transcriptional responses to environmental fluctuations, especially for genes involved in photosynthesis and development. Nevertheless, variation in solar radiation and temperature at the timescale of hours had reproducible effects across environmental contexts. These results provide a basis for broad-based predictive modeling of plant gene expression in the field. DOI: http://dx.doi.org/10.7554/eLife.08411.001 PMID:26609814

[Application of dhfr gene negative Chinese hamster ovary cell line to express hepatitis B virus surface antigen].

PubMed

Yi, Y; Zhang, M; Liu, C

2001-06-01

To set up an efficient expressing system for recombinant hepatitis B virus surface antigen (HBsAg) in dhfr gene negative CHO cell line. HBsAg gene expressing plasmid pCI-dhfr-S was constructed by integrating HBsAg gene into plasmid pCI which carries dhfr gene. The HBsAg expressing cell line was set up by transfection of plasmid pCI-dhfr-S into dhfr gene negative CHO cell line in the way of lipofectin. Under the selective pressure of MTX, 18 of 28 clonized cell lines expressed HBsAg, 4 of them reached a high titer of 1:32 and protein content 1-3 micrograms/ml. In this study, the high level expression of HBsAg demonstrated that the dhfr negative mammalian cell line when recombined with plasmid harboring the corresponding deleted gene can efficiently express the foreign gene. The further steps toward building optimum conditions of the expressing system and the increase of expressed product are under study.

Identification and Evaluation of Reliable Reference Genes for Quantitative Real-Time PCR Analysis in Tea Plant (Camellia sinensis (L.) O. Kuntze)

PubMed Central

Hao, Xinyuan; Horvath, David P.; Chao, Wun S.; Yang, Yajun; Wang, Xinchao; Xiao, Bin

2014-01-01

Reliable reference selection for the accurate quantification of gene expression under various experimental conditions is a crucial step in qRT-PCR normalization. To date, only a few housekeeping genes have been identified and used as reference genes in tea plant. The validity of those reference genes are not clear since their expression stabilities have not been rigorously examined. To identify more appropriate reference genes for qRT-PCR studies on tea plant, we examined the expression stability of 11 candidate reference genes from three different sources: the orthologs of Arabidopsis traditional reference genes and stably expressed genes identified from whole-genome GeneChip studies, together with three housekeeping gene commonly used in tea plant research. We evaluated the transcript levels of these genes in 94 experimental samples. The expression stabilities of these 11 genes were ranked using four different computation programs including geNorm, Normfinder, BestKeeper, and the comparative ∆CT method. Results showed that the three commonly used housekeeping genes of CsTUBULIN1, CsACINT1 and Cs18S rRNA1 together with CsUBQ1 were the most unstable genes in all sample ranking order. However, CsPTB1, CsEF1, CsSAND1, CsCLATHRIN1 and CsUBC1 were the top five appropriate reference genes for qRT-PCR analysis in complex experimental conditions. PMID:25474086

Exploring valid internal-control genes in Porphyra yezoensis (Bangiaceae) during stress response conditions

NASA Astrophysics Data System (ADS)

Wang, Wenlei; Wu, Xiaojie; Wang, Chao; Jia, Zhaojun; He, Linwen; Wei, Yifan; Niu, Jianfeng; Wang, Guangce

2014-07-01

To screen the stable expression genes related to the stress (strong light, dehydration and temperature shock) we applied Absolute real-time PCR technology to determine the transcription numbers of the selected test genes in P orphyra yezoensis, which has been regarded as a potential model species responding the stress conditions in the intertidal. Absolute real-time PCR technology was applied to determine the transcription numbers of the selected test genes in P orphyra yezoensis, which has been regarded as a potential model species in stress responding. According to the results of photosynthesis parameters, we observed that Y(II) and F v/ F m were significantly affected when stress was imposed on the thalli of P orphyra yezoensis, but underwent almost completely recovered under normal conditions, which were collected for the following experiments. Then three samples, which were treated with different grade stresses combined with salinity, irradiation and temperature, were collected. The transcription numbers of seven constitutive expression genes in above samples were determined after RNA extraction and cDNA synthesis. Finally, a general insight into the selection of internal control genes during stress response was obtained. We found that there were no obvious effects in terms of salinity stress (at salinity 90) on transcription of most genes used in the study. The 18S ribosomal RNA gene had the highest expression level, varying remarkably among different tested groups. RPS8 expression showed a high irregular variance between samples. GAPDH presented comparatively stable expression and could thus be selected as the internal control. EF-1α showed stable expression during the series of multiple-stress tests. Our research provided available references for the selection of internal control genes for transcripts determination of P. yezoensis.

Identification of novel Cyclooxygenase-2-dependent genes in Helicobacter pylori infection in vivo

PubMed Central

Walduck, Anna K; Weber, Matthias; Wunder, Christian; Juettner, Stefan; Stolte, Manfred; Vieth, Michael; Wiedenmann, Bertram; Meyer, Thomas F; Naumann, Michael; Hoecker, Michael

2009-01-01

Background Helicobacter pylori is a crucial determining factor in the pathogenesis of benign and neoplastic gastric diseases. Cyclooxygenase-2 (Cox-2) is the inducible key enzyme of arachidonic acid metabolism and is a central mediator in inflammation and cancer. Expression of the Cox-2 gene is up-regulated in the gastric mucosa during H. pylori infection but the pathobiological consequences of this enhanced Cox-2 expression are not yet characterized. The aim of this study was to identify novel genes down-stream of Cox-2 in an in vivo model, thereby identifying potential targets for the study of the role of Cox- 2 in H. pylori pathogenesis and the initiation of pre- cancerous changes. Results Gene expression profiles in the gastric mucosa of mice treated with a specific Cox-2 inhibitor (NS398) or vehicle were analysed at different time points (6, 13 and 19 wk) after H. pylori infection. H. pylori infection affected the expression of 385 genes over the experimental period, including regulators of gastric physiology, proliferation, apoptosis and mucosal defence. Under conditions of Cox-2 inhibition, 160 target genes were regulated as a result of H. pylori infection. The Cox-2 dependent subset included those influencing gastric physiology (Gastrin, Galr1), epithelial barrier function (Tjp1, connexin45, Aqp5), inflammation (Icam1), apoptosis (Clu) and proliferation (Gdf3, Igf2). Treatment with NS398 alone caused differential expression of 140 genes, 97 of which were unique, indicating that these genes are regulated under conditions of basal Cox-2 expression. Conclusion This study has identified a panel of novel Cox-2 dependent genes influenced under both normal and the inflammatory conditions induced by H. pylori infection. These data provide important new links between Cox-2 and inflammatory processes, epithelial repair and integrity. PMID:19317916

Evaluation and Validation of Reference Genes for qRT-PCR Normalization in Frankliniella occidentalis (Thysanoptera:Thripidae)

PubMed Central

Zheng, Yu-Tao; Li, Hong-Bo; Lu, Ming-Xing; Du, Yu-Zhou

2014-01-01

Quantitative real time PCR (qRT-PCR) has emerged as a reliable and reproducible technique for studying gene expression analysis. For accurate results, the normalization of data with reference genes is particularly essential. Once the transcriptome sequencing of Frankliniella occidentalis was completed, numerous unigenes were identified and annotated. Unfortunately, there are no studies on the stability of reference genes used in F. occidentalis. In this work, seven candidate reference genes, including actin, 18S rRNA, H3, tubulin, GAPDH, EF-1 and RPL32, were evaluated for their suitability as normalization genes under different experimental conditions using the statistical software programs BestKeeper, geNorm, Normfinder and the comparative ΔCt method. Because the rankings of the reference genes provided by each of the four programs were different, we chose a user-friendly web-based comprehensive tool RefFinder to get the final ranking. The result demonstrated that EF-1 and RPL32 displayed the most stable expression in different developmental stages; RPL32 and GAPDH showed the most stable expression at high temperatures, while 18S and EF-1 exhibited the most stable expression at low temperatures. In this study, we validated the suitable reference genes in F. occidentalis for gene expression profiling under different experimental conditions. The choice of internal standard is very important in the normalization of the target gene expression levels, thus validating and selecting the best genes will help improve the quality of gene expression data of F. occidentalis. What is more, these validated reference genes could serve as the basis for the selection of candidate reference genes in other insects. PMID:25356721

Evaluation and validation of reference genes for qRT-PCR normalization in Frankliniella occidentalis (Thysanoptera: Thripidae).

PubMed

Zheng, Yu-Tao; Li, Hong-Bo; Lu, Ming-Xing; Du, Yu-Zhou

2014-01-01

Quantitative real time PCR (qRT-PCR) has emerged as a reliable and reproducible technique for studying gene expression analysis. For accurate results, the normalization of data with reference genes is particularly essential. Once the transcriptome sequencing of Frankliniella occidentalis was completed, numerous unigenes were identified and annotated. Unfortunately, there are no studies on the stability of reference genes used in F. occidentalis. In this work, seven candidate reference genes, including actin, 18S rRNA, H3, tubulin, GAPDH, EF-1 and RPL32, were evaluated for their suitability as normalization genes under different experimental conditions using the statistical software programs BestKeeper, geNorm, Normfinder and the comparative ΔCt method. Because the rankings of the reference genes provided by each of the four programs were different, we chose a user-friendly web-based comprehensive tool RefFinder to get the final ranking. The result demonstrated that EF-1 and RPL32 displayed the most stable expression in different developmental stages; RPL32 and GAPDH showed the most stable expression at high temperatures, while 18S and EF-1 exhibited the most stable expression at low temperatures. In this study, we validated the suitable reference genes in F. occidentalis for gene expression profiling under different experimental conditions. The choice of internal standard is very important in the normalization of the target gene expression levels, thus validating and selecting the best genes will help improve the quality of gene expression data of F. occidentalis. What is more, these validated reference genes could serve as the basis for the selection of candidate reference genes in other insects.

Selection of reference genes for expression analyses of red-fleshed sweet orange (Citrus sinensis).

PubMed

Pinheiro, T T; Nishimura, D S; De Nadai, F B; Figueira, A; Latado, R R

2015-12-28

Red-fleshed oranges (Citrus sinensis) contain high levels of carotenoids and lycopene. The growing consumer demand for products with health benefits has increased interest in these types of Citrus cultivars as a potential source of nutraceuticals. However, little is known about the physiology of these cultivars under Brazilian conditions. Transcriptome and gene expression analyses are important tools in the breeding and management of red-fleshed sweet orange cultivars. Reverse transcription quantitative polymerase chain reaction is a method of quantifying gene expression, but various standardizations are required to obtain precise, accurate, and specific results. Among the standardizations required, the choice of suitable stable reference genes is fundamental. The objective of this study was to evaluate the stability of 11 candidate genes using various tissue and organ samples from healthy plants or leaves from citrus greening disease (Huanglongbing)-symptomatic plants of a Brazilian red-fleshed cultivar ('Sanguínea de Mombuca'), in order to select the most suitable reference gene for investigating gene expression under these conditions. geNorm and NormFinder identified genes that encoded translation initiation factor 3, ribosomal protein L35, and translation initiation factor 5A as the most stable genes under the biological conditions tested, and genes coding actin (ACT) and the subunit of the PSI reaction center subunit III were the least stable. Phosphatase, malate dehydrogenase, and ACT were the most stable genes in the leaf samples of infected plants.

OsNucleolin1-L Expression in Arabidopsis Enhances Photosynthesis via Transcriptome Modification under Salt Stress Conditions.

PubMed

Udomchalothorn, Thanikarn; Plaimas, Kitiporn; Sripinyowanich, Siriporn; Boonchai, Chutamas; Kojonna, Thammaporn; Chutimanukul, Panita; Comai, Luca; Buaboocha, Teerapong; Chadchawan, Supachitra

2017-04-01

OsNUC1 encodes rice nucleolin, which has been shown to be involved in salt stress responses. Expression of the full-length OsNUC1 gene in Arabidopsis resulted in hypersensitivity to ABA during germination. Transcriptome analysis of the transgenic lines, in comparison with the wild type, revealed that the RNA abundance of >1,900 genes was significantly changed under normal growth conditions, while under salt stress conditions the RNAs of 999 genes were found to be significantly regulated. Gene enrichment analysis showed that under normal conditions OsNUC1 resulted in repression of genes involved in photosynthesis, while in salt stress conditions OsNUC1 increased expression of the genes involved in the light-harvesting complex. Correspondingly, the net rate of photosynthesis of the transgenic lines was increased under salt stress. Transgenic rice lines with overexpression of the OsNUC1-L gene were generated and tested for photosynthetic performance under salt stress conditions. The transgenic rice lines treated with salt stress at the booting stage had a higher photosynthetic rate and stomatal conductance in flag leaves and second leaves than the wild type. Moreover, higher contents of Chl a and carotenoids were found in flag leaves of the transgenic rice. These results suggest a role for OsNUC1 in the modification of the transcriptome, especially the gene transcripts responsible for photosynthesis, leading to stabilization of photosynthesis under salt stress conditions. © The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Improvement of experimental testing and network training conditions with genome-wide microarrays for more accurate predictions of drug gene targets

PubMed Central

2014-01-01

Background Genome-wide microarrays have been useful for predicting chemical-genetic interactions at the gene level. However, interpreting genome-wide microarray results can be overwhelming due to the vast output of gene expression data combined with off-target transcriptional responses many times induced by a drug treatment. This study demonstrates how experimental and computational methods can interact with each other, to arrive at more accurate predictions of drug-induced perturbations. We present a two-stage strategy that links microarray experimental testing and network training conditions to predict gene perturbations for a drug with a known mechanism of action in a well-studied organism. Results S. cerevisiae cells were treated with the antifungal, fluconazole, and expression profiling was conducted under different biological conditions using Affymetrix genome-wide microarrays. Transcripts were filtered with a formal network-based method, sparse simultaneous equation models and Lasso regression (SSEM-Lasso), under different network training conditions. Gene expression results were evaluated using both gene set and single gene target analyses, and the drug’s transcriptional effects were narrowed first by pathway and then by individual genes. Variables included: (i) Testing conditions – exposure time and concentration and (ii) Network training conditions – training compendium modifications. Two analyses of SSEM-Lasso output – gene set and single gene – were conducted to gain a better understanding of how SSEM-Lasso predicts perturbation targets. Conclusions This study demonstrates that genome-wide microarrays can be optimized using a two-stage strategy for a more in-depth understanding of how a cell manifests biological reactions to a drug treatment at the transcription level. Additionally, a more detailed understanding of how the statistical model, SSEM-Lasso, propagates perturbations through a network of gene regulatory interactions is achieved. PMID:24444313

Lhx2 Determines Odorant Receptor Expression Frequency in Mature Olfactory Sensory Neurons

PubMed Central

Zhang, Guangfan; Titlow, William B.; Biecker, Stephanie M.; Stromberg, Arnold J.

2016-01-01

Abstract A developmental program of epigenetic repression prepares each mammalian olfactory sensory neuron (OSN) to strongly express one allele from just one of hundreds of odorant receptor (OR) genes, but what completes this process of OR gene choice by driving the expression of this allele is incompletely understood. Conditional deletion experiments in mice demonstrate that Lhx2 is necessary for normal expression frequencies of nearly all ORs and all trace amine-associated receptors, irrespective of whether the deletion of Lhx2 is initiated in immature or mature OSNs. Given previous evidence that Lhx2 binds OR gene control elements, these findings indicate that Lhx2 is directly involved in driving OR expression. The data also support the conclusion that OR expression is necessary to allow immature OSNs to complete differentiation and become mature. In contrast to the robust effects of conditional deletion of Lhx2, the loss of Emx2 has much smaller effects and more often causes increased expression frequencies. Lhx2:Emx2 double mutants show opposing effects on Olfr15 expression that reveal independent effects of these two transcription factors. While Lhx2 is necessary for OR expression that supports OR gene choice, Emx2 can act differently; perhaps by helping to control the availability of OR genes for expression. PMID:27822500

Conditional transgenic mouse models: from the basics to genome-wide sets of knockouts and current studies of tissue regeneration.

PubMed

Bockamp, Ernesto; Sprengel, Rolf; Eshkind, Leonid; Lehmann, Thomas; Braun, Jan M; Emmrich, Frank; Hengstler, Jan G

2008-03-01

Many mouse models are currently available, providing avenues to elucidate gene function and to recapitulate specific pathological conditions. To a large extent, successful translation of clinical evidence or analytical data into appropriate mouse models is possible through progress in transgenic or gene-targeting technology. Beginning with a review of standard mouse transgenics and conventional gene targeting, this article will move on to discussing the basics of conditional gene expression: the tetracycline (tet)-off and tet-on systems based on the transactivators tet-controlled transactivator (Tta) and reverse tet-on transactivator (rtTA) that allow downregulation or induction of gene expression; Cre or Flp recombinase-mediated modifications, including excision, inversion, insertion and interchromosomal translocation; combination of the tet and Cre systems, permitting inducible knockout, reporter gene activation or activation of point mutations; the avian retroviral system based on delivery of rtTA specifically into cells expressing the avian retroviral receptor, which enables cell type-specific, inducible gene expression; the tamoxifen system, one of the most frequently applied steroid receptor-based systems, allows rapid activation of a fusion protein between the gene of interest and a mutant domain of the estrogen receptor, whereby activation does not depend on transcription; and techniques for cell type-specific ablation. The diphtheria toxin receptor system offers the advantage that it can be combined with the 'zoo' of Cre recombinase driver mice. Having described the basics we move on to the cutting edge: generation of genome-wide sets of conditional knockout mice. To this end, large ongoing projects apply two strategies: gene trapping based on random integration of trapping vectors into introns leading to truncation of the transcript, and gene targeting, representing the directed approach using homologous recombination. It can be expected that in the near future genome-wide sets of such mice will be available. Finally, the possibilities of conditional expression systems for investigating gene function in tissue regeneration will be illustrated by examples for neurodegenerative disease, liver regeneration and wound healing of the skin.

BFDCA: A Comprehensive Tool of Using Bayes Factor for Differential Co-Expression Analysis.

PubMed

Wang, Duolin; Wang, Juexin; Jiang, Yuexu; Liang, Yanchun; Xu, Dong

2017-02-03

Comparing the gene-expression profiles between biological conditions is useful for understanding gene regulation underlying complex phenotypes. Along this line, analysis of differential co-expression (DC) has gained attention in the recent years, where genes under one condition have different co-expression patterns compared with another. We developed an R package Bayes Factor approach for Differential Co-expression Analysis (BFDCA) for DC analysis. BFDCA is unique in integrating various aspects of DC patterns (including Shift, Cross, and Re-wiring) into one uniform Bayes factor. We tested BFDCA using simulation data and experimental data. Simulation results indicate that BFDCA outperforms existing methods in accuracy and robustness of detecting DC pairs and DC modules. Results of using experimental data suggest that BFDCA can cluster disease-related genes into functional DC subunits and estimate the regulatory impact of disease-related genes well. BFDCA also achieves high accuracy in predicting case-control phenotypes by using significant DC gene pairs as markers. BFDCA is publicly available at http://dx.doi.org/10.17632/jdz4vtvnm3.1. Copyright © 2016 Elsevier Ltd. All rights reserved.

Expression profiling of Ribosomal Protein gene family in dehydration stress responses and characterization of transgenic rice plants overexpressing RPL23A for water-use efficiency and tolerance to drought and salt stresses

NASA Astrophysics Data System (ADS)

Moin, Mazahar; Bakshi, Achala; Madhav, M. S.; Kirti, P. B.

2017-11-01

Our previous findings on the screening of a large-pool of activation tagged rice plants grown under limited water conditions revealed the activation of Ribosomal Protein Large (RPL) subunit genes, RPL6 and RPL23A in two mutants that exhibited high water-use efficiency (WUE) with the genes getting activated by the integrated 4x enhancers (Moin et al., 2016a). In continuation of these findings, we have comprehensively characterized the Ribosomal Protein (RP) gene family including both small (RPS) and large (RPL) subunits, which have been identified to be encoded by at least 70 representative genes; RP-genes exist as multiple expressed copies with high nucleotide and amino acid sequence similarity. The differential expression of all the representative genes in rice was performed under limited water and drought conditions at progressive time intervals in the present study. More than 50% of the RP genes were upregulated in both shoot and root tissues. Some of them exhibited an overlap in the upregulation under both the treatments indicating that they might have a common role in inducing tolerance under limited water and drought conditions. Among the genes that became significantly upregulated in both the tissues and under both the treatments are RPL6, 7, 23A, 24 and 31 and RPS4, 10 and 18a. To further validate the role of RP genes in WUE and inducing tolerance to other stresses, we have raised transgenic plants overexpressing RPL23A in rice. The high expression lines of RPL23A exhibited low Δ13C, increased quantum efficiency along with suitable growth and yield parameters with respect to negative control under the conditions of limited water availability. The constitutive expression of RPL23A was also associated with transcriptional upregulation of many other RPL and RPS genes. The seedlings of RPL23A high expression lines also showed a significant increase in fresh weight, root length, proline and chlorophyll contents under simulated drought and salt stresses. Taken together, our findings provide a secure basis for the RPL gene family expression as a potential resource for exploring abiotic stress tolerant properties in rice.

Joint mapping of genes and conditions via multidimensional unfolding analysis

PubMed Central

Van Deun, Katrijn; Marchal, Kathleen; Heiser, Willem J; Engelen, Kristof; Van Mechelen, Iven

2007-01-01

Background Microarray compendia profile the expression of genes in a number of experimental conditions. Such data compendia are useful not only to group genes and conditions based on their similarity in overall expression over profiles but also to gain information on more subtle relations between genes and conditions. Getting a clear visual overview of all these patterns in a single easy-to-grasp representation is a useful preliminary analysis step: We propose to use for this purpose an advanced exploratory method, called multidimensional unfolding. Results We present a novel algorithm for multidimensional unfolding that overcomes both general problems and problems that are specific for the analysis of gene expression data sets. Applying the algorithm to two publicly available microarray compendia illustrates its power as a tool for exploratory data analysis: The unfolding analysis of a first data set resulted in a two-dimensional representation which clearly reveals temporal regulation patterns for the genes and a meaningful structure for the time points, while the analysis of a second data set showed the algorithm's ability to go beyond a mere identification of those genes that discriminate between different patient or tissue types. Conclusion Multidimensional unfolding offers a useful tool for preliminary explorations of microarray data: By relying on an easy-to-grasp low-dimensional geometric framework, relations among genes, among conditions and between genes and conditions are simultaneously represented in an accessible way which may reveal interesting patterns in the data. An additional advantage of the method is that it can be applied to the raw data without necessitating the choice of suitable genewise transformations of the data. PMID:17550582

Transcriptome-Wide Identification of Reference Genes for Expression Analysis of Soybean Responses to Drought Stress along the Day.

PubMed

Marcolino-Gomes, Juliana; Rodrigues, Fabiana Aparecida; Fuganti-Pagliarini, Renata; Nakayama, Thiago Jonas; Ribeiro Reis, Rafaela; Bouças Farias, Jose Renato; Harmon, Frank G; Correa Molinari, Hugo Bruno; Correa Molinari, Mayla Daiane; Nepomuceno, Alexandre

2015-01-01

The soybean transcriptome displays strong variation along the day in optimal growth conditions and also in response to adverse circumstances, like drought stress. However, no study conducted to date has presented suitable reference genes, with stable expression along the day, for relative gene expression quantification in combined studies on drought stress and diurnal oscillations. Recently, water deficit responses have been associated with circadian clock oscillations at the transcription level, revealing the existence of hitherto unknown processes and increasing the demand for studies on plant responses to drought stress and its oscillation during the day. We performed data mining from a transcriptome-wide background using microarrays and RNA-seq databases to select an unpublished set of candidate reference genes, specifically chosen for the normalization of gene expression in studies on soybean under both drought stress and diurnal oscillations. Experimental validation and stability analysis in soybean plants submitted to drought stress and sampled during a 24 h timecourse showed that four of these newer reference genes (FYVE, NUDIX, Golgin-84 and CYST) indeed exhibited greater expression stability than the conventionally used housekeeping genes (ELF1-β and β-actin) under these conditions. We also demonstrated the effect of using reference candidate genes with different stability values to normalize the relative expression data from a drought-inducible soybean gene (DREB5) evaluated in different periods of the day.

Biochemical characteristics and gene expression profiles of two paralogous luciferases from the Japanese firefly Pyrocoelia atripennis (Coleoptera, Lampyridae, Lampyrinae): insight into the evolution of firefly luciferase genes.

PubMed

Bessho-Uehara, Manabu; Konishi, Kaori; Oba, Yuichi

2017-08-09

Two paralogous genes of firefly luciferase, Luc1 and Luc2, have been isolated from the species in two subfamilies, Luciolinae and Photurinae, of the family Lampyridae. The gene expression profiles have previously been examined only in the species of Luciolinae. Here we isolated Luc1 and Luc2 genes from the Japanese firefly Pyrocoelia atripennis. This is the first report of the presence of both Luc1 and Luc2 genes in the species of the subfamily Lampyrinae and of the exon-intron structure of Luc2 in the family Lampyridae. The luminescence of both gene products peaked at 547 nm under neutral buffer conditions, and the spectrum of Luc1, but not Luc2, was red-shifted under acidic conditions, as observed for Luc2 in the Luciolinae species. The semi-quantitative reverse transcription-polymerase chain reaction suggested that Luc1 was expressed in lanterns of all the stages except eggs, while Luc2 was expressed in the non-lantern bodies of eggs, prepupae, pupae, and female adults. These expression profiles are consistent with those in the Luciolinae species. Considering the distant phylogenetic relationship between Lampyrinae and Luciolinae in Lampyridae, we propose that fireflies generally possess two different luciferase genes and the biochemical properties and gene expression profiles for each paralog are conserved among lampyrid species.

Pervasive Effects of Aging on Gene Expression in Wild Wolves.

PubMed

Charruau, Pauline; Johnston, Rachel A; Stahler, Daniel R; Lea, Amanda; Snyder-Mackler, Noah; Smith, Douglas W; vonHoldt, Bridgett M; Cole, Steven W; Tung, Jenny; Wayne, Robert K

2016-08-01

Gene expression levels change as an individual ages and responds to environmental conditions. With the exception of humans, such patterns have principally been studied under controlled conditions, overlooking the array of developmental and environmental influences that organisms encounter under conditions in which natural selection operates. We used high-throughput RNA sequencing (RNA-Seq) of whole blood to assess the relative impacts of social status, age, disease, and sex on gene expression levels in a natural population of gray wolves (Canis lupus). Our findings suggest that age is broadly associated with gene expression levels, whereas other examined factors have minimal effects on gene expression patterns. Further, our results reveal evolutionarily conserved signatures of senescence, such as immunosenescence and metabolic aging, between wolves and humans despite major differences in life history and environment. The effects of aging on gene expression levels in wolves exhibit conservation with humans, but the more rapid expression differences observed in aging wolves is evolutionarily appropriate given the species' high level of extrinsic mortality due to intraspecific aggression. Some expression changes that occur with age can facilitate physical age-related changes that may enhance fitness in older wolves. However, the expression of these ancestral patterns of aging in descendant modern dogs living in highly modified domestic environments may be maladaptive and cause disease. This work provides evolutionary insight into aging patterns observed in domestic dogs and demonstrates the applicability of studying natural populations to investigate the mechanisms of aging. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Hypothermia reduces VEGF-165 expression, but not osteogenic differentiation of human adipose stem cells under hypoxia

PubMed Central

Bakker, Astrid D.; Hogervorst, Jolanda M. A.; Nolte, Peter A.; Klein-Nulend, Jenneke

2017-01-01

Cryotherapy is successfully used in the clinic to reduce pain and inflammation after musculoskeletal damage, and might prevent secondary tissue damage under the prevalent hypoxic conditions. Whether cryotherapy reduces mesenchymal stem cell (MSC) number and differentiation under hypoxic conditions, causing impaired callus formation is unknown. We aimed to determine whether hypothermia modulates proliferation, apoptosis, nitric oxide production, VEGF gene and protein expression, and osteogenic/chondrogenic differentiation of human MSCs under hypoxia. Human adipose MSCs were cultured under hypoxia (37°C, 1% O2), hypothermia and hypoxia (30°C, 1% O2), or control conditions (37°C, 20% O2). Total DNA, protein, nitric oxide production, alkaline phosphatase activity, gene expression, and VEGF protein concentration were measured up to day 8. Hypoxia enhanced KI67 expression at day 4. The combination of hypothermia and hypoxia further enhanced KI67 gene expression compared to hypoxia alone, but was unable to prevent the 1.2-fold reduction in DNA amount caused by hypoxia at day 4. Addition of hypothermia to hypoxic cells did not alter the effect of hypoxia alone on BAX-to-BCL-2 ratio, alkaline phosphatase activity, gene expression of SOX9, COL1, or osteocalcin, or nitric oxide production. Hypothermia decreased the stimulating effect of hypoxia on VEGF-165 gene expression by 6-fold at day 4 and by 2-fold at day 8. Hypothermia also decreased VEGF protein expression under hypoxia by 2.9-fold at day 8. In conclusion, hypothermia decreased VEGF-165 gene and protein expression, but did not affect differentiation, or apoptosis of MSCs cultured under hypoxia. These in vitro results implicate that hypothermia treatment in vivo, applied to alleviate pain and inflammation, is not likely to harm early stages of callus formation. PMID:28166273

Effects of bright light exposure during daytime on peripheral clock gene expression in humans.

PubMed

Sato, Maki; Wakamura, Tomoko; Morita, Takeshi; Okamoto, Akihiko; Akashi, Makoto; Matsui, Takuya; Sato, Motohiko

2017-06-01

Light is the strongest synchronizer controlling circadian rhythms. The intensity and duration of light change throughout the year, thereby influencing body weight, food preferences, and melatonin secretion in humans and animals. Although the expression of clock genes has been examined using human samples, it currently remains unknown whether bright light during the daytime affects the expression of these genes in humans. Therefore, we herein investigated the effects of bright light exposure during the daytime on clock gene expression in the hair follicular and root cells of the human scalp. Seven healthy men (20.4 ± 2.2 years old; 172.3 ± 5.8 cm; 64.3 ± 8.5 kg; BMI 21.7 ± 3.1 kg/m 2 , mean ± SD) participated in this study. Subjects completed 3-day experimental sessions twice in 1 month during which they were exposed to bright and dim light conditions. The mRNA expression of Per1-3, Cry1-2, Rev-erb-α (Nr1d1), Rev-erb-β (Nr1d2), and Dec1 was analyzed using branched DNA probes. No significant changes were observed in the expression of Per1, Per2, Per3, Cry1, Cry2, Rev-erb-α (Nr1d1), or Dec1 following exposure to bright light conditions. However, the expression of Rev-erb-β (Nr1d2) tended to be stronger under bright light than dim light conditions. These results suggest that the bright light stimulus did not influence the expression of clock genes in humans. Long-lasting bright light exposure during the daytime may be required to change the expression of clock genes in humans.

Gene expression of commensal Lactobacillus johnsonii strain NCC533 during in vitro growth and in the murine gut.

PubMed

Denou, Emmanuel; Berger, Bernard; Barretto, Caroline; Panoff, Jean-Michel; Arigoni, Fabrizio; Brüssow, Harald

2007-11-01

Work with pathogens like Vibrio cholerae has shown major differences between genes expressed in bacteria grown in vitro and in vivo. To explore this subject for commensals, we investigated the transcription of the Lactobacillus johnsonii NCC533 genome during in vitro and in vivo growth using the microarray technology. During broth growth, 537, 626, and 277 of the 1,756 tested genes were expressed during exponential phase, "adaptation" (early stationary phase), and stationary phase, respectively. One hundred one, 150, and 33 genes, respectively, were specifically transcribed in these three phases. To explore the in vivo transcription program, we fed L. johnsonii containing a resistance plasmid to antibiotic-treated mice. After a 2-day washout phase, we determined the viable-cell counts of lactobacilli that were in the lumina and associated with the mucosae of different gut segments. While the cell counts showed a rather uniform distribution along the gut, we observed marked differences with respect to the expression of the Lactobacillus genome. The largest number of transcribed genes was in the stomach (n = 786); the next-largest numbers occurred in the cecum (n = 391) and the jejunum (n = 296), while only 26 Lactobacillus genes were transcribed in the colon. In vitro and in vivo transcription programs overlapped only partially. One hundred ninety-one of the transcripts from the lactobacilli in the stomach were not detected during in vitro growth; 202 and 213 genes, respectively, were transcribed under all in vitro and in vivo conditions; but the core transcriptome for all growth conditions comprised only 103 genes. Forty-four percent of the NCC533 genes were not detectably transcribed under any of the investigated conditions. Nontranscribed genes were clustered on the genome and enriched in the variable-genome part. Our data revealed not only major differences between in vitro- and in vivo-expressed genes in a Lactobacillus gut commensal organism but also marked changes in the expression of genes along the digestive tract.

Season-dependent effects of photoperiod and temperature on circadian rhythm of arylalkylamine N-acetyltransferase2 gene expression in pineal organ of an air-breathing catfish, Clarias gariepinus.

PubMed

Singh, Kshetrimayum Manisana; Saha, Saurav; Gupta, Braj Bansh Prasad

2017-08-01

Arylalkylamine N-acetyltransferase (AANAT) activity, aanat gene expression and melatonin production have been reported to exhibit prominent circadian rhythm in the pineal organ of most species of fish. Three types of aanat genes are expressed in fish, but the fish pineal organ predominantly expresses aanat2 gene. Increase and decrease in daylength is invariably associated with increase and decrease in temperature, respectively. But so far no attempt has been made to delineate the role of photoperiod and temperature in regulation of the circadian rhythm of aanat2 gene expression in the pineal organ of any fish with special reference to seasons. Therefore, we studied effects of various lighting regimes (12L-12D, 16L-8D, 8L-16D, LL and DD) at a constant temperature (25°C) and effects of different temperatures (15°, 25° and 35°C) under a common photoperiod 12L-12D on circadian rhythm of aanat2 gene expression in the pineal organ of Clarias gariepinus during summer and winter seasons. Aanat2 gene expression in fish pineal organ was studied by measuring aanat2 mRNA levels using Real-Time PCR. Our findings indicate that the pineal organ of C. gariepinus exhibits a prominent circadian rhythm of aanat2 gene expression irrespective of photoperiods, temperatures and seasons, and the circadian rhythm of aanat2 gene expression responds differently to different photoperiods and temperatures in a season-dependent manner. Existence of circadian rhythm of aanat2 gene expression in pineal organs maintained in vitro under 12L-12D and DD conditions as well as a free running rhythm of the gene expression in pineal organ of the fish maintained under LL and DD conditions suggest that the fish pineal organ possesses an endogenous circadian oscillator, which is entrained by light-dark cycle. Copyright © 2017 Elsevier B.V. All rights reserved.

Effects of methionine supplementation on the expression of oxidative stress-related genes in acute heat stress-exposed broilers.

PubMed

Del Vesco, Ana Paula; Gasparino, Eliane; Grieser, Daiane de Oliveira; Zancanela, Vittor; Soares, Maria Amélia Menck; Neto, Adhemar Rodrigues de Oliveira

2015-02-28

The aim of the present study was to evaluate the effects of heat stress (HS) and methionine supplementation on the markers of stress and on the gene expression levels of uncoupling proteins (UCP), betaine-homocysteine methyltransferase (BHMT), cystathionine β-synthase (CBS), glutathione synthetase (GSS) and glutathione peroxidase 7 (GPx7). Broilers from 1 to 21 d and from 22 to 42 d of age were divided into three treatment groups related to methionine supplementation: without methionine supplementation (MD); recommended level of methionine supplementation (DL1); excess methionine supplementation (DL2). The broilers were either kept at a comfortable thermal temperature or exposed to HS (38°C for 24 h). During the starter period, we observed the effects of the interaction between diet and environment on the gene expression levels of UCP, BHMT and GSS. Higher gene expression levels of UCP and BHMT were observed in broilers that were maintained at thermal comfort conditions and received the MD diet. HS broilers fed the DL1 and DL2 diets had the highest expression level of GSS. The expression levels of the CBS and GPx7 genes were influenced by both the environment and methionine supplementation. During the grower period, the gene expression levels of BHMT, CBS, GSS and GPx7 were affected by the diet × environment interaction. A higher expression level of BHMT was observed in broilers maintained at thermal comfort conditions and on the MD diet. HS induced higher expression levels of CBS, GSS and GPx7 in broilers that received the DL1 and DL2 diets. The present results suggest that under HS conditions, methionine supplementation could mitigate the effects of stress, since methionine contributed to the increased expression levels of genes related to antioxidant activity.

A Stationary Wavelet Entropy-Based Clustering Approach Accurately Predicts Gene Expression

PubMed Central

Nguyen, Nha; Vo, An; Choi, Inchan

2015-01-01

Abstract Studying epigenetic landscapes is important to understand the condition for gene regulation. Clustering is a useful approach to study epigenetic landscapes by grouping genes based on their epigenetic conditions. However, classical clustering approaches that often use a representative value of the signals in a fixed-sized window do not fully use the information written in the epigenetic landscapes. Clustering approaches to maximize the information of the epigenetic signals are necessary for better understanding gene regulatory environments. For effective clustering of multidimensional epigenetic signals, we developed a method called Dewer, which uses the entropy of stationary wavelet of epigenetic signals inside enriched regions for gene clustering. Interestingly, the gene expression levels were highly correlated with the entropy levels of epigenetic signals. Dewer separates genes better than a window-based approach in the assessment using gene expression and achieved a correlation coefficient above 0.9 without using any training procedure. Our results show that the changes of the epigenetic signals are useful to study gene regulation. PMID:25383910

Genome-wide identification of suitable zebrafish Danio rerio reference genes for normalization of gene expression data by RT-qPCR.

PubMed

Xu, H; Li, C; Zeng, Q; Agrawal, I; Zhu, X; Gong, Z

2016-06-01

In this study, to systematically identify the most stably expressed genes for internal reference in zebrafish Danio rerio investigations, 37 D. rerio transcriptomic datasets (both RNA sequencing and microarray data) were collected from gene expression omnibus (GEO) database and unpublished data, and gene expression variations were analysed under three experimental conditions: tissue types, developmental stages and chemical treatments. Forty-four putative candidate genes were identified with the c.v. <0·2 from all datasets. Following clustering into different functional groups, 21 genes, in addition to four conventional housekeeping genes (eef1a1l1, b2m, hrpt1l and actb1), were selected from different functional groups for further quantitative real-time (qrt-)PCR validation using 25 RNA samples from different adult tissues, developmental stages and chemical treatments. The qrt-PCR data were then analysed using the statistical algorithm refFinder for gene expression stability. Several new candidate genes showed better expression stability than the conventional housekeeping genes in all three categories. It was found that sep15 and metap1 were the top two stable genes for tissue types, ube2a and tmem50a the top two for different developmental stages, and rpl13a and rp1p0 the top two for chemical treatments. Thus, based on the extensive transcriptomic analyses and qrt-PCR validation, these new reference genes are recommended for normalization of D. rerio qrt-PCR data respectively for the three different experimental conditions. © 2016 The Fisheries Society of the British Isles.

Neutral buoyancy and sleep-deprived serum factors alter expression of cytokines regulating osteogenesis

NASA Astrophysics Data System (ADS)

Gorczynski, Reginald M.; Gorczynski, Christopher P.; Gorczynski, Laura Y.; Hu, Jiang; Lu, Jin; Manuel, Justin; Lee, Lydia

2005-05-01

We examined expression of genes associated with cytokine production, and genes implicated in regulating bone metabolism, in bone stromal and osteoblast cells incubated under standard ground conditions and under conditions of neutral buoyancy, and in the presence/absence of serum from normal or sleep-deprived mice. We observed a clear interaction between these two conditions (exposure to neutral buoyancy and serum stimulation) in promoting enhanced osteoclastogenesis. Both conditions independently altered expression of a number of cytokines implicated in the regulation of bone metabolism. However, using stromal cells from IL-1 and TNF α cytokine r KO mice, we concluded that the increased bone loss under microgravity conditions was not primarily cytokine mediated.

Manufacturing porcine islets: culture at 22 °C has no advantage above culture at 37 °C: a gene expression evaluation.

PubMed

Mueller, Kate R; Martins, Kyra V; Murtaugh, Michael P; Schuurman, Henk-Jan; Papas, Klearchos K

2013-01-01

The manufacturing process of islets includes a culture step which was originally introduced to ease the logistics of procedures in preparing the graft and transplant recipient. It has been suggested that culture at room temperature has an advantage over culture at 37 °C, in part by reducing immunogenicity via preferential elimination of contaminating cells (such as passenger leukocytes) within islets. We investigated this using islets isolated from pancreata of adult pigs. Porcine islets were isolated from three donors and cultured at 37 °C for 1 day, and then under three different conditions: 37 °C for 6 days (condition A); 22 °C for 6 days (condition B); or 22 °C for 5 days followed by 37 °C for 1 day (condition C). Recovery was assessed by DNA measurement, viability by oxygen consumption rate normalized for DNA (OCR/DNA), and gene expression by RT-PCR for a series of 9 lymphocyte markers, 11 lymphokines and chemokines, and 14 apoptotic and stress markers. Post-culture islet recoveries were similar for the three culture conditions. Average OCR/DNA values were 129-159 nmol/min·mgDNA before culture, and 259-291, 204-212, and 207-228 nmol/min·mgDNA, respectively, for culture under conditions A, B, and C, respectively. Irrespective of culture condition, examined gene expression in all three series of lymphocyte markers, lymphokines and chemokines, and apoptotic and stress markers manifested a statistically significant decrease upon culture for 7 days. This decrease was most dramatic for condition A: in particular, most of lymphocyte markers showed a >10-fold reduction and also six markers in the lymphokine and chemokine series; these reductions are consistent with the elimination of immune cells present within islets during culture. The reduction was less for apoptotic and stress markers. For culture under condition B, the reduction in gene expression was less, and culture under condition C resulted in gene expression levels similar to those under condition A: this indicates that 24 h at 37 °C is sufficient to re-equilibrate gene expression levels from those in islets cultured at 22 °C to those in islets cultured at 37 °C. Results were consistent among the preparations from the three donors. Culture of porcine islets at 37 °C provides benefits over culture at 22 °C with respect to OCR/DNA outcomes and reduced expression of genes encoding lymphocyte markers, lymphokines and chemokines, and markers for apoptosis and stress. © 2013 John Wiley & Sons A/S.

Responses of plant seedlings to hypergravity: cellular and molecular aspects

NASA Astrophysics Data System (ADS)

Hoson, T.; Yoshioka, R.; Soga, K.; Wakabayashi, K.; Takeba, G.

Hypergravity produced by centrifugation has been used to analyze the responses of plant seedlings to gravity stimulus. Elongation growth of stem organs is suppressed by hypergravity, which can be recognized as a way for plants to resist gravitational force. The mechanisms inducing growth suppression under hypergravity conditions were analyzed at cellular and molecular levels. When growth was suppressed by hypergravity, a decrease in the cell wall extensibility was brought about in various plants. Hypergravity also induced a cell wall thickening and an increase in the molecular mass of the certain hemicellulosic polysaccharides. Both a decrease in the activities hydrolyzing such polysaccharides and an increase in the apoplast pH were involved in such changes in the cell wall constituents. Thus, the cell wall metabolism is greatly modified under hypergravity conditions, which causes a decrease in the cell wall extensibility, thereby inhibiting elongation growth in stem organs. On the other hand, to identify genes involved in hypergravity-induced growth suppression, changes in gene expression by hypergravity treatment were analyzed in Arabidopsis hypocotyls by differential display method. Sixty-two genes were expressed differentially: expression levels of 39 genes increased, whereas those of 23 genes decreased under hypergravity conditions. The expression of these genes was further analyzed using RT-PCR. One of genes upregulated by hypergravity encoded hydroxymethylglutaryl-CoA reductase (HMGR), which catalyzes a reaction producing mevalonic acid, a key precursor of hormones such as gibberellic acid and abscisic acid. The expression of HMGR gene increased within several hours after hypergravity treatment. Also, compactin, an inhibitor of HMGR activity, prevented hypergravity-induced growth suppression, suggesting that HMGR is involved in suppression of Arabidopsis hypocotyl growth by hypergravity. In addition, hypergravity increased the expression levels of CCR1 and ERD15, which were shown to take part in the signaling pathway of environmental stimuli such as temperature and water. These cellular and molecular changes appear to be involved in a series of events leading to growth suppression of stem organs under hypergravity conditions.

Hypoxia triggers angiogenesis by increasing expression of LOX genes in 3-D culture of ASCs and ECs.

PubMed

Xie, Qiang; Xie, Jiamin; Tian, Taoran; Ma, Quanquan; Zhang, Qi; Zhu, Bofeng; Cai, Xiaoxiao

2017-03-01

This study aimed to investigate the expression changes of LOX (lysyl oxidase) family genes, VEGFA, and VEGFB under hypoxic conditions in a co-culture system of ASCs (adipose-derived stromal cells) and ECs (endothelial cells). ASCs and ECs were co-cultured under hypoxic and normal oxygen conditions in a 1:1 ratio, and the formation of vessel-like was detected at 7 days. The transwell co-culture system was used and cell lysates were collected at 7 days after co-culture in hypoxic and normal oxygen condition. Semi-quantitative PCR was performed to quantify the mRNA expression of VEGFA, VEGFB, and the LOX genes (LOX, LOXL-1, LOXL-2, LOXL-3, and LOXL-4). Expression changes were determined by densitomery. Enhanced angiogenesis was detected in the co-culture of ASCs and ECs under hypoxic conditions. Among the genes screened, VEGFA, VEGFB, LOXL-1, and LOXL-3 in ECs, both mono-cultured and co-cultured, were significantly enhanced after culturing under hypoxic conditions. In ASCs, VEGFB, LOXL-1, and LOXL-3 were upregulated. Contact co-culture between ASCs and ECs promotes angiogenesis under hypoxia. LOXL-1 and LOXL-3 expressions were increased in both ASCs and ECs and might play important roles in the enhanced angiogenesis promoted by hypoxia. Copyright © 2017 Elsevier Inc. All rights reserved.

Scleral gene expression during recovery from myopia compared with expression during myopia development in tree shrew.

PubMed

Guo, Lin; Frost, Michael R; Siegwart, John T; Norton, Thomas T

2014-01-01

During postnatal refractive development, the sclera receives retinally generated signals that regulate its biochemical properties. Hyperopic refractive error causes the retina to produce "GO" signals that, through the direct emmetropization pathway, cause scleral remodeling that increases the axial elongation rate of the eye, reducing the hyperopia. Myopia causes the retina to generate "STOP" signals that produce scleral remodeling, slowing the axial elongation rate and reducing the myopia. Our aim was to compare the pattern of gene expression produced in the sclera by the STOP signals with the GO gene expression signature we described previously. The GO gene expression signature was produced by monocular -5 diopter (D) lens wear for 2 days (ML-2) or 4 days (ML-4); an additional "STAY" condition was examined after eyes had fully compensated for a -5 D lens after 11 days of lens wear (ML-11). After 11 days of -5 D lens wear had produced full refractive compensation, gene expression in the STOP condition was examined during recovery (without the lens) for 2 days (REC-2) or 4 days (REC-4). The untreated contralateral eyes served as a control in all groups. Two age-matched normal groups provided a comparison with the treated groups. Quantitative real-time PCR was used to measure mRNA levels for 55 candidate genes. The STAY group compensated fully for the lens (treated eye versus control eye, -5.1±0.2 D). Wearing the lens, the hyperopic signal for elongation had dissipated (-0.3±0.3 D). In the STOP groups, the refraction in the recovering eyes became less myopic relative to the control eyes (REC-2, +1.3±0.3 D; REC-4, +2.6±0.4 D). In the STAY group, three genes showed significant downregulation. However, many genes that were significantly altered in GO showed smaller, nonsignificant, expression differences in the same direction in STAY, suggesting the gene expression signature in STAY is a greatly weakened form of the GO signature. In the STOP groups, a different gene expression pattern was observed, characterized by mostly upregulation with larger fold differences after 4 days than after 2 days of recovery. Eleven of the 55 genes examined showed significant bidirectional GO/STOP regulation in the ML-2 and REC-2 groups, and 13 genes showed bidirectional regulation in the ML-4 and REC-4 groups. Eight of these genes (NPR3, CAPNS1, NGEF, TGFB1, CTGF, NOV, TIMP1, and HS6ST1) were bidirectionally regulated at both time points in the GO and STOP conditions. An additional 15 genes showed significant regulation in either GO or STOP conditions but not in both. Many genes are involved in scleral remodeling and the control of axial length. The STOP (recovery) gene expression signature in the sclera involves some of the same genes, bidirectionally regulated, as the GO signature. However, other genes, regulated in GO, are not differentially regulated in STOP, and others show differential regulation only in STOP.

Stress-Related Gene Expression Reflects Morphophysiological Responses to Water Deficit.

PubMed

Rymaszewski, Wojciech; Vile, Denis; Bediee, Alexis; Dauzat, Myriam; Luchaire, Nathalie; Kamrowska, Dominika; Granier, Christine; Hennig, Jacek

2017-07-01

Acclimation to water deficit (WD) enables plants to maintain growth under unfavorable environmental conditions, although the mechanisms are not completely understood. In this study, the natural variation of long-term acclimation to moderate and severe soil WD was investigated in 18 Arabidopsis ( Arabidopsis thaliana ) accessions using PHENOPSIS, an automated phenotyping platform. Soil water content was adjusted at an early stage of plant development and maintained at a constant level until reproductive age was achieved. The accessions were selected based on the expression levels of ANNEXIN1 , a drought-related marker. Severe WD conditions had a greater effect on most of the measured morphophysiological traits than moderate WD conditions. Multivariate analyses indicated that trait responses associated with plant size and water management drove most of the variation. Accessions with similar responses at these two levels were grouped in clusters that displayed different response strategies to WD The expression levels of selected stress-response genes revealed large natural variation under WD conditions. Responses of morphophysiological traits, such as projected rosette area, transpiration rate, and rosette water content, were correlated with changes in the expression of stress-related genes, such as NINE-CIS-EPOXYCAROTENOID DIOXYGENASE3 and N-MYC DOWNREGULATED-LIKE1 ( NDL1 ), in response to WD Interestingly, the morphophysiological acclimation response to WD also was reflected in the gene expression levels (most notably those of NDL1 , CHALCONE SYNTHASE , and MYB DOMAIN PROTEIN44 ) in plants cultivated under well-watered conditions. Our results may lead to the development of biomarkers and predictors of plant morphophysiological responses based on gene expression patterns. © 2017 American Society of Plant Biologists. All Rights Reserved.

Erwinia amylovora Expresses Fast and Simultaneously hrp/dsp Virulence Genes during Flower Infection on Apple Trees

PubMed Central

Pester, Doris; Milčevičová, Renáta; Schaffer, Johann; Wilhelm, Eva; Blümel, Sylvia

2012-01-01

Background Pathogen entry through host blossoms is the predominant infection pathway of the Gram-negative bacterium Erwinia amylovora leading to manifestation of the disease fire blight. Like in other economically important plant pathogens, E. amylovora pathogenicity depends on a type III secretion system encoded by hrp genes. However, timing and transcriptional order of hrp gene expression during flower infections are unknown. Methodology/Principal Findings Using quantitative real-time PCR analyses, we addressed the questions of how fast, strong and uniform key hrp virulence genes and the effector dspA/E are expressed when bacteria enter flowers provided with the full defense mechanism of the apple plant. In non-invasive bacterial inoculations of apple flowers still attached to the tree, E. amylovora activated expression of key type III secretion genes in a narrow time window, mounting in a single expression peak of all investigated hrp/dspA/E genes around 24–48 h post inoculation (hpi). This single expression peak coincided with a single depression in the plant PR-1 expression at 24 hpi indicating transient manipulation of the salicylic acid pathway as one target of E. amylovora type III effectors. Expression of hrp/dspA/E genes was highly correlated to expression of the regulator hrpL and relative transcript abundances followed the ratio: hrpA>hrpN>hrpL>dspA/E. Acidic conditions (pH 4) in flower infections led to reduced virulence/effector gene expression without the typical expression peak observed under natural conditions (pH 7). Conclusion/Significance The simultaneous expression of hrpL, hrpA, hrpN, and the effector dspA/E during early floral infection indicates that speed and immediate effector transmission is important for successful plant invasion. When this delicate balance is disturbed, e.g., by acidic pH during infection, virulence gene expression is reduced, thus partly explaining the efficacy of acidification in fire blight control on a molecular level. PMID:22412891

Division rate, cell size and proteome allocation: impact on gene expression noise and implications for the dynamics of genetic circuits

PubMed Central

2018-01-01

The cell division rate, size and gene expression programmes change in response to external conditions. These global changes impact on average concentrations of biomolecule and their variability or noise. Gene expression is inherently stochastic, and noise levels of individual proteins depend on synthesis and degradation rates as well as on cell-cycle dynamics. We have modelled stochastic gene expression inside growing and dividing cells to study the effect of division rates on noise in mRNA and protein expression. We use assumptions and parameters relevant to Escherichia coli, for which abundant quantitative data are available. We find that coupling of transcription, but not translation rates to the rate of cell division can result in protein concentration and noise homeostasis across conditions. Interestingly, we find that the increased cell size at fast division rates, observed in E. coli and other unicellular organisms, buffers noise levels even for proteins with decreased expression at faster growth. We then investigate the functional importance of these regulations using gene regulatory networks that exhibit bi-stability and oscillations. We find that network topology affects robustness to changes in division rate in complex and unexpected ways. In particular, a simple model of persistence, based on global physiological feedback, predicts increased proportion of persister cells at slow division rates. Altogether, our study reveals how cell size regulation in response to cell division rate could help controlling gene expression noise. It also highlights that understanding circuits' robustness across growth conditions is key for the effective design of synthetic biological systems. PMID:29657814

The Global Error Assessment (GEA) model for the selection of differentially expressed genes in microarray data.

PubMed

Mansourian, Robert; Mutch, David M; Antille, Nicolas; Aubert, Jerome; Fogel, Paul; Le Goff, Jean-Marc; Moulin, Julie; Petrov, Anton; Rytz, Andreas; Voegel, Johannes J; Roberts, Matthew-Alan

2004-11-01

Microarray technology has become a powerful research tool in many fields of study; however, the cost of microarrays often results in the use of a low number of replicates (k). Under circumstances where k is low, it becomes difficult to perform standard statistical tests to extract the most biologically significant experimental results. Other more advanced statistical tests have been developed; however, their use and interpretation often remain difficult to implement in routine biological research. The present work outlines a method that achieves sufficient statistical power for selecting differentially expressed genes under conditions of low k, while remaining as an intuitive and computationally efficient procedure. The present study describes a Global Error Assessment (GEA) methodology to select differentially expressed genes in microarray datasets, and was developed using an in vitro experiment that compared control and interferon-gamma treated skin cells. In this experiment, up to nine replicates were used to confidently estimate error, thereby enabling methods of different statistical power to be compared. Gene expression results of a similar absolute expression are binned, so as to enable a highly accurate local estimate of the mean squared error within conditions. The model then relates variability of gene expression in each bin to absolute expression levels and uses this in a test derived from the classical ANOVA. The GEA selection method is compared with both the classical and permutational ANOVA tests, and demonstrates an increased stability, robustness and confidence in gene selection. A subset of the selected genes were validated by real-time reverse transcription-polymerase chain reaction (RT-PCR). All these results suggest that GEA methodology is (i) suitable for selection of differentially expressed genes in microarray data, (ii) intuitive and computationally efficient and (iii) especially advantageous under conditions of low k. The GEA code for R software is freely available upon request to authors.

Predicting gene regulatory networks of soybean nodulation from RNA-Seq transcriptome data.

PubMed

Zhu, Mingzhu; Dahmen, Jeremy L; Stacey, Gary; Cheng, Jianlin

2013-09-22

High-throughput RNA sequencing (RNA-Seq) is a revolutionary technique to study the transcriptome of a cell under various conditions at a systems level. Despite the wide application of RNA-Seq techniques to generate experimental data in the last few years, few computational methods are available to analyze this huge amount of transcription data. The computational methods for constructing gene regulatory networks from RNA-Seq expression data of hundreds or even thousands of genes are particularly lacking and urgently needed. We developed an automated bioinformatics method to predict gene regulatory networks from the quantitative expression values of differentially expressed genes based on RNA-Seq transcriptome data of a cell in different stages and conditions, integrating transcriptional, genomic and gene function data. We applied the method to the RNA-Seq transcriptome data generated for soybean root hair cells in three different development stages of nodulation after rhizobium infection. The method predicted a soybean nodulation-related gene regulatory network consisting of 10 regulatory modules common for all three stages, and 24, 49 and 70 modules separately for the first, second and third stage, each containing both a group of co-expressed genes and several transcription factors collaboratively controlling their expression under different conditions. 8 of 10 common regulatory modules were validated by at least two kinds of validations, such as independent DNA binding motif analysis, gene function enrichment test, and previous experimental data in the literature. We developed a computational method to reliably reconstruct gene regulatory networks from RNA-Seq transcriptome data. The method can generate valuable hypotheses for interpreting biological data and designing biological experiments such as ChIP-Seq, RNA interference, and yeast two hybrid experiments.

Genome-wide profiling of diel and circadian gene expression in the malaria vector Anopheles gambiae.

PubMed

Rund, Samuel S C; Hou, Tim Y; Ward, Sarah M; Collins, Frank H; Duffield, Giles E

2011-08-09

Anopheles gambiae, the primary African vector of malaria parasites, exhibits numerous rhythmic behaviors including flight activity, swarming, mating, host seeking, egg laying, and sugar feeding. However, little work has been performed to elucidate the molecular basis for these daily rhythms. To study how gene expression is regulated globally by diel and circadian mechanisms, we have undertaken a DNA microarray analysis of An. gambiae under light/dark cycle (LD) and constant dark (DD) conditions. Adult mated, non-blood-fed female mosquitoes were collected every 4 h for 48 h, and samples were processed with DNA microarrays. Using a cosine wave-fitting algorithm, we identified 1,293 and 600 rhythmic genes with a period length of 20-28 h in the head and body, respectively, under LD conditions, representing 9.7 and 4.5% of the An. gambiae gene set. A majority of these genes was specific to heads or bodies. Examination of mosquitoes under DD conditions revealed that rhythmic programming of the transcriptome is dependent on an interaction between the endogenous clock and extrinsic regulation by the LD cycle. A subset of genes, including the canonical clock components, was expressed rhythmically under both environmental conditions. A majority of genes had peak expression clustered around the day/night transitions, anticipating dawn and dusk. Genes cover diverse biological processes such as transcription/translation, metabolism, detoxification, olfaction, vision, cuticle regulation, and immunity, and include rate-limiting steps in the pathways. This study highlights the fundamental roles that both the circadian clock and light play in the physiology of this important insect vector and suggests targets for intervention.

Validation of reference genes for quantitative expression analysis by real-time RT-PCR in Saccharomyces cerevisiae

PubMed Central

Teste, Marie-Ange; Duquenne, Manon; François, Jean M; Parrou, Jean-Luc

2009-01-01

Background Real-time RT-PCR is the recommended method for quantitative gene expression analysis. A compulsory step is the selection of good reference genes for normalization. A few genes often referred to as HouseKeeping Genes (HSK), such as ACT1, RDN18 or PDA1 are among the most commonly used, as their expression is assumed to remain unchanged over a wide range of conditions. Since this assumption is very unlikely, a geometric averaging of multiple, carefully selected internal control genes is now strongly recommended for normalization to avoid this problem of expression variation of single reference genes. The aim of this work was to search for a set of reference genes for reliable gene expression analysis in Saccharomyces cerevisiae. Results From public microarray datasets, we selected potential reference genes whose expression remained apparently invariable during long-term growth on glucose. Using the algorithm geNorm, ALG9, TAF10, TFC1 and UBC6 turned out to be genes whose expression remained stable, independent of the growth conditions and the strain backgrounds tested in this study. We then showed that the geometric averaging of any subset of three genes among the six most stable genes resulted in very similar normalized data, which contrasted with inconsistent results among various biological samples when the normalization was performed with ACT1. Normalization with multiple selected genes was therefore applied to transcriptional analysis of genes involved in glycogen metabolism. We determined an induction ratio of 100-fold for GPH1 and 20-fold for GSY2 between the exponential phase and the diauxic shift on glucose. There was no induction of these two genes at this transition phase on galactose, although in both cases, the kinetics of glycogen accumulation was similar. In contrast, SGA1 expression was independent of the carbon source and increased by 3-fold in stationary phase. Conclusion In this work, we provided a set of genes that are suitable reference genes for quantitative gene expression analysis by real-time RT-PCR in yeast biological samples covering a large panel of physiological states. In contrast, we invalidated and discourage the use of ACT1 as well as other commonly used reference genes (PDA1, TDH3, RDN18, etc) as internal controls for quantitative gene expression analysis in yeast. PMID:19874630

Validation of reference genes for quantitative expression analysis by real-time RT-PCR in Saccharomyces cerevisiae.

PubMed

Teste, Marie-Ange; Duquenne, Manon; François, Jean M; Parrou, Jean-Luc

2009-10-30

Real-time RT-PCR is the recommended method for quantitative gene expression analysis. A compulsory step is the selection of good reference genes for normalization. A few genes often referred to as HouseKeeping Genes (HSK), such as ACT1, RDN18 or PDA1 are among the most commonly used, as their expression is assumed to remain unchanged over a wide range of conditions. Since this assumption is very unlikely, a geometric averaging of multiple, carefully selected internal control genes is now strongly recommended for normalization to avoid this problem of expression variation of single reference genes. The aim of this work was to search for a set of reference genes for reliable gene expression analysis in Saccharomyces cerevisiae. From public microarray datasets, we selected potential reference genes whose expression remained apparently invariable during long-term growth on glucose. Using the algorithm geNorm, ALG9, TAF10, TFC1 and UBC6 turned out to be genes whose expression remained stable, independent of the growth conditions and the strain backgrounds tested in this study. We then showed that the geometric averaging of any subset of three genes among the six most stable genes resulted in very similar normalized data, which contrasted with inconsistent results among various biological samples when the normalization was performed with ACT1. Normalization with multiple selected genes was therefore applied to transcriptional analysis of genes involved in glycogen metabolism. We determined an induction ratio of 100-fold for GPH1 and 20-fold for GSY2 between the exponential phase and the diauxic shift on glucose. There was no induction of these two genes at this transition phase on galactose, although in both cases, the kinetics of glycogen accumulation was similar. In contrast, SGA1 expression was independent of the carbon source and increased by 3-fold in stationary phase. In this work, we provided a set of genes that are suitable reference genes for quantitative gene expression analysis by real-time RT-PCR in yeast biological samples covering a large panel of physiological states. In contrast, we invalidated and discourage the use of ACT1 as well as other commonly used reference genes (PDA1, TDH3, RDN18, etc) as internal controls for quantitative gene expression analysis in yeast.

Drought responsive gene expression regulatory divergence between upland and lowland ecotypes of a perennial C4 grass.

PubMed

Lovell, John T; Schwartz, Scott; Lowry, David B; Shakirov, Eugene V; Bonnette, Jason E; Weng, Xiaoyu; Wang, Mei; Johnson, Jenifer; Sreedasyam, Avinash; Plott, Christopher; Jenkins, Jerry; Schmutz, Jeremy; Juenger, Thomas E

2016-04-01

Climatic adaptation is an example of a genotype-by-environment interaction (G×E) of fitness. Selection upon gene expression regulatory variation can contribute to adaptive phenotypic diversity; however, surprisingly few studies have examined how genome-wide patterns of gene expression G×E are manifested in response to environmental stress and other selective agents that cause climatic adaptation. Here, we characterize drought-responsive expression divergence between upland (drought-adapted) and lowland (mesic) ecotypes of the perennial C4 grass,Panicum hallii, in natural field conditions. Overall, we find that cis-regulatory elements contributed to gene expression divergence across 47% of genes, 7.2% of which exhibit drought-responsive G×E. While less well-represented, we observe 1294 genes (7.8%) with transeffects.Trans-by-environment interactions are weaker and much less common than cis G×E, occurring in only 0.7% oft rans-regulated genes. Finally, gene expression heterosis is highly enriched in expression phenotypes with significant G×E. As such, modes of inheritance that drive heterosis, such as dominance or overdominance, may be common among G×E genes. Interestingly, motifs specific to drought-responsive transcription factors are highly enriched in the promoters of genes exhibiting G×E and transregulation, indicating that expression G×E and heterosis may result from the evolution of transcription factors or their binding sites.P. hallii serves as the genomic model for its close relative and emerging biofuel crop, switchgrass (Panicum virgatum). Accordingly, the results here not only aid in the discovery of the genetic mechanisms that underlie local adaptation but also provide a foundation to improve switchgrass yield under water-limited conditions. © 2016 Lovell et al.; Published by Cold Spring Harbor Laboratory Press.

Drought responsive gene expression regulatory divergence between upland and lowland ecotypes of a perennial C4 grass

PubMed Central

Lovell, John T.; Schwartz, Scott; Lowry, David B.; Shakirov, Eugene V.; Bonnette, Jason E.; Weng, Xiaoyu; Wang, Mei; Johnson, Jenifer; Sreedasyam, Avinash; Plott, Christopher; Jenkins, Jerry; Schmutz, Jeremy; Juenger, Thomas E.

2016-01-01

Climatic adaptation is an example of a genotype-by-environment interaction (G×E) of fitness. Selection upon gene expression regulatory variation can contribute to adaptive phenotypic diversity; however, surprisingly few studies have examined how genome-wide patterns of gene expression G×E are manifested in response to environmental stress and other selective agents that cause climatic adaptation. Here, we characterize drought-responsive expression divergence between upland (drought-adapted) and lowland (mesic) ecotypes of the perennial C4 grass, Panicum hallii, in natural field conditions. Overall, we find that cis-regulatory elements contributed to gene expression divergence across 47% of genes, 7.2% of which exhibit drought-responsive G×E. While less well-represented, we observe 1294 genes (7.8%) with trans effects. Trans-by-environment interactions are weaker and much less common than cis G×E, occurring in only 0.7% of trans-regulated genes. Finally, gene expression heterosis is highly enriched in expression phenotypes with significant G×E. As such, modes of inheritance that drive heterosis, such as dominance or overdominance, may be common among G×E genes. Interestingly, motifs specific to drought-responsive transcription factors are highly enriched in the promoters of genes exhibiting G×E and trans regulation, indicating that expression G×E and heterosis may result from the evolution of transcription factors or their binding sites. P. hallii serves as the genomic model for its close relative and emerging biofuel crop, switchgrass (Panicum virgatum). Accordingly, the results here not only aid in the discovery of the genetic mechanisms that underlie local adaptation but also provide a foundation to improve switchgrass yield under water-limited conditions. PMID:26953271

Global Transcriptional Responses to Osmotic, Oxidative, and Imipenem Stress Conditions in Pseudomonas putida

PubMed Central

Bojanovič, Klara; D'Arrigo, Isotta

2017-01-01

ABSTRACT Bacteria cope with and adapt to stress by modulating gene expression in response to specific environmental cues. In this study, the transcriptional response of Pseudomonas putida KT2440 to osmotic, oxidative, and imipenem stress conditions at two time points was investigated via identification of differentially expressed mRNAs and small RNAs (sRNAs). A total of 440 sRNA transcripts were detected, of which 10% correspond to previously annotated sRNAs, 40% to novel intergenic transcripts, and 50% to novel transcripts antisense to annotated genes. Each stress elicits a unique response as far as the extent and dynamics of the transcriptional changes. Nearly 200 protein-encoding genes exhibited significant changes in all stress types, implicating their participation in a general stress response. Almost half of the sRNA transcripts were differentially expressed under at least one condition, suggesting possible functional roles in the cellular response to stress conditions. The data show a larger fraction of differentially expressed sRNAs than of mRNAs with >5-fold expression changes. The work provides detailed insights into the mechanisms through which P. putida responds to different stress conditions and increases understanding of bacterial adaptation in natural and industrial settings. IMPORTANCE This study maps the complete transcriptional response of P. putida KT2440 to osmotic, oxidative, and imipenem stress conditions at short and long exposure times. Over 400 sRNA transcripts, consisting of both intergenic and antisense transcripts, were detected, increasing the number of identified sRNA transcripts in the strain by a factor of 10. Unique responses to each type of stress are documented, including both the extent and dynamics of the gene expression changes. The work adds rich detail to previous knowledge of stress response mechanisms due to the depth of the RNA sequencing data. Almost half of the sRNAs exhibit significant expression changes under at least one condition, suggesting their involvement in adaptation to stress conditions and identifying interesting candidates for further functional characterization. PMID:28130298

Stocking impacts the expression of candidate genes and physiological condition in introgressed brook charr (Salvelinus fontinalis) populations

PubMed Central

Lamaze, Fabien C; Garant, Dany; Bernatchez, Louis

2013-01-01

Translocation of plants and animal populations between environments is one of the major forms of anthropogenic perturbation experienced by pristine populations, and consequently, human-mediated hybridization by stocking practices between wild and exogenous conspecifics is of increasing concern. In this study, we compared the expression of seven candidate genes involved in multifactorial traits and regulatory pathways for growth as a function of level of introgressive hybridization between wild and domestic brook charr to test the null hypothesis of no effect of introgression on wild fish. Our analyses revealed that the expression of two of the genes tested, cytochrome c oxidase VIIa and the growth hormone receptor isoform I, was positively correlated with the level of introgression. We also observed a positive relationship between the extent of introgression and physiological status quantified by the Fulton's condition index. The expression of other genes was influenced by other variables, including year of sampling (reflecting different thermal conditions), sampling method and lake of origin. This is the first demonstration in nature that introgression from stocked populations has an impact on the expression of genes playing a role in important biological functions that may be related with fitness in wild introgressed populations. PMID:23467764

Expression analysis in response to drought stress in soybean: Shedding light on the regulation of metabolic pathway genes.

PubMed

Guimarães-Dias, Fábia; Neves-Borges, Anna Cristina; Viana, Antonio Americo Barbosa; Mesquita, Rosilene Oliveira; Romano, Eduardo; de Fátima Grossi-de-Sá, Maria; Nepomuceno, Alexandre Lima; Loureiro, Marcelo Ehlers; Alves-Ferreira, Márcio

2012-06-01

Metabolomics analysis of wild type Arabidopsis thaliana plants, under control and drought stress conditions revealed several metabolic pathways that are induced under water deficit. The metabolic response to drought stress is also associated with ABA dependent and independent pathways, allowing a better understanding of the molecular mechanisms in this model plant. Through combining an in silico approach and gene expression analysis by quantitative real-time PCR, the present work aims at identifying genes of soybean metabolic pathways potentially associated with water deficit. Digital expression patterns of Arabidopsis genes, which were selected based on the basis of literature reports, were evaluated under drought stress condition by Genevestigator. Genes that showed strong induction under drought stress were selected and used as bait to identify orthologs in the soybean genome. This allowed us to select 354 genes of putative soybean orthologs of 79 Arabidopsis genes belonging to 38 distinct metabolic pathways. The expression pattern of the selected genes was verified in the subtractive libraries available in the GENOSOJA project. Subsequently, 13 genes from different metabolic pathways were selected for validation by qPCR experiments. The expression of six genes was validated in plants undergoing drought stress in both pot-based and hydroponic cultivation systems. The results suggest that the metabolic response to drought stress is conserved in Arabidopsis and soybean plants.

Hypothyroidism alters the expression of Bcl-2 family genes to induce enhanced apoptosis in the developing cerebellum.

PubMed

Singh, R; Upadhyay, G; Kumar, S; Kapoor, A; Kumar, A; Tiwari, M; Godbole, M M

2003-01-01

Thyroid hormone (TH) deficiency results in delayed proliferation and migration of cerebellar granule cells. Although extensive cell loss during the development of the cerebellum under hypothyroid conditions is known, its nature and its mechanism are poorly understood. Bcl-2 family gene expression is known to determine the fate of cells to undergo apoptosis. We evaluated the effect of hypothyroidism on Bcl-2 family gene expression in the developing rat cerebellum. Electrophoresis and Western blotting were used to analyze DNA fragmentation and expression of DNA fragmentation factor (DFF-45), Bcl-2, Bcl-xL and Bax genes respectively. In the hypothyroid condition, extensive DNA fragmentation and enhanced cleavage of DFF-45 were seen throughout development (postnatal day 0 to day 24) and adulthood whereas they were absent in the euthyroid state. The anti-apoptotic genes Bcl-2 and Bcl-xL were down-regulated and the pro-apoptotic gene Bax was expressed at higher levels compared with the euthyroid state. These results suggest that normal levels of TH prevent cerebellar apoptosis to a large extent, whereas hypothyroidism not only increases the extent but also the duration of apoptosis by down-regulating the anti-apoptotic genes and maintaining a high level of the pro-apoptotic gene Bax.

Validation of Suitable Reference Genes for Expression Normalization in Echinococcus spp. Larval Stages

PubMed Central

Espínola, Sergio Martin; Ferreira, Henrique Bunselmeyer; Zaha, Arnaldo

2014-01-01

In recent years, a significant amount of sequence data (both genomic and transcriptomic) for Echinococcus spp. has been published, thereby facilitating the analysis of genes expressed during a specific stage or involved in parasite development. To perform a suitable gene expression quantification analysis, the use of validated reference genes is strongly recommended. Thus, the aim of this work was to identify suitable reference genes to allow reliable expression normalization for genes of interest in Echinococcus granulosus sensu stricto (s.s.) (G1) and Echinococcus ortleppi upon induction of the early pre-adult development. Untreated protoscoleces (PS) and pepsin-treated protoscoleces (PSP) from E. granulosus s.s. (G1) and E. ortleppi metacestode were used. The gene expression stability of eleven candidate reference genes (βTUB, NDUFV2, RPL13, TBP, CYP-1, RPII, EF-1α, βACT-1, GAPDH, ETIF4A-III and MAPK3) was assessed using geNorm, Normfinder, and RefFinder. Our qPCR data showed a good correlation with the recently published RNA-seq data. Regarding expression stability, EF-1α and TBP were the most stable genes for both species. Interestingly, βACT-1 (the most commonly used reference gene), and GAPDH and ETIF4A-III (previously identified as housekeeping genes) did not behave stably in our assay conditions. We propose the use of EF-1α as a reference gene for studies involving gene expression analysis in both PS and PSP experimental conditions for E. granulosus s.s. and E. ortleppi. To demonstrate its applicability, EF-1α was used as a normalizer gene in the relative quantification of transcripts from genes coding for antigen B subunits. The same EF-1α reference gene may be used in studies with other Echinococcus sensu lato species. This report validates suitable reference genes for species of class Cestoda, phylum Platyhelminthes, thus providing a foundation for further validation in other epidemiologically important cestode species, such as those from the Taenia genus. PMID:25014071

Validation of suitable reference genes for expression normalization in Echinococcus spp. larval stages.

PubMed

Espínola, Sergio Martin; Ferreira, Henrique Bunselmeyer; Zaha, Arnaldo

2014-01-01

In recent years, a significant amount of sequence data (both genomic and transcriptomic) for Echinococcus spp. has been published, thereby facilitating the analysis of genes expressed during a specific stage or involved in parasite development. To perform a suitable gene expression quantification analysis, the use of validated reference genes is strongly recommended. Thus, the aim of this work was to identify suitable reference genes to allow reliable expression normalization for genes of interest in Echinococcus granulosus sensu stricto (s.s.) (G1) and Echinococcus ortleppi upon induction of the early pre-adult development. Untreated protoscoleces (PS) and pepsin-treated protoscoleces (PSP) from E. granulosus s.s. (G1) and E. ortleppi metacestode were used. The gene expression stability of eleven candidate reference genes (βTUB, NDUFV2, RPL13, TBP, CYP-1, RPII, EF-1α, βACT-1, GAPDH, ETIF4A-III and MAPK3) was assessed using geNorm, Normfinder, and RefFinder. Our qPCR data showed a good correlation with the recently published RNA-seq data. Regarding expression stability, EF-1α and TBP were the most stable genes for both species. Interestingly, βACT-1 (the most commonly used reference gene), and GAPDH and ETIF4A-III (previously identified as housekeeping genes) did not behave stably in our assay conditions. We propose the use of EF-1α as a reference gene for studies involving gene expression analysis in both PS and PSP experimental conditions for E. granulosus s.s. and E. ortleppi. To demonstrate its applicability, EF-1α was used as a normalizer gene in the relative quantification of transcripts from genes coding for antigen B subunits. The same EF-1α reference gene may be used in studies with other Echinococcus sensu lato species. This report validates suitable reference genes for species of class Cestoda, phylum Platyhelminthes, thus providing a foundation for further validation in other epidemiologically important cestode species, such as those from the Taenia genus.

Phosphorus starvation induces post-transcriptional CHS gene silencing in Petunia corolla.

PubMed

Hosokawa, Munetaka; Yamauchi, Takayoshi; Takahama, Masayoshi; Goto, Mariko; Mikano, Sachiko; Yamaguchi, Yuki; Tanaka, Yoshiyuki; Ohno, Sho; Koeda, Sota; Doi, Motoaki; Yazawa, Susumu

2013-05-01

The corolla of Petunia 'Magic Samba' exhibits unstable anthocyanin expression depending on its phosphorus content. Phosphorus deficiency enhanced post-transcriptional gene silencing of chalcone synthase - A in the corolla. Petunia (Petunia hybrida) 'Magic Samba' has unstable red-white bicolored corollas that respond to nutrient deficiency. We grew this cultivar hydroponically using solutions that lacked one or several nutrients to identify the specific nutrient related to anthocyanin expression in corolla. The white area of the corolla widened under phosphorus (P)-deficient conditions. When the P content of the corolla grown under P-deficient conditions dropped to <2,000 ppm, completely white corollas continued to develop in >40 corollas until the plants died. Other elemental deficiencies had no clear effects on anthocyanin suppression in the corolla. After phosphate was resupplied to the P-deficient plants, anthocyanin was restored in the corollas. The expression of chalcone synthase-A (CHS-A) was suppressed in the white area that widened under P-suppressed conditions, whereas the expression of several other genes related to anthocyanin biosynthesis was enhanced more in the white area than in the red area. Reddish leaves and sepals developed under the P-deficient condition, which is a typical P-deficiency symptom. Two genes related to anthocyanin biosynthesis were enhanced in the reddish organs. Small interfering RNA analysis of CHS-A showed that the suppression resulted from post-transcriptional gene silencing (PTGS). Thus, it was hypothesized that the enhancement of anthocyanin biosynthetic gene expression due to P-deficiency triggered PTGS of CHS-A, which resulted in white corolla development.

Microbial forensics: predicting phenotypic characteristics and environmental conditions from large-scale gene expression profiles.

PubMed

Kim, Minseung; Zorraquino, Violeta; Tagkopoulos, Ilias

2015-03-01

A tantalizing question in cellular physiology is whether the cellular state and environmental conditions can be inferred by the expression signature of an organism. To investigate this relationship, we created an extensive normalized gene expression compendium for the bacterium Escherichia coli that was further enriched with meta-information through an iterative learning procedure. We then constructed an ensemble method to predict environmental and cellular state, including strain, growth phase, medium, oxygen level, antibiotic and carbon source presence. Results show that gene expression is an excellent predictor of environmental structure, with multi-class ensemble models achieving balanced accuracy between 70.0% (±3.5%) to 98.3% (±2.3%) for the various characteristics. Interestingly, this performance can be significantly boosted when environmental and strain characteristics are simultaneously considered, as a composite classifier that captures the inter-dependencies of three characteristics (medium, phase and strain) achieved 10.6% (±1.0%) higher performance than any individual models. Contrary to expectations, only 59% of the top informative genes were also identified as differentially expressed under the respective conditions. Functional analysis of the respective genetic signatures implicates a wide spectrum of Gene Ontology terms and KEGG pathways with condition-specific information content, including iron transport, transferases, and enterobactin synthesis. Further experimental phenotypic-to-genotypic mapping that we conducted for knock-out mutants argues for the information content of top-ranked genes. This work demonstrates the degree at which genome-scale transcriptional information can be predictive of latent, heterogeneous and seemingly disparate phenotypic and environmental characteristics, with far-reaching applications.

Sequential Metabolic Phases as a Means to Optimize Cellular Output in a Constant Environment

PubMed Central

Bockmayr, Alexander; Holzhütter, Hermann-Georg

2015-01-01

Temporal changes of gene expression are a well-known regulatory feature of all cells, which is commonly perceived as a strategy to adapt the proteome to varying external conditions. However, temporal (rhythmic and non-rhythmic) changes of gene expression are also observed under virtually constant external conditions. Here we hypothesize that such changes are a means to render the synthesis of the metabolic output more efficient than under conditions of constant gene activities. In order to substantiate this hypothesis, we used a flux-balance model of the cellular metabolism. The total time span spent on the production of a given set of target metabolites was split into a series of shorter time intervals (metabolic phases) during which only selected groups of metabolic genes are active. The related flux distributions were calculated under the constraint that genes can be either active or inactive whereby the amount of protein related to an active gene is only controlled by the number of active genes: the lower the number of active genes the more protein can be allocated to the enzymes carrying non-zero fluxes. This concept of a predominantly protein-limited efficiency of gene expression clearly differs from other concepts resting on the assumption of an optimal gene regulation capable of allocating to all enzymes and transporters just that fraction of protein necessary to prevent rate limitation. Applying this concept to a simplified metabolic network of the central carbon metabolism with glucose or lactate as alternative substrates, we demonstrate that switching between optimally chosen stationary flux modes comprising different sets of active genes allows producing a demanded amount of target metabolites in a significantly shorter time than by a single optimal flux mode at fixed gene activities. Our model-based findings suggest that temporal expression of metabolic genes can be advantageous even under conditions of constant external substrate supply. PMID:25786979

Spatial fluctuations in expression of the heterocyst differentiation regulatory gene hetR in Anabaena filaments.

PubMed

Corrales-Guerrero, Laura; Tal, Asaf; Arbel-Goren, Rinat; Mariscal, Vicente; Flores, Enrique; Herrero, Antonia; Stavans, Joel

2015-04-01

Under nitrogen deprivation, filaments of the cyanobacterium Anabaena undergo a process of development, resulting in a one-dimensional pattern of nitrogen-fixing heterocysts separated by about ten photosynthetic vegetative cells. Many aspects of gene expression before nitrogen deprivation and during the developmental process remain to be elucidated. Furthermore, the coupling of gene expression fluctuations between cells along a multicellular filament is unknown. We studied the statistics of fluctuations of gene expression of HetR, a transcription factor essential for heterocyst differentiation, both under steady-state growth in nitrogen-rich conditions and at different times following nitrogen deprivation, using a chromosomally-encoded translational hetR-gfp fusion. Statistical analysis of fluorescence at the individual cell level in wild-type and mutant filaments demonstrates that expression fluctuations of hetR in nearby cells are coupled, with a characteristic spatial range of circa two to three cells, setting the scale for cellular interactions along a filament. Correlations between cells predominantly arise from intercellular molecular transfer and less from cell division. Fluctuations after nitrogen step-down can build up on those under nitrogen-replete conditions. We found that under nitrogen-rich conditions, basal, steady-state expression of the HetR inhibitor PatS, cell-cell communication influenced by the septal protein SepJ and positive HetR auto-regulation are essential determinants of fluctuations in hetR expression and its distribution along filaments. A comparison between the expression of hetR-gfp under nitrogen-rich and nitrogen-poor conditions highlights the differences between the two HetR inhibitors PatS and HetN, as well as the differences in specificity between the septal proteins SepJ and FraC/FraD. Activation, inhibition and cell-cell communication lie at the heart of developmental processes. Our results show that proteins involved in these basic ingredients combine together in the presence of inevitable stochasticity in gene expression, to control the coupled fluctuations of gene expression that give rise to a one-dimensional developmental pattern in this organism.

Transcriptomic response of the Antarctic pteropod Limacina helicina antarctica to ocean acidification.

PubMed

Johnson, Kevin M; Hofmann, Gretchen E

2017-10-23

Ocean acidification (OA), a change in ocean chemistry due to the absorption of atmospheric CO 2 into surface oceans, challenges biogenic calcification in many marine organisms. Ocean acidification is expected to rapidly progress in polar seas, with regions of the Southern Ocean expected to experience severe OA within decades. Biologically, the consequences of OA challenge calcification processes and impose an energetic cost. In order to better characterize the response of a polar calcifier to conditions of OA, we assessed differential gene expression in the Antarctic pteropod, Limacina helicina antarctica. Experimental levels of pCO 2 were chosen to create both contemporary pH conditions, and to mimic future pH expected in OA scenarios. Significant changes in the transcriptome were observed when juvenile L. h. antarctica were acclimated for 21 days to low-pH (7.71), mid-pH (7.9) or high-pH (8.13) conditions. Differential gene expression analysis of individuals maintained in the low-pH treatment identified down-regulation of genes involved in cytoskeletal structure, lipid transport, and metabolism. High pH exposure led to increased expression and enrichment for genes involved in shell formation, calcium ion binding, and DNA binding. Significant differential gene expression was observed in four major cellular and physiological processes: shell formation, the cellular stress response, metabolism, and neural function. Across these functional groups, exposure to conditions that mimic ocean acidification led to rapid suppression of gene expression. Results of this study demonstrated that the transcriptome of the juvenile pteropod, L. h. antarctica, was dynamic and changed in response to different levels of pCO 2 . In a global change context, exposure of L. h. antarctica to the low pH, high pCO 2 OA conditions resulted in a suppression of transcripts for genes involved in key physiological processes: calcification, metabolism, and the cellular stress response. The transcriptomic response at both acute and longer-term acclimation time frames indicated that contemporary L. h. antarctica may not have the physiological plasticity necessary for adaptation to OA conditions expected in future decades. Lastly, the differential gene expression results further support the role of shelled pteropods such as L. h. antarctica as sentinel organisms for the impacts of ocean acidification.

Phylogeny and expression profiling of CAD and CAD-like genes in hybrid Populus (P. deltoides × P. nigra): evidence from herbivore damage for subfunctionalization and functional divergence

PubMed Central

2010-01-01

Background Cinnamyl Alcohol Dehydrogenase (CAD) proteins function in lignin biosynthesis and play a critical role in wood development and plant defense against stresses. Previous phylogenetic studies did not include genes from seedless plants and did not reflect the deep evolutionary history of this gene family. We reanalyzed the phylogeny of CAD and CAD-like genes using a representative dataset including lycophyte and bryophyte sequences. Many CAD/CAD-like genes do not seem to be associated with wood development under normal growth conditions. To gain insight into the functional evolution of CAD/CAD-like genes, we analyzed their expression in Populus plant tissues in response to feeding damage by gypsy moth larvae (Lymantria dispar L.). Expression of CAD/CAD-like genes in Populus tissues (xylem, leaves, and barks) was analyzed in herbivore-treated and non-treated plants by real time quantitative RT-PCR. Results CAD family genes were distributed in three classes based on sequence conservation. All the three classes are represented by seedless as well as seed plants, including the class of bona fide lignin pathway genes. The expression of some CAD/CAD-like genes that are not associated with xylem development were induced following herbivore damage in leaves, while other genes were induced in only bark or xylem tissues. Five of the CAD/CAD-like genes, however, showed a shift in expression from one tissue to another between non-treated and herbivore-treated plants. Systemic expression of the CAD/CAD-like genes was generally suppressed. Conclusions Our results indicated a correlation between the evolution of the CAD gene family and lignin and that the three classes of genes may have evolved in the ancestor of land plants. Our results also suggest that the CAD/CAD-like genes have evolved a diversity of expression profiles and potentially different functions, but that they are nonetheless co-regulated under stress conditions. PMID:20509918

Gene expression of bovine embryos developing at the air-liquid interface on oviductal epithelial cells (ALI-BOEC).

PubMed

van der Weijden, Vera A; Chen, Shuai; Bauersachs, Stefan; Ulbrich, Susanne E; Schoen, Jennifer

2017-11-25

We recently developed an air-liquid interface long-term culture of differentiated bovine oviductal epithelial cells (ALI-BOEC). This ex vivo oviduct epithelium is capable of supporting embryo development in co-culture up to the blastocyst stage without addition of embryo culture medium. However, blastocyst rates in co-culture were markedly lower than in conventional in vitro embryo production procedures. In the present study, we assessed target gene expression of ALI-BOEC derived embryos to test their similarity to embryos from conventional in vitro embryo culture. We screened previously published data from developing bovine embryos and selected 41 genes which are either differentially expressed during embryo development, or reflect differences between various in vitro culture conditions or in vitro and in vivo embryos. Target gene expression was measured in 8-cell embryos and blastocysts using a 48.48 Dynamic Array™ on a Biomark HD instrument. For comparison with the ALI-BOEC system, we generated embryos by two different standard IVP protocols. The culture conditions lead to differential gene expression in both 8-cell embryos and blastocysts. Across the expression of all target genes the embryos developing on ALI-BOEC did not depart from conventional IVP embryos. These first results prove that gene expression in ALI-BOEC embryos is not largely aberrant. However, there was no clear indication for a more in vivo-like target gene expression of these embryos. This calls for further optimization of the ALI-BOEC system to increase its efficiency both quantitatively and qualitatively.

From Coexpression to Coregulation: An Approach to Inferring Transcriptional Regulation Among Gene Classes from Large-Scale Expression Data

NASA Technical Reports Server (NTRS)

Mjolsness, Eric; Castano, Rebecca; Mann, Tobias; Wold, Barbara

2000-01-01

We provide preliminary evidence that existing algorithms for inferring small-scale gene regulation networks from gene expression data can be adapted to large-scale gene expression data coming from hybridization microarrays. The essential steps are (I) clustering many genes by their expression time-course data into a minimal set of clusters of co-expressed genes, (2) theoretically modeling the various conditions under which the time-courses are measured using a continuous-time analog recurrent neural network for the cluster mean time-courses, (3) fitting such a regulatory model to the cluster mean time courses by simulated annealing with weight decay, and (4) analysing several such fits for commonalities in the circuit parameter sets including the connection matrices. This procedure can be used to assess the adequacy of existing and future gene expression time-course data sets for determining transcriptional regulatory relationships such as coregulation.

Heterogeneous expression pattern of tandem duplicated sHsps genes during fruit ripening in two tomato species

NASA Astrophysics Data System (ADS)

Arce, DP; Krsticevic, FJ; Ezpeleta, J.; Ponce, SD; Pratta, GR; Tapia, E.

2016-04-01

The small heat shock proteins (sHSPs) have been found to play a critical role in physiological stress conditions in protecting proteins from irreversible aggregation. To characterize the gene expression profile of four sHsps with a tandem gene structure arrangement in the domesticated Solanum lycopersicum (Heinz 1706) genome and its wild close relative Solanum pimpinellifolium (LA1589), differential gene expression analysis using RNA-Seq was conducted in three ripening stages in both cultivars fruits. Gene promoter analysis was performed to explain the heterogeneous pattern of gene expression found for these tandem duplicated sHsps. In silico analysis results contribute to refocus wet experiment analysis in tomato sHsp family proteins.

Identification of warm day and cool night conditions induced flowering-related genes in a Phalaenopsis orchid hybrid by suppression subtractive hybridization.

PubMed

Li, D M; Lü, F B; Zhu, G F; Sun, Y B; Xu, Y C; Jiang, M D; Liu, J W; Wang, Z

2014-02-14

The influence of warm day and cool night conditions on induction of spikes in Phalaenopsis orchids has been studied with respect to photosynthetic efficiency, metabolic cycles and physiology. However, molecular events involved in spike emergence induced by warm day and cool night conditions are not clearly understood. We examined gene expression induced by warm day and cool night conditions in the Phalaenopsis hybrid Fortune Saltzman through suppression subtractive hybridization, which allowed identification of flowering-related genes in warm day and cool night conditions in spikes and leaves at vegetative phase grown under warm daily temperatures. In total, 450 presumably regulated expressed sequence tags (ESTs) were identified and classified into functional categories, including metabolism, development, transcription factor, signal transduction, transportation, cell defense, and stress. Furthermore, database comparisons revealed a notable number of Phalaenopsis hybrid Fortune Saltzman ESTs that matched genes with unknown function. The expression profiles of 24 genes (from different functional categories) have been confirmed by quantitative real-time PCR in induced spikes and juvenile apical leaves. The results of the real-time PCR showed that, compared to the vegetative apical leaves, the transcripts of genes encoding flowering locus T, AP1, AP2, KNOX1, knotted1-like homeobox protein, R2R3-like MYB, adenosine kinase 2, S-adenosylmethionine synthetase, dihydroflavonol 4-reductase, and naringenin 3-dioxygenase accumulated significantly higher levels, and genes encoding FCA, retrotransposon protein Ty3 and C3HC4-type RING finger protein accumulated remarkably lower levels in spikes of early developmental stages. These results suggested that the genes of two expression changing trends may play positive and negative roles in the early floral transition of Phalaenopsis orchids. In conclusion, spikes induced by warm day and cool night conditions were complex in Phalaenopsis orchids; nevertheless, several molecular flowering pathway-related genes were found. The acquired data form the basis for a molecular understanding of spike induction by warm day and cool night conditions in Phalaenopsis orchids.

Plasticity of the Chemoreceptor Repertoire in Drosophila melanogaster

PubMed Central

Zhou, Shanshan; Stone, Eric A.; Mackay, Trudy F. C.; Anholt, Robert R. H.

2009-01-01

For most organisms, chemosensation is critical for survival and is mediated by large families of chemoreceptor proteins, whose expression must be tuned appropriately to changes in the chemical environment. We asked whether expression of chemoreceptor genes that are clustered in the genome would be regulated independently; whether expression of certain chemoreceptor genes would be especially sensitive to environmental changes; whether groups of chemoreceptor genes undergo coordinated rexpression; and how plastic the expression of chemoreceptor genes is with regard to sex, development, reproductive state, and social context. To answer these questions we used Drosophila melanogaster, because its chemosensory systems are well characterized and both the genotype and environment can be controlled precisely. Using customized cDNA microarrays, we showed that chemoreceptor genes that are clustered in the genome undergo independent transcriptional regulation at different developmental stages and between sexes. Expression of distinct subgroups of chemoreceptor genes is sensitive to reproductive state and social interactions. Furthermore, exposure of flies only to odor of the opposite sex results in altered transcript abundance of chemoreceptor genes. These genes are distinct from those that show transcriptional plasticity when flies are allowed physical contact with same or opposite sex members. We analyzed covariance in transcript abundance of chemosensory genes across all environmental conditions and found that they segregated into 20 relatively small, biologically relevant modules of highly correlated transcripts. This finely pixilated modular organization of the chemosensory subgenome enables fine tuning of the expression of the chemoreceptor repertoire in response to ecologically relevant environmental and physiological conditions. PMID:19816562

Validation of reference genes for accurate normalization of gene expression for real time-quantitative PCR in strawberry fruits using different cultivars and osmotic stresses.

PubMed

Galli, Vanessa; Borowski, Joyce Moura; Perin, Ellen Cristina; Messias, Rafael da Silva; Labonde, Julia; Pereira, Ivan dos Santos; Silva, Sérgio Delmar Dos Anjos; Rombaldi, Cesar Valmor

2015-01-10

The increasing demand of strawberry (Fragaria×ananassa Duch) fruits is associated mainly with their sensorial characteristics and the content of antioxidant compounds. Nevertheless, the strawberry production has been hampered due to its sensitivity to abiotic stresses. Therefore, to understand the molecular mechanisms highlighting stress response is of great importance to enable genetic engineering approaches aiming to improve strawberry tolerance. However, the study of expression of genes in strawberry requires the use of suitable reference genes. In the present study, seven traditional and novel candidate reference genes were evaluated for transcript normalization in fruits of ten strawberry cultivars and two abiotic stresses, using RefFinder, which integrates the four major currently available software programs: geNorm, NormFinder, BestKeeper and the comparative delta-Ct method. The results indicate that the expression stability is dependent on the experimental conditions. The candidate reference gene DBP (DNA binding protein) was considered the most suitable to normalize expression data in samples of strawberry cultivars and under drought stress condition, and the candidate reference gene HISTH4 (histone H4) was the most stable under osmotic stresses and salt stress. The traditional genes GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and 18S (18S ribosomal RNA) were considered the most unstable genes in all conditions. The expression of phenylalanine ammonia lyase (PAL) and 9-cis epoxycarotenoid dioxygenase (NCED1) genes were used to further confirm the validated candidate reference genes, showing that the use of an inappropriate reference gene may induce erroneous results. This study is the first survey on the stability of reference genes in strawberry cultivars and osmotic stresses and provides guidelines to obtain more accurate RT-qPCR results for future breeding efforts. Copyright © 2014 Elsevier B.V. All rights reserved.

Expression of cholera toxin under non-AKI conditions in Vibrio cholerae El Tor induced by increasing the exposed surface of cultures.

PubMed

Sánchez, Joaquín; Medina, Gerardo; Buhse, Thomas; Holmgren, Jan; Soberón-Chavez, Gloria

2004-03-01

The regulatory systems controlling expression of the ctxAB genes encoding cholera toxin (CT) in the classical and El Tor biotypes of pathogenic Vibrio cholerae have been characterized and found to be almost identical. Notwithstanding this, special in vitro conditions, called AKI conditions, are required for El Tor bacteria to produce CT. The AKI conditions involve biphasic cultures. In phase 1 the organism is grown in a still tube for 4 h. In phase 2 the medium is poured into a flask to continue growth with shaking. Virtually no expression of CT occurs if this protocol is not followed. Here we demonstrated that CT expression takes place in single-phase still cultures if the volume-to-surface-area ratio is decreased, both under air and under an inert atmosphere. The expression of key genes involved in the regulation of CT production was analyzed, and we found that the expression pattern closely resembles the in vivo expression pattern.

Selection and Validation of Reference Genes for qRT-PCR Expression Analysis of Candidate Genes Involved in Olfactory Communication in the Butterfly Bicyclus anynana

PubMed Central

Arun, Alok; Baumlé, Véronique; Amelot, Gaël; Nieberding, Caroline M.

2015-01-01

Real-time quantitative reverse transcription PCR (qRT-PCR) is a technique widely used to quantify the transcriptional expression level of candidate genes. qRT-PCR requires the selection of one or several suitable reference genes, whose expression profiles remain stable across conditions, to normalize the qRT-PCR expression profiles of candidate genes. Although several butterfly species (Lepidoptera) have become important models in molecular evolutionary ecology, so far no study aimed at identifying reference genes for accurate data normalization for any butterfly is available. The African bush brown butterfly Bicyclus anynana has drawn considerable attention owing to its suitability as a model for evolutionary ecology, and we here provide a maiden extensive study to identify suitable reference gene in this species. We monitored the expression profile of twelve reference genes: eEF-1α, FK506, UBQL40, RpS8, RpS18, HSP, GAPDH, VATPase, ACT3, TBP, eIF2 and G6PD. We tested the stability of their expression profiles in three different tissues (wings, brains, antennae), two developmental stages (pupal and adult) and two sexes (male and female), all of which were subjected to two food treatments (food stress and control feeding ad libitum). The expression stability and ranking of twelve reference genes was assessed using two algorithm-based methods, NormFinder and geNorm. Both methods identified RpS8 as the best suitable reference gene for expression data normalization. We also showed that the use of two reference genes is sufficient to effectively normalize the qRT-PCR data under varying tissues and experimental conditions that we used in B. anynana. Finally, we tested the effect of choosing reference genes with different stability on the normalization of the transcript abundance of a candidate gene involved in olfactory communication in B. anynana, the Fatty Acyl Reductase 2, and we confirmed that using an unstable reference gene can drastically alter the expression profile of the target candidate genes. PMID:25793735

Selection and validation of reference genes for qRT-PCR expression analysis of candidate genes involved in olfactory communication in the butterfly Bicyclus anynana.

PubMed

Arun, Alok; Baumlé, Véronique; Amelot, Gaël; Nieberding, Caroline M

2015-01-01

Real-time quantitative reverse transcription PCR (qRT-PCR) is a technique widely used to quantify the transcriptional expression level of candidate genes. qRT-PCR requires the selection of one or several suitable reference genes, whose expression profiles remain stable across conditions, to normalize the qRT-PCR expression profiles of candidate genes. Although several butterfly species (Lepidoptera) have become important models in molecular evolutionary ecology, so far no study aimed at identifying reference genes for accurate data normalization for any butterfly is available. The African bush brown butterfly Bicyclus anynana has drawn considerable attention owing to its suitability as a model for evolutionary ecology, and we here provide a maiden extensive study to identify suitable reference gene in this species. We monitored the expression profile of twelve reference genes: eEF-1α, FK506, UBQL40, RpS8, RpS18, HSP, GAPDH, VATPase, ACT3, TBP, eIF2 and G6PD. We tested the stability of their expression profiles in three different tissues (wings, brains, antennae), two developmental stages (pupal and adult) and two sexes (male and female), all of which were subjected to two food treatments (food stress and control feeding ad libitum). The expression stability and ranking of twelve reference genes was assessed using two algorithm-based methods, NormFinder and geNorm. Both methods identified RpS8 as the best suitable reference gene for expression data normalization. We also showed that the use of two reference genes is sufficient to effectively normalize the qRT-PCR data under varying tissues and experimental conditions that we used in B. anynana. Finally, we tested the effect of choosing reference genes with different stability on the normalization of the transcript abundance of a candidate gene involved in olfactory communication in B. anynana, the Fatty Acyl Reductase 2, and we confirmed that using an unstable reference gene can drastically alter the expression profile of the target candidate genes.

Increased gene expression noise in human cancers is correlated with low p53 and immune activities as well as late stage cancer.

PubMed

Han, Rongfei; Huang, Guanqun; Wang, Yejun; Xu, Yafei; Hu, Yueming; Jiang, Wenqi; Wang, Tianfu; Xiao, Tian; Zheng, Duo

2016-11-01

Gene expression in metazoans is delicately organized. As genetic information transmits from DNA to RNA and protein, expression noise is inevitably generated. Recent studies begin to unveil the mechanisms of gene expression noise control, but the changes of gene expression precision in pathologic conditions like cancers are unknown. Here we analyzed the transcriptomic data of human breast, liver, lung and colon cancers, and found that the expression noise of more than 74.9% genes was increased in cancer tissues as compared to adjacent normal tissues. This suggested that gene expression precision controlling collapsed during cancer development. A set of 269 genes with noise increased more than 2-fold were identified across different cancer types. These genes were involved in cell adhesion, catalytic and metabolic functions, implying the vulnerability of deregulation of these processes in cancers. We also observed a tendency of increased expression noise in patients with low p53 and immune activity in breast, liver and lung caners but not in colon cancers, which indicated the contributions of p53 signaling and host immune surveillance to gene expression noise in cancers. Moreover, more than 53.7% genes had increased noise in patients with late stage than early stage cancers, suggesting that gene expression precision was associated with cancer outcome. Together, these results provided genomic scale explorations of gene expression noise control in human cancers.

Conditioned medium from persistently RSV-infected macrophages alters transcriptional profile and inflammatory response of non-infected macrophages.

PubMed

Rivera-Toledo, Evelyn; Salido-Guadarrama, Iván; Rodríguez-Dorantes, Mauricio; Torres-González, Laura; Santiago-Olivares, Carlos; Gómez, Beatriz

2017-02-15

Cells susceptible to persistent viral infections undergo important changes in their biological functions as a consequence of the expression of viral gene products that are capable of altering the gene expression profile of the host cell. Previously, we reported that persistence of the RSV genome in a mouse macrophage cell line induces important alterations in cell homeostasis, including constitutive expression of IFN-β and other pro-inflammatory cytokines. Here, we postulated that changes in the homeostasis of non-infected macrophages could be induced by soluble factors secreted by persistently RSV- infected macrophages. To test this hypothesis, non-infected mouse macrophages were treated with conditioned medium (CM) collected from cultures of persistently RSV-infected macrophages. Total RNA was extracted and a microarray-based gene expression analysis was performed. Non-infected macrophages, treated under similar conditions with CM obtained from cultures of non-infected macrophages, were used as a control to establish differential gene expression between the two conditions. Results showed that CM from the persistently RSV-infected cultures altered expression of a total of 95 genes in non-infected macrophages, resulting in an antiviral gene-transcription profile along with inhibition of the inflammatory response, since some inflammatory genes were down-regulated, including Nlrp3 and Il-1 β, both related to the inflammasome pathway. However, down-regulation of Nlrp3 and Il-1 β was reversible upon acute RSV infection. Additionally, we observed that the inflammatory response, evaluated by secreted IL-1 β, a final product of the inflammasome activity, was enhanced during acute RSV infection in macrophages treated with CM from persistently RSV-infected cultures, compared to that in macrophages treated with the control CM. This suggests that soluble factors secreted during RSV persistence may induce an exacerbated inflammatory response in non-infected cells. Copyright © 2017 Elsevier B.V. All rights reserved.

Selection of suitable reference genes from bone cells in large gradient high magnetic field based on GeNorm algorithm.

PubMed

Di, Shengmeng; Tian, Zongcheng; Qian, Airong; Gao, Xiang; Yu, Dan; Brandi, Maria Luisa; Shang, Peng

2011-12-01

Studies of animals and humans subjected to spaceflight demonstrate that weightlessness negatively affects the mass and mechanical properties of bone tissue. Bone cells could sense and respond to the gravity unloading, and genes sensitive to gravity change were considered to play a critical role in the mechanotransduction of bone cells. To evaluate the fold-change of gene expression, appropriate reference genes should be identified because there is no housekeeping gene having stable expression in all experimental conditions. Consequently, expression stability of ten candidate housekeeping genes were examined in osteoblast-like MC3T3-E1, osteocyte-like MLO-Y4, and preosteoclast-like FLG29.1 cells under different apparent gravities (μg, 1 g, and 2 g) in the high-intensity gradient magnetic field produced by a superconducting magnet. The results showed that the relative expression of these ten candidate housekeeping genes was different in different bone cells; Moreover, the most suitable reference genes of the same cells in altered gravity conditions were also different from that in strong magnetic field. It demonstrated the importance of selecting suitable reference genes in experimental set-ups. Furthermore, it provides an alternative choice to the traditionally accepted housekeeping genes used so far about studies of gravitational biology and magneto biology.

Transmembrane transporter expression regulated by the glucosylceramide pathway in Cryptococcus neoformans.

PubMed

Singh, Arpita; Rella, Antonella; Schwacke, John; Vacchi-Suzzi, Caterina; Luberto, Chiara; Del Poeta, Maurizio

2015-11-16

The sphingolipid glucosylceramide (GlcCer) and factors involved in the fungal GlcCer pathways were shown earlier to be an integral part of fungal virulence, especially in fungal replication at 37 °C, in neutral/alkaline pH and 5 % CO2 environments (e.g. alveolar spaces). Two mutants, ∆gcs 1 lacking glucosylceramide synthase 1 gene (GCS1) which catalyzes the formation of sphingolipid GlcCer from the C9-methyl ceramide and ∆smt1 lacking sphingolipid C9 methyltransferase gene (SMT1), which adds a methyl group to position nine of the sphingosine backbone of ceramide, of this pathway were attenuated in virulence and have a growth defect at the above-mentioned conditions. These mutants with either no or structurally modified GlcCer located on the cell-membrane have reduced membrane rigidity, which may have altered not only the physical location of membrane proteins but also their expression, as the pathogen's mode of adaptation to changing need. Importantly, pathogens are known to adapt themselves to the changing host environments by altering their patterns of gene expression. By transcriptional analysis of gene expression, we identified six genes whose expression was changed from their wild-type counterpart grown in the same conditions, i.e. they became either down regulated or up regulated in these two mutants. The microarray data was validated by real-time PCR, which confirmed their fold change in gene expression. All the six genes we identified, viz siderochrome-iron transporter (CNAG_02083), monosaccharide transporter (CNAG_05340), glucose transporter (CNAG_03772), membrane protein (CNAG_03912), membrane transport protein (CNAG_00539), and sugar transporter (CNAG_06963), are membrane-localized and have significantly altered gene expression levels. Therefore, we hypothesize that these genes function either independently or in tandem with a structurally modified cell wall/plasma membrane resulting from the modifications of the GlcCer pathway and thus possibly disrupt transmembrane signaling complex, which in turn contributes to cryptococcal osmotic, pH, ion homeostasis and its pathobiology. Six genes identified from gene expression microarrays by gene set enrichment analysis and validated by RT-PCR, are membrane located and associated with the growth defect at neutral-alkaline pH due to the absence and or presence of a structurally modified GlcCer. They may be involved in the transmembrane signaling network in Cryptococcus neoformans, and therefore the pathobiology of the fungus in these conditions.

ABA signaling in stress-response and seed development.

PubMed

Nakashima, Kazuo; Yamaguchi-Shinozaki, Kazuko

2013-07-01

KEY MESSAGE : We review the recent progress on ABA signaling, especially ABA signaling for ABA-dependent gene expression, including the AREB/ABF regulon, SnRK2 protein kinase, 2C-type protein phosphatases and ABA receptors. Drought negatively impacts plant growth and the productivity of crops. Drought causes osmotic stress to organisms, and the osmotic stress causes dehydration in plant cells. Abscisic acid (ABA) is produced under osmotic stress conditions, and it plays an important role in the stress response and tolerance of plants. ABA regulates many genes under osmotic stress conditions. It also regulates gene expression during seed development and germination. The ABA-responsive element (ABRE) is the major cis-element for ABA-responsive gene expression. ABRE-binding protein (AREB)/ABRE-binding factor (ABF) transcription factors (TFs) regulate ABRE-dependent gene expression. Other TFs are also involved in ABA-responsive gene expression. SNF1-related protein kinases 2 are the key regulators of ABA signaling including the AREB/ABF regulon. Recently, ABA receptors and group A 2C-type protein phosphatases were shown to govern the ABA signaling pathway. Moreover, recent studies have suggested that there are interactions between the major ABA signaling pathway and other signaling factors in stress-response and seed development. The control of the expression of ABA signaling factors may improve tolerance to environmental stresses.

Mindfulness-Based Stress Reduction training reduces loneliness and pro-inflammatory gene expression in older adults: a small randomized controlled trial.

PubMed

Creswell, J David; Irwin, Michael R; Burklund, Lisa J; Lieberman, Matthew D; Arevalo, Jesusa M G; Ma, Jeffrey; Breen, Elizabeth Crabb; Cole, Steven W

2012-10-01

Lonely older adults have increased expression of pro-inflammatory genes as well as increased risk for morbidity and mortality. Previous behavioral treatments have attempted to reduce loneliness and its concomitant health risks, but have had limited success. The present study tested whether the 8-week Mindfulness-Based Stress Reduction (MBSR) program (compared to a Wait-List control group) reduces loneliness and downregulates loneliness-related pro-inflammatory gene expression in older adults (N = 40). Consistent with study predictions, mixed effect linear models indicated that the MBSR program reduced loneliness, compared to small increases in loneliness in the control group (treatment condition × time interaction: F(1,35) = 7.86, p = .008). Moreover, at baseline, there was an association between reported loneliness and upregulated pro-inflammatory NF-κB-related gene expression in circulating leukocytes, and MBSR downregulated this NF-κB-associated gene expression profile at post-treatment. Finally, there was a trend for MBSR to reduce C Reactive Protein (treatment condition × time interaction: (F(1,33) = 3.39, p = .075). This work provides an initial indication that MBSR may be a novel treatment approach for reducing loneliness and related pro-inflammatory gene expression in older adults. Copyright © 2012 Elsevier Inc. All rights reserved.

Mindfulness-Based Stress Reduction Training Reduces Loneliness and Pro-Inflammatory Gene Expression in Older Adults: A Small Randomized Controlled Trial

PubMed Central

Creswell, J. David; Irwin, Michael R.; Burklund, Lisa J.; Lieberman, Matthew D.; Arevalo, Jesusa M. G.; Ma, Jeffrey; Breen, Elizabeth Crabb; Cole, Steven W.

2013-01-01

Lonely older adults have increased expression of pro-inflammatory genes as well as increased risk for morbidity and mortality. Previous behavioral treatments have attempted to reduce loneliness and its concomitant health risks, but have had limited success. The present study tested whether the 8-week Mindfulness-Based Stress Reduction (MBSR) program (compared to a Wait-List control group) reduces loneliness and downregulates loneliness-related pro-inflammatory gene expression in older adults (N=40). Consistent with study predictions, mixed effect linear models indicated that the MBSR program reduced loneliness, compared to small increases in loneliness in the control group (treatment condition × time interaction: F(1,35)=7.86, p=.008). Moreover, at baseline, there was an association between reported loneliness and upregulated pro-inflammatory NF-κB-related gene expression in circulating leukocytes, and MBSR downregulated this NF-κB-associated gene expression profile at post-treatment. Finally, there was a trend for MBSR to reduce C Reactive Protein (treatment condition × time interaction: (F(1,33)=3.39, p=.075). This work provides an initial indication that MBSR may be a novel treatment approach for reducing loneliness and related pro-inflammatory gene expression in older adults. PMID:22820409

Interaction of rearing environment and reproductive tactic on gene expression profiles in Atlantic salmon

USGS Publications Warehouse

Aubin-Horth, N.; Letcher, B.H.; Hofmann, H.A.

2005-01-01

Organisms that share the same genotype can develop into divergent phenotypes, depending on environmental conditions. In Atlantic salmon, young males of the same age can be found either as sneakers or immature males that are future anadromous fish. Just as the organism-level phenotype varies between divergent male developmental trajectories, brain gene expression is expected to vary as well. We hypothesized that rearing environment can also have an important effect on gene expression in the brain and possibly interact with the reproductive tactic adopted. We tested this hypothesis by comparing brain gene expression profiles of the two male tactics in fish from the same population that were reared in either a natural stream or under laboratory conditions. We found that expression of certain genes was affected by rearing environment only, while others varied between male reproductive tactics independent of rearing environment. Finally, more than half of all genes that showed variable expression varied between the two male tactics only in one environment. Thus, in these fish, very different molecular pathways can give rise to similar macro-phenotypes depending on rearing environment. This result gives important insights into the molecular underpinnings of developmental plasticity in relationship to the environment. ?? 2005 The American Genetic Association.

Validation of endogenous reference genes for qRT-PCR analysis of human visceral adipose samples

PubMed Central

2010-01-01

Background Given the epidemic proportions of obesity worldwide and the concurrent prevalence of metabolic syndrome, there is an urgent need for better understanding the underlying mechanisms of metabolic syndrome, in particular, the gene expression differences which may participate in obesity, insulin resistance and the associated series of chronic liver conditions. Real-time PCR (qRT-PCR) is the standard method for studying changes in relative gene expression in different tissues and experimental conditions. However, variations in amount of starting material, enzymatic efficiency and presence of inhibitors can lead to quantification errors. Hence the need for accurate data normalization is vital. Among several known strategies for data normalization, the use of reference genes as an internal control is the most common approach. Recent studies have shown that both obesity and presence of insulin resistance influence an expression of commonly used reference genes in omental fat. In this study we validated candidate reference genes suitable for qRT-PCR profiling experiments using visceral adipose samples from obese and lean individuals. Results Cross-validation of expression stability of eight selected reference genes using three popular algorithms, GeNorm, NormFinder and BestKeeper found ACTB and RPII as most stable reference genes. Conclusions We recommend ACTB and RPII as stable reference genes most suitable for gene expression studies of human visceral adipose tissue. The use of these genes as a reference pair may further enhance the robustness of qRT-PCR in this model system. PMID:20492695

Validation of endogenous reference genes for qRT-PCR analysis of human visceral adipose samples.

PubMed

Mehta, Rohini; Birerdinc, Aybike; Hossain, Noreen; Afendy, Arian; Chandhoke, Vikas; Younossi, Zobair; Baranova, Ancha

2010-05-21

Given the epidemic proportions of obesity worldwide and the concurrent prevalence of metabolic syndrome, there is an urgent need for better understanding the underlying mechanisms of metabolic syndrome, in particular, the gene expression differences which may participate in obesity, insulin resistance and the associated series of chronic liver conditions. Real-time PCR (qRT-PCR) is the standard method for studying changes in relative gene expression in different tissues and experimental conditions. However, variations in amount of starting material, enzymatic efficiency and presence of inhibitors can lead to quantification errors. Hence the need for accurate data normalization is vital. Among several known strategies for data normalization, the use of reference genes as an internal control is the most common approach. Recent studies have shown that both obesity and presence of insulin resistance influence an expression of commonly used reference genes in omental fat. In this study we validated candidate reference genes suitable for qRT-PCR profiling experiments using visceral adipose samples from obese and lean individuals. Cross-validation of expression stability of eight selected reference genes using three popular algorithms, GeNorm, NormFinder and BestKeeper found ACTB and RPII as most stable reference genes. We recommend ACTB and RPII as stable reference genes most suitable for gene expression studies of human visceral adipose tissue. The use of these genes as a reference pair may further enhance the robustness of qRT-PCR in this model system.

Cloning and expression analysis of carboxyltransferase of acetyl-coA carboxylase from Jatropha curcas.

PubMed

Xie, Wu-Wei; Gao, Shun; Wang, Sheng-Hua; Zhu, Jin-Qiu; Xu, Ying; Tang, Lin; Chen, Fang

2010-01-01

A full-length cDNA of the carboxyltransferase (accA) gene of acetyl-coenzym A (acetyl-CoA) carboxylase from Jatropha curcas was cloned and sequenced. The gene with an open reading frame (ORF) of 1149 bp encodes a polypeptide of 383 amino acids, with a molecular mass of 41.9 kDa. Utilizing fluorogenic real-time polymerase chain reaction (RT-PCR), the expression levels of the accA gene in leaves and fruits at early, middle and late stages under pH 7.0/8.0 and light/darkness stress were investigated. The expression levels of the accA gene in leaves at early, middle and late stages increased significantly under pH 8.0 stress compared to pH 7.0. Similarly, the expression levels in fruits showed a significant increase under darkness condition compared to the control. Under light stress, the expression levels in the fruits at early, middle and late stages showed the largest fluctuations compared to those of the control. These findings suggested that the expression levels of the accA gene are closely related to the growth conditions and developmental stages in the leaves and fruits of Jatropha curcas.

Comparative Transcriptomic Analysis Reveals Similarities and Dissimilarities in Saccharomyces cerevisiae Wine Strains Response to Nitrogen Availability

PubMed Central

Barbosa, Catarina; García-Martínez, José; Pérez-Ortín, José E.; Mendes-Ferreira, Ana

2015-01-01

Nitrogen levels in grape-juices are of major importance in winemaking ensuring adequate yeast growth and fermentation performance. Here we used a comparative transcriptome analysis to uncover wine yeasts responses to nitrogen availability during fermentation. Gene expression was assessed in three genetically and phenotypically divergent commercial wine strains (CEG, VL1 and QA23), under low (67 mg/L) and high nitrogen (670 mg/L) regimes, at three time points during fermentation (12h, 24h and 96h). Two-way ANOVA analysis of each fermentation condition led to the identification of genes whose expression was dependent on strain, fermentation stage and on the interaction of both factors. The high fermenter yeast strain QA23 was more clearly distinct from the other two strains, by differential expression of genes involved in flocculation, mitochondrial functions, energy generation and protein folding and stabilization. For all strains, higher transcriptional variability due to fermentation stage was seen in the high nitrogen fermentations. A positive correlation between maximum fermentation rate and the expression of genes involved in stress response was observed. The finding of common genes correlated with both fermentation activity and nitrogen up-take underlies the role of nitrogen on yeast fermentative fitness. The comparative analysis of genes differentially expressed between both fermentation conditions at 12h, where the main difference was the level of nitrogen available, showed the highest variability amongst strains revealing strain-specific responses. Nevertheless, we were able to identify a small set of genes whose expression profiles can quantitatively assess the common response of the yeast strains to varying nitrogen conditions. The use of three contrasting yeast strains in gene expression analysis prompts the identification of more reliable, accurate and reproducible biomarkers that will facilitate the diagnosis of deficiency of this nutrient in the grape-musts and the development of strategies to optimize yeast performance in industrial fermentations. PMID:25884705

An alternative methionine aminopeptidase, MAP-A, is required for nitrogen starvation and high-light acclimation in the cyanobacterium Synechocystis sp. PCC 6803.

PubMed

Drath, Miriam; Baier, Kerstin; Forchhammer, Karl

2009-05-01

Methionine aminopeptidases (MetAPs or MAPs, encoded by map genes) are ubiquitous and pivotal enzymes for protein maturation in all living organisms. Whereas most bacteria harbour only one map gene, many cyanobacterial genomes contain two map paralogues, the genome of Synechocystis sp. PCC 6803 even three. The physiological function of multiple map paralogues remains elusive so far. This communication reports for the first time differential MetAP function in a cyanobacterium. In Synechocystis sp. PCC 6803, the universally conserved mapC gene (sll0555) is predominantly expressed in exponentially growing cells and appears to be a housekeeping gene. By contrast, expression of mapA (slr0918) and mapB (slr0786) genes increases during stress conditions. The mapB paralogue is only transiently expressed, whereas the widely distributed mapA gene appears to be the major MetAP during stress conditions. A mapA-deficient Synechocystis mutant shows a subtle impairment of photosystem II properties even under non-stressed conditions. In particular, the binding site for the quinone Q(B) is affected, indicating specific N-terminal methionine processing requirements of photosystem II components. MAP-A-specific processing becomes essential under certain stress conditions, since the mapA-deficient mutant is severely impaired in surviving conditions of prolonged nitrogen starvation and high light exposure.

YY1 Regulates Melanocyte Development and Function by Cooperating with MITF

PubMed Central

Bell, Robert J. A.; Tran, Thanh-Nga T.; Haq, Rizwan; Liu, Huifei; Love, Kevin T.; Langer, Robert; Anderson, Daniel G.; Larue, Lionel; Fisher, David E.

2012-01-01

Studies of coat color mutants have greatly contributed to the discovery of genes that regulate melanocyte development and function. Here, we generated Yy1 conditional knockout mice in the melanocyte-lineage and observed profound melanocyte deficiency and premature gray hair, similar to the loss of melanocytes in human piebaldism and Waardenburg syndrome. Although YY1 is a ubiquitous transcription factor, YY1 interacts with M-MITF, the Waardenburg Syndrome IIA gene and a master transcriptional regulator of melanocytes. YY1 cooperates with M-MITF in regulating the expression of piebaldism gene KIT and multiple additional pigmentation genes. Moreover, ChIP–seq identified genome-wide YY1 targets in the melanocyte lineage. These studies mechanistically link genes implicated in human conditions of melanocyte deficiency and reveal how a ubiquitous factor (YY1) gains lineage-specific functions by co-regulating gene expression with a lineage-restricted factor (M-MITF)—a general mechanism which may confer tissue-specific gene expression in multiple lineages. PMID:22570637

A novel gene whose expression in Medicago truncatula roots is suppressed in response to colonization by vesicular-arbuscular mycorrhizal (VAM) fungi and to phosphate nutrition.

PubMed

Burleigh, S H; Harrison, M J

1997-05-01

A cDNA clone (Mt4) was isolated as a result of a differential screen to identify genes showing altered expression during the interaction between Medicago truncatula and the vesicular-arbuscular mycorrhizal (VAM) fungus Glomus versiforme. Mt4 represents a M. truncatula mRNA that contains numerous short open reading frames, the two longest of which are predicted to encode polypeptides of 51 amino acids each. One of these open reading frames shares a short region of identity with a phosphate starvation-inducible gene from tomato. Mt4 gene expression is regulated in response to colonization by mycorrhizal fungi: transcripts were detected in non-colonized roots and levels decreased in both M. truncatula and M. sativa (alfalfa) roots after colonization by G. versiforme. Transcript levels also decreased during the incomplete interaction between G. versiforme and a M. sativa mycorrhizal minus (myc-) line, indicating that the down-regulation of this gene occurs early during the interaction between the fungus and its host plant. Phosphate levels in the nutrient media also affected the expression of the Mt4 gene: transcripts were present in the roots of plants grown under phosphate-deficient conditions, but were undetectable in the roots of plants grown under phosphate sufficient conditions. Furthermore, expression was only observed when plants were grown under nitrogen-sufficient conditions. Northern blot analyses indicate that Mt4 transcripts are present primarily in roots and barely detectable in stems or leaves. Thus, Mt4 represents a M. truncatula gene whose expression is regulated in response to both colonization by mycorrhizal fungi and to the phosphate status of the plant.

Gene expression signatures in tree shrew choroid in response to three myopiagenic conditions

PubMed Central

He, Li; Frost, Michael R.; Siegwart, John T.; Norton, Thomas T.

2014-01-01

We examined gene expression in tree shrew choroid in response to three different myopiagenic conditions: minus lens (ML) wear, form deprivation (FD), and continuous darkness (DK). Four groups of tree shrews (n = 7 per group) were used. Starting 24 days after normal eye opening (days of visual experience [DVE]), the ML group wore a monocular −5 D lens for 2 days. The FD group wore a monocular translucent diffuser for 2 days. The DK group experienced continuous darkness binocularly for 11 days, starting at 17 DVE. An age-matched normal group was examined at 26 DVE. Quantitative PCR was used to measure the relative (treated eye vs. control eye) differences in mRNA levels in the choroid for 77 candidate genes. Small myopic changes were observed in the treated eyes (relative to the control eyes) of the ML group (−1.0 ± 0.2 D; mean ± SEM) and FD group (−1.9 ± 0.2 D). A larger myopia developed in the DK group (−4.4 ± 1.0 D) relative to Normal eyes (both groups, mean of right and left eyes). In the ML group, 28 genes showed significant differential mRNA expression; eighteen were down-regulated. A very similar pattern occurred in the FD group; twenty-seven of the same genes were similarly regulated, along with five additional genes. Fewer expression differences in the DK group were significant compared to normal or the control eyes of the ML and FD groups, but the pattern was similar to that of the ML and FD differential expression patterns. These data suggest that, at the level of the choroid, the gene expression signatures produced by “GO” emmetropization signals are highly similar despite the different visual conditions. PMID:25072854

Analysis of Gene Expression in Escherichia coli in Response to Changes of Growth-Limiting Nutrient in Chemostat Cultures

PubMed Central

Hua, Qiang; Yang, Chen; Oshima, Taku; Mori, Hirotada; Shimizu, Kazuyuki

2004-01-01

Studies of steady-state metabolic fluxes in Escherichia coli grown in nutrient-limited chemostat cultures suggest remarkable flux alterations in response to changes of growth-limiting nutrient in the medium (Hua et al., J. Bacteriol. 185:7053-7067, 2003). To elucidate the physiological adaptation of cells to the nutrient condition through the flux change and understand the molecular mechanisms underlying the change in the flux, information on gene expression is of great importance. DNA microarray analysis was performed to investigate the global transcriptional responses of steady-state cells grown in chemostat cultures with limited glucose or ammonia while other environmental conditions and the growth rate were kept constant. In slow-growing cells (specific growth rate of 0.10 h−1), 9.8% of a total of 4,071 genes investigated, especially those involved in amino acid metabolism, central carbon and energy metabolism, transport system and cell envelope, were observed to be differentially expressed between the two nutrient-limited cultures. One important characteristic of E. coli grown under nutrient limitation was its capacity to scavenge carbon or nitrogen from the medium through elevating the expression of the corresponding transport and assimilation genes. The number of differentially expressed genes in faster-growing cells (specific growth rate of 0.55 h−1), however, decreased to below half of that in slow-growing cells, which could be explained by diverse transcriptional responses to the growth rate under different nutrient limitations. Independent of the growth rate, 92 genes were identified as being differentially expressed. Genes tightly related to the culture conditions were highlighted, some of which may be used to characterize nutrient-limited growth. PMID:15066832

Identification of Reference Genes for Normalizing Quantitative Real-Time PCR in Urechis unicinctus

NASA Astrophysics Data System (ADS)

Bai, Yajiao; Zhou, Di; Wei, Maokai; Xie, Yueyang; Gao, Beibei; Qin, Zhenkui; Zhang, Zhifeng

2018-06-01

The reverse transcription quantitative real-time PCR (RT-qPCR) has become one of the most important techniques of studying gene expression. A set of valid reference genes are essential for the accurate normalization of data. In this study, five candidate genes were analyzed with geNorm, NormFinder, BestKeeper and ΔCt methods to identify the genes stably expressed in echiuran Urechis unicinctus, an important commercial marine benthic worm, under abiotic (sulfide stress) and normal (adult tissues, embryos and larvae at different development stages) conditions. The comprehensive results indicated that the expression of TBP was the most stable at sulfide stress and in developmental process, while the expression of EF- 1- α was the most stable at sulfide stress and in various tissues. TBP and EF- 1- α were recommended as a suitable reference gene combination to accurately normalize the expression of target genes at sulfide stress; and EF- 1- α, TBP and TUB were considered as a potential reference gene combination for normalizing the expression of target genes in different tissues. No suitable gene combination was obtained among these five candidate genes for normalizing the expression of target genes for developmental process of U. unicinctus. Our results provided a valuable support for quantifying gene expression using RT-qPCR in U. unicinctus.

Bioinformatics approach for choosing the correct reference genes when studying gene expression in human keratinocytes.

PubMed

Beer, Lucian; Mlitz, Veronika; Gschwandtner, Maria; Berger, Tanja; Narzt, Marie-Sophie; Gruber, Florian; Brunner, Patrick M; Tschachler, Erwin; Mildner, Michael

2015-10-01

Reverse transcription polymerase chain reaction (qRT-PCR) has become a mainstay in many areas of skin research. To enable quantitative analysis, it is necessary to analyse expression of reference genes (RGs) for normalization of target gene expression. The selection of reliable RGs therefore has an important impact on the experimental outcome. In this study, we aimed to identify and validate the best suited RGs for qRT-PCR in human primary keratinocytes (KCs) over a broad range of experimental conditions using the novel bioinformatics tool 'RefGenes', which is based on a manually curated database of published microarray data. Expression of 6 RGs identified by RefGenes software and 12 commonly used RGs were validated by qRT-PCR. We assessed whether these 18 markers fulfilled the requirements for a valid RG by the comprehensive ranking of four bioinformatics tools and the coefficient of variation (CV). In an overall ranking, we found GUSB to be the most stably expressed RG, whereas the expression values of the commonly used RGs, GAPDH and B2M were significantly affected by varying experimental conditions. Our results identify RefGenes as a powerful tool for the identification of valid RGs and suggest GUSB as the most reliable RG for KCs. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Transcriptome-wide identification of reference genes for expression analysis of soybean responses to drought stress along the day

USDA-ARS?s Scientific Manuscript database

The soybean transcriptome displays strong variation along the day in optimal growth conditions and also in response to adverse circumstances, like drought stress. However, no study conducted to date has presented suitable reference genes, with stable expression along the day, for relative gene expre...

Identification and evaluation of reliable reference genes for quantitative real-time PCR analysis in tea plant (Camellia sinensis (L.) O. Kuntze)

USDA-ARS?s Scientific Manuscript database

Quantitative real-time polymerase chain reaction (qRT-PCR) is a commonly used technique for measuring gene expression levels due to its simplicity, specificity, and sensitivity. Reliable reference selection for the accurate quantification of gene expression under various experimental conditions is a...

Manufacturing porcine islets: culture at 22°C has no advantage above culture at 37°C

PubMed Central

Mueller, Kate R; Martins, Kyra V; Murtaugh, Michael P; Schuurman, Henk-Jan; Papas, Klearchos K

2013-01-01

Background The manufacturing process of islets includes a culture step which was originally introduced to ease the logistics of procedures in preparing the graft and transplant recipient. It has been suggested that culture at room temperature has an advantage over culture at 37°C, in part by reducing immunogenicity via preferential elimination of contaminating cells (such as passenger leukocytes) within islets. We investigated this using islets isolated from pancreata of adult pigs. Methods Porcine islets were isolated from three donors and cultured at 37°C for 1 day, and then under three different conditions: 37°C for 6 days (condition A); 22°C for 6 days (condition B); or 22°C for 5 days followed by 37°C for 1 day (condition C). Recovery was assessed by DNA measurement, viability by oxygen consumption rate normalized for DNA (OCR/DNA), and gene expression by RT-PCR for a series of 9 lymphocyte markers, 11 lymphokines and chemokines, and 14 apoptotic and stress markers. Results Post-culture islet recoveries were similar for the three culture conditions. Average OCR/DNA values were 129–159 nmol/min.mgDNA before culture, and 259–291, 204–212, and 207–228 nmol/min•mgDNA, respectively, for culture under conditions A, B, and C, respectively. Irrespective of culture condition, examined gene expression in all three series of lymphocyte markers, lymphokines and chemokines, and apoptotic and stress markers manifested a statistically significant decrease upon culture for 7 days. This decrease was most dramatic for condition A: in particular most of lymphocyte markers showed a >10-fold reduction and also 6 markers in the lymphokine and chemokine series: these reductions are consistent with the elimination of immune cells present within islets during culture. The reduction was less for apoptotic and stress markers. For culture under condition B the reduction in gene expression was less, and culture under condition C resulted in gene expression levels similar to those under condition A: this indicates that 24 hours at 37°C is sufficient to re-equilibrate gene expression levels from those in islets cultured at 22°C to those in islets cultured at 37°C. Results were consistent among the preparations from the three donors. Conclusions Culture of porcine islets at 37°C provides benefits over culture at 22°C with respect to OCR/DNA outcomes and reduced expression of genes encoding lymphocyte markers, lymphokines and chemokines, and markers for apoptosis and stress. PMID:23941232

Material and methods to increase plant growth and yield

DOEpatents

Kirst, Matias

2015-09-15

The present invention relates to materials and methods for modulating growth rates, yield, and/or resistance to drought conditions in plants. In one embodiment, a method of the invention comprises increasing expression of an hc1 gene (or a homolog thereof that provides for substantially the same activity), or increasing expression or activity of the protein encoded by an hc1 gene thereof, in a plant, wherein expression of the hc1 gene or expression or activity of the protein encoded by an hc1 gene results in increased growth rate, yield, and/or drought resistance in the plant.

Materials and methods to increase plant growth and yield

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kirst, Matias

The present invention relates to materials and methods for modulating growth rates, yield, and/or resistance to drought conditions in plants. In one embodiment, a method of the invention comprises increasing expression of an hc1 gene (or a homolog thereof that provides for substantially the same activity), or increasing expression or activity of the protein encoded by an hc1 gene thereof, in a plant, wherein expression of the hc1 gene or expression or activity of the protein encoded by an hc1 gene results in increased growth rate, yield, and/or drought resistance in the plant.

Selection and Evaluation of Potential Reference Genes for Gene Expression Analysis in the Brown Planthopper, Nilaparvata lugens (Hemiptera: Delphacidae) Using Reverse-Transcription Quantitative PCR

PubMed Central

Zhu, Xun; Wan, Hu; Shakeel, Muhammad; Zhan, Sha; Jin, Byung-Rae; Li, Jianhong

2014-01-01

The brown planthopper (BPH), Nilaparvata lugens (Hemiptera, Delphacidae), is one of the most important rice pests. Abundant genetic studies on BPH have been conducted using reverse-transcription quantitative real-time PCR (qRT-PCR). Using qRT-PCR, the expression levels of target genes are calculated on the basis of endogenous controls. These genes need to be appropriately selected by experimentally assessing whether they are stably expressed under different conditions. However, such studies on potential reference genes in N. lugens are lacking. In this paper, we presented a systematic exploration of eight candidate reference genes in N. lugens, namely, actin 1 (ACT), muscle actin (MACT), ribosomal protein S11 (RPS11), ribosomal protein S15e (RPS15), alpha 2-tubulin (TUB), elongation factor 1 delta (EF), 18S ribosomal RNA (18S), and arginine kinase (AK) and used four alternative methods (BestKeeper, geNorm, NormFinder, and the delta Ct method) to evaluate the suitability of these genes as endogenous controls. We examined their expression levels among different experimental factors (developmental stage, body part, geographic population, temperature variation, pesticide exposure, diet change, and starvation) following the MIQE (Minimum Information for publication of Quantitative real time PCR Experiments) guidelines. Based on the results of RefFinder, which integrates four currently available major software programs to compare and rank the tested candidate reference genes, RPS15, RPS11, and TUB were found to be the most suitable reference genes in different developmental stages, body parts, and geographic populations, respectively. RPS15 was the most suitable gene under different temperature and diet conditions, while RPS11 was the most suitable gene under different pesticide exposure and starvation conditions. This work sheds light on establishing a standardized qRT-PCR procedure in N. lugens, and serves as a starting point for screening for reference genes for expression studies of related insects. PMID:24466124

Selection and evaluation of potential reference genes for gene expression analysis in the brown planthopper, Nilaparvata lugens (Hemiptera: Delphacidae) using reverse-transcription quantitative PCR.

PubMed

Yuan, Miao; Lu, Yanhui; Zhu, Xun; Wan, Hu; Shakeel, Muhammad; Zhan, Sha; Jin, Byung-Rae; Li, Jianhong

2014-01-01

The brown planthopper (BPH), Nilaparvata lugens (Hemiptera, Delphacidae), is one of the most important rice pests. Abundant genetic studies on BPH have been conducted using reverse-transcription quantitative real-time PCR (qRT-PCR). Using qRT-PCR, the expression levels of target genes are calculated on the basis of endogenous controls. These genes need to be appropriately selected by experimentally assessing whether they are stably expressed under different conditions. However, such studies on potential reference genes in N. lugens are lacking. In this paper, we presented a systematic exploration of eight candidate reference genes in N. lugens, namely, actin 1 (ACT), muscle actin (MACT), ribosomal protein S11 (RPS11), ribosomal protein S15e (RPS15), alpha 2-tubulin (TUB), elongation factor 1 delta (EF), 18S ribosomal RNA (18S), and arginine kinase (AK) and used four alternative methods (BestKeeper, geNorm, NormFinder, and the delta Ct method) to evaluate the suitability of these genes as endogenous controls. We examined their expression levels among different experimental factors (developmental stage, body part, geographic population, temperature variation, pesticide exposure, diet change, and starvation) following the MIQE (Minimum Information for publication of Quantitative real time PCR Experiments) guidelines. Based on the results of RefFinder, which integrates four currently available major software programs to compare and rank the tested candidate reference genes, RPS15, RPS11, and TUB were found to be the most suitable reference genes in different developmental stages, body parts, and geographic populations, respectively. RPS15 was the most suitable gene under different temperature and diet conditions, while RPS11 was the most suitable gene under different pesticide exposure and starvation conditions. This work sheds light on establishing a standardized qRT-PCR procedure in N. lugens, and serves as a starting point for screening for reference genes for expression studies of related insects.

Expression of genes associated with stress conditions by Listeria monocytogenes in interaction with nisin producer Lactococcus lactis.

PubMed

Miranda, Rodrigo Otávio; Campos-Galvão, Maria Emilene Martino; Nero, Luís Augusto

2018-03-01

The use of nisin producers in foods is considered a mitigation strategy to control foodborne pathogens growth, such as Listeria monocytogenes, due to the production of this bacteriocin in situ. However, when the bacteriocin does not reach an adequate concentration, the target bacteria can develop a cross-response to different stress conditions in food, such as acid, thermal and osmotic. This study aimed to evaluate the interaction of a nisin-producing strain of Lactococcus lactis DY-13 and L. monocytogenes in BHI and skim milk, and its influence on general (sigB), acid (gadD2), thermal (groEL) and osmotic (gbu) stress-related genes of the pathogen. L. monocytogenes populations decreased approximately 2log in BHI and 1log in milk after 24h in co-culture with the nisin producer L. lactis, coherent with the increasing expression of nisK. Expression of stress-related genes by L. monocytogenes presented lower oscillation in BHI than in milk, indicating its better ability to survive in milk, despite the higher nisin production. Stress-related genes presented a varied expression by L. monocytogenes in the tested conditions: sigB expression remained stable or reduced over time; gadD2 presented high expression in milk; groEL presented low expression in BHI when compared to milk, trending to decrease overtime; gbu expression in milk after 24h was lower than in BHI. The presented study demonstrated the growth of a nisin producer L. lactis can affect the expression of stress-related genes by L. monocytogenes, and understating these mechanisms is crucial to enhance the conservation methods employed in foods. Copyright © 2017 Elsevier Ltd. All rights reserved.

Exploiting the full power of temporal gene expression profiling through a new statistical test: application to the analysis of muscular dystrophy data.

PubMed

Vinciotti, Veronica; Liu, Xiaohui; Turk, Rolf; de Meijer, Emile J; 't Hoen, Peter A C

2006-04-03

The identification of biologically interesting genes in a temporal expression profiling dataset is challenging and complicated by high levels of experimental noise. Most statistical methods used in the literature do not fully exploit the temporal ordering in the dataset and are not suited to the case where temporal profiles are measured for a number of different biological conditions. We present a statistical test that makes explicit use of the temporal order in the data by fitting polynomial functions to the temporal profile of each gene and for each biological condition. A Hotelling T2-statistic is derived to detect the genes for which the parameters of these polynomials are significantly different from each other. We validate the temporal Hotelling T2-test on muscular gene expression data from four mouse strains which were profiled at different ages: dystrophin-, beta-sarcoglycan and gamma-sarcoglycan deficient mice, and wild-type mice. The first three are animal models for different muscular dystrophies. Extensive biological validation shows that the method is capable of finding genes with temporal profiles significantly different across the four strains, as well as identifying potential biomarkers for each form of the disease. The added value of the temporal test compared to an identical test which does not make use of temporal ordering is demonstrated via a simulation study, and through confirmation of the expression profiles from selected genes by quantitative PCR experiments. The proposed method maximises the detection of the biologically interesting genes, whilst minimising false detections. The temporal Hotelling T2-test is capable of finding relatively small and robust sets of genes that display different temporal profiles between the conditions of interest. The test is simple, it can be used on gene expression data generated from any experimental design and for any number of conditions, and it allows fast interpretation of the temporal behaviour of genes. The R code is available from V.V. The microarray data have been submitted to GEO under series GSE1574 and GSE3523.

Exploiting the full power of temporal gene expression profiling through a new statistical test: Application to the analysis of muscular dystrophy data

PubMed Central

Vinciotti, Veronica; Liu, Xiaohui; Turk, Rolf; de Meijer, Emile J; 't Hoen, Peter AC

2006-01-01

Background The identification of biologically interesting genes in a temporal expression profiling dataset is challenging and complicated by high levels of experimental noise. Most statistical methods used in the literature do not fully exploit the temporal ordering in the dataset and are not suited to the case where temporal profiles are measured for a number of different biological conditions. We present a statistical test that makes explicit use of the temporal order in the data by fitting polynomial functions to the temporal profile of each gene and for each biological condition. A Hotelling T2-statistic is derived to detect the genes for which the parameters of these polynomials are significantly different from each other. Results We validate the temporal Hotelling T2-test on muscular gene expression data from four mouse strains which were profiled at different ages: dystrophin-, beta-sarcoglycan and gamma-sarcoglycan deficient mice, and wild-type mice. The first three are animal models for different muscular dystrophies. Extensive biological validation shows that the method is capable of finding genes with temporal profiles significantly different across the four strains, as well as identifying potential biomarkers for each form of the disease. The added value of the temporal test compared to an identical test which does not make use of temporal ordering is demonstrated via a simulation study, and through confirmation of the expression profiles from selected genes by quantitative PCR experiments. The proposed method maximises the detection of the biologically interesting genes, whilst minimising false detections. Conclusion The temporal Hotelling T2-test is capable of finding relatively small and robust sets of genes that display different temporal profiles between the conditions of interest. The test is simple, it can be used on gene expression data generated from any experimental design and for any number of conditions, and it allows fast interpretation of the temporal behaviour of genes. The R code is available from V.V. The microarray data have been submitted to GEO under series GSE1574 and GSE3523. PMID:16584545

LINCS Canvas Browser: interactive web app to query, browse and interrogate LINCS L1000 gene expression signatures.

PubMed

Duan, Qiaonan; Flynn, Corey; Niepel, Mario; Hafner, Marc; Muhlich, Jeremy L; Fernandez, Nicolas F; Rouillard, Andrew D; Tan, Christopher M; Chen, Edward Y; Golub, Todd R; Sorger, Peter K; Subramanian, Aravind; Ma'ayan, Avi

2014-07-01

For the Library of Integrated Network-based Cellular Signatures (LINCS) project many gene expression signatures using the L1000 technology have been produced. The L1000 technology is a cost-effective method to profile gene expression in large scale. LINCS Canvas Browser (LCB) is an interactive HTML5 web-based software application that facilitates querying, browsing and interrogating many of the currently available LINCS L1000 data. LCB implements two compacted layered canvases, one to visualize clustered L1000 expression data, and the other to display enrichment analysis results using 30 different gene set libraries. Clicking on an experimental condition highlights gene-sets enriched for the differentially expressed genes from the selected experiment. A search interface allows users to input gene lists and query them against over 100 000 conditions to find the top matching experiments. The tool integrates many resources for an unprecedented potential for new discoveries in systems biology and systems pharmacology. The LCB application is available at http://www.maayanlab.net/LINCS/LCB. Customized versions will be made part of the http://lincscloud.org and http://lincs.hms.harvard.edu websites. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Revealing cell cycle control by combining model-based detection of periodic expression with novel cis-regulatory descriptors

PubMed Central

Andersson, Claes R; Hvidsten, Torgeir R; Isaksson, Anders; Gustafsson, Mats G; Komorowski, Jan

2007-01-01

Background We address the issue of explaining the presence or absence of phase-specific transcription in budding yeast cultures under different conditions. To this end we use a model-based detector of gene expression periodicity to divide genes into classes depending on their behavior in experiments using different synchronization methods. While computational inference of gene regulatory circuits typically relies on expression similarity (clustering) in order to find classes of potentially co-regulated genes, this method instead takes advantage of known time profile signatures related to the studied process. Results We explain the regulatory mechanisms of the inferred periodic classes with cis-regulatory descriptors that combine upstream sequence motifs with experimentally determined binding of transcription factors. By systematic statistical analysis we show that periodic classes are best explained by combinations of descriptors rather than single descriptors, and that different combinations correspond to periodic expression in different classes. We also find evidence for additive regulation in that the combinations of cis-regulatory descriptors associated with genes periodically expressed in fewer conditions are frequently subsets of combinations associated with genes periodically expression in more conditions. Finally, we demonstrate that our approach retrieves combinations that are more specific towards known cell-cycle related regulators than the frequently used clustering approach. Conclusion The results illustrate how a model-based approach to expression analysis may be particularly well suited to detect biologically relevant mechanisms. Our new approach makes it possible to provide more refined hypotheses about regulatory mechanisms of the cell cycle and it can easily be adjusted to reveal regulation of other, non-periodic, cellular processes. PMID:17939860

Environmental effects on resistance gene expression in milk stage popcorn kernels and associations with mycotoxin production.

PubMed

Dowd, Patrick F; Johnson, Eric T

2015-05-01

Like other forms of maize, popcorn is subject to increased levels of contamination by a variety of different mycotoxins under stress conditions, although levels generally are less than dent maize under comparable stress. Gene array analysis was used to determine expression differences of disease resistance-associated genes in milk stage kernels from commercial popcorn fields over 3 years. Relatively lower expression of resistance gene types was noted in years with higher temperatures and lower rainfall, which was consistent with prior results for many previously identified resistance response-associated genes. The lower rates of expression occurred for genes such as chitinases, protease inhibitors, and peroxidases; enzymes involved in the synthesis of cell wall barriers and secondary metabolites; and regulatory proteins. However, expression of several specific resistance genes previously associated with mycotoxins, such as aflatoxin in dent maize, was not affected. Insect damage altered the spectrum of resistance gene expression differences compared to undamaged ears. Correlation analyses showed expression differences of some previously reported resistance genes that were highly associated with mycotoxin levels and included glucanases, protease inhibitors, peroxidases, and thionins.

Juvenile hormone and colony conditions differentially influence cytochrome P450 gene expression in the termite Reticulitermes flavipes.

PubMed

Zhou, X; Song, C; Grzymala, T L; Oi, F M; Scharf, M E

2006-12-01

In lower termites, the worker caste is a totipotent immature stage that is capable of differentiating into other adult caste phenotypes. We investigated the diversity of family 4 cytochrome P450 (CYP4) genes in Reticulitermes flavipes workers, with the specific goal of identifying P450s potentially involved in regulating caste differentiation. Seven novel CYP4 genes were identified. Quantitative real-time PCR revealed the tissue distribution of expression for the seven CYP4s, as well as temporal expression changes in workers in association with a release from colony influences and during juvenile hormone (JH)-induced soldier caste differentiation. Several fat-body-related CYP4 genes were differentially expressed after JH treatment. Still other genes changed expression in association with removal from colony influences, suggesting that primer pheromones and/or other colony influences impact their expression. These findings add to a growing database of candidate termite caste-regulatory genes, and provide explicit evidence that colony factors influence termite gene expression.

Conditioned taste aversion dependent regulation of amygdala gene expression.

PubMed

Panguluri, Siva K; Kuwabara, Nobuyuki; Kang, Yi; Cooper, Nigel; Lundy, Robert F

2012-02-28

The present experiments investigated gene expression in the amygdala following contingent taste/LiCl treatment that supports development of conditioned taste aversion (CTA). The use of whole genome chips and stringent data set filtering led to the identification of 168 genes regulated by CTA compared to non-contingent LiCl treatment that does not support CTA learning. Seventy-six of these genes were eligible for network analysis. Such analysis identified "behavior" as the top biological function, which was represented by 15 of the 76 genes. These genes included several neuropeptides, G protein-coupled receptors, ion channels, kinases, and phosphatases. Subsequent qRT-PCR analyses confirmed changes in mRNA expression for 5 of 7 selected genes. We were able to demonstrate directionally consistent changes in protein level for 3 of these genes; insulin 1, oxytocin, and major histocompatibility complex class I-C. Behavioral analyses demonstrated that blockade of central insulin receptors produced a weaker CTA that was less resistant to extinction. Together, these results support the notion that we have identified downstream genes in the amygdala that contribute to CTA learning. Copyright © 2011 Elsevier Inc. All rights reserved.

Gene expression changes governing extreme dehydration tolerance in an Antarctic insect

PubMed Central

Teets, Nicholas M.; Peyton, Justin T.; Colinet, Herve; Renault, David; Kelley, Joanna L.; Kawarasaki, Yuta; Lee, Richard E.; Denlinger, David L.

2012-01-01

Among terrestrial organisms, arthropods are especially susceptible to dehydration, given their small body size and high surface area to volume ratio. This challenge is particularly acute for polar arthropods that face near-constant desiccating conditions, as water is frozen and thus unavailable for much of the year. The molecular mechanisms that govern extreme dehydration tolerance in insects remain largely undefined. In this study, we used RNA sequencing to quantify transcriptional mechanisms of extreme dehydration tolerance in the Antarctic midge, Belgica antarctica, the world’s southernmost insect and only insect endemic to Antarctica. Larvae of B. antarctica are remarkably tolerant of dehydration, surviving losses up to 70% of their body water. Gene expression changes in response to dehydration indicated up-regulation of cellular recycling pathways including the ubiquitin-mediated proteasome and autophagy, with concurrent down-regulation of genes involved in general metabolism and ATP production. Metabolomics results revealed shifts in metabolite pools that correlated closely with changes in gene expression, indicating that coordinated changes in gene expression and metabolism are a critical component of the dehydration response. Finally, using comparative genomics, we compared our gene expression results with a transcriptomic dataset for the Arctic collembolan, Megaphorura arctica. Although B. antarctica and M. arctica are adapted to similar environments, our analysis indicated very little overlap in expression profiles between these two arthropods. Whereas several orthologous genes showed similar expression patterns, transcriptional changes were largely species specific, indicating these polar arthropods have developed distinct transcriptional mechanisms to cope with similar desiccating conditions. PMID:23197828

Gene expression changes governing extreme dehydration tolerance in an Antarctic insect.

PubMed

Teets, Nicholas M; Peyton, Justin T; Colinet, Herve; Renault, David; Kelley, Joanna L; Kawarasaki, Yuta; Lee, Richard E; Denlinger, David L

2012-12-11

Among terrestrial organisms, arthropods are especially susceptible to dehydration, given their small body size and high surface area to volume ratio. This challenge is particularly acute for polar arthropods that face near-constant desiccating conditions, as water is frozen and thus unavailable for much of the year. The molecular mechanisms that govern extreme dehydration tolerance in insects remain largely undefined. In this study, we used RNA sequencing to quantify transcriptional mechanisms of extreme dehydration tolerance in the Antarctic midge, Belgica antarctica, the world's southernmost insect and only insect endemic to Antarctica. Larvae of B. antarctica are remarkably tolerant of dehydration, surviving losses up to 70% of their body water. Gene expression changes in response to dehydration indicated up-regulation of cellular recycling pathways including the ubiquitin-mediated proteasome and autophagy, with concurrent down-regulation of genes involved in general metabolism and ATP production. Metabolomics results revealed shifts in metabolite pools that correlated closely with changes in gene expression, indicating that coordinated changes in gene expression and metabolism are a critical component of the dehydration response. Finally, using comparative genomics, we compared our gene expression results with a transcriptomic dataset for the Arctic collembolan, Megaphorura arctica. Although B. antarctica and M. arctica are adapted to similar environments, our analysis indicated very little overlap in expression profiles between these two arthropods. Whereas several orthologous genes showed similar expression patterns, transcriptional changes were largely species specific, indicating these polar arthropods have developed distinct transcriptional mechanisms to cope with similar desiccating conditions.

JRmGRN: Joint reconstruction of multiple gene regulatory networks with common hub genes using data from multiple tissues or conditions.

PubMed

Deng, Wenping; Zhang, Kui; Liu, Sanzhen; Zhao, Patrick; Xu, Shizhong; Wei, Hairong

2018-04-30

Joint reconstruction of multiple gene regulatory networks (GRNs) using gene expression data from multiple tissues/conditions is very important for understanding common and tissue/condition-specific regulation. However, there are currently no computational models and methods available for directly constructing such multiple GRNs that not only share some common hub genes but also possess tissue/condition-specific regulatory edges. In this paper, we proposed a new graphic Gaussian model for joint reconstruction of multiple gene regulatory networks (JRmGRN), which highlighted hub genes, using gene expression data from several tissues/conditions. Under the framework of Gaussian graphical model, JRmGRN method constructs the GRNs through maximizing a penalized log likelihood function. We formulated it as a convex optimization problem, and then solved it with an alternating direction method of multipliers (ADMM) algorithm. The performance of JRmGRN was first evaluated with synthetic data and the results showed that JRmGRN outperformed several other methods for reconstruction of GRNs. We also applied our method to real Arabidopsis thaliana RNA-seq data from two light regime conditions in comparison with other methods, and both common hub genes and some conditions-specific hub genes were identified with higher accuracy and precision. JRmGRN is available as a R program from: https://github.com/wenpingd. hairong@mtu.edu. Proof of theorem, derivation of algorithm and supplementary data are available at Bioinformatics online.

An inducible HSP70 gene from the midge Chironomus dilutus: Characterization and transcription profile under environmental stress

USGS Publications Warehouse

Karouna-Renier, N. K.; Rao, K.R.

2009-01-01

In the present study, we identified and characterized an inducible heat shock protein 70 (HSP70) from the midge Chironomus dilutus and investigated the transcriptional profile of the gene under baseline and environmentally stressful conditions. Using real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR), we observed increased expression of CD-HSP70-1 in response to both heat shock and copper stress. We also investigated the expression of this gene during midge development. All C. dilutus developmental stages expressed CD-HSP70-1 under normal conditions, although at extremely low levels. Phylogenetic analysis of the amino acid sequence demonstrated distinct clustering of this gene with inducible HSP70s from other insect species. ?? 2008 The Authors.

Extensive circadian and light regulation of the transcriptome in the malaria mosquito Anopheles gambiae

PubMed Central

2013-01-01

Background Mosquitoes exhibit 24 hr rhythms in flight activity, feeding, reproduction and development. To better understand the molecular basis for these rhythms in the nocturnal malaria vector Anopheles gambiae, we have utilized microarray analysis on time-of-day specific collections of mosquitoes over 48 hr to explore the coregulation of gene expression rhythms by the circadian clock and light, and compare these with the 24 hr rhythmic gene expression in the diurnal Aedes aegypti dengue vector mosquito. Results In time courses from An. gambiae head and body collected under light:dark cycle (LD) and constant dark (DD) conditions, we applied three algorithms that detect sinusoidal patterns and an algorithm that detects spikes in expression. This revealed across four experimental conditions 393 probes newly scored as rhythmic. These genes correspond to functions such as metabolic detoxification, immunity and nutrient sensing. This includes glutathione S-transferase GSTE5, whose expression pattern and chromosomal location are shared with other genes, suggesting shared chromosomal regulation; and pulsatile expression of the gene encoding CYP6M2, a cytochrome P450 that metabolizes pyrethroid insecticides. We explored the interaction of light and the circadian clock and highlight the regulation of odorant binding proteins (OBPs), important components of the olfactory system. We reveal that OBPs have unique expression patterns as mosquitoes make the transition from LD to DD conditions. We compared rhythmic expression between An. gambiae and Ae. aegypti heads collected under LD conditions using a single cosine fitting algorithm, and report distinct similarities and differences in the temporal regulation of genes involved in tRNA priming, the vesicular-type ATPase, olfaction and vision between the two species. Conclusions These data build on our previous analyses of time-of-day specific regulation of the An. gambiae transcriptome to reveal additional rhythmic genes, an improved understanding of the co-regulation of rhythms in gene expression by the circadian clock and by light, and an understanding of the time-of-day specific regulation of some of these rhythmic processes in comparison with a different species of mosquito. Improved understanding of biological timing at the molecular level that underlies key physiological aspects of mosquito vectors may prove to be important to successful implementation of established and novel insect control methods. PMID:23552056

Effects of P limitation and molecules from peanut root exudates on pqqE gene expression and pqq promoter activity in the phosphate-solubilizing strain Serratia sp. S119.

PubMed

Ludueña, Liliana M; Anzuay, Maria S; Magallanes-Noguera, Cynthia; Tonelli, Maria L; Ibañez, Fernando J; Angelini, Jorge G; Fabra, Adriana; McIntosh, Matthew; Taurian, Tania

2017-10-01

The mineral phosphate-solubilizing phenotype in bacteria is attributed predominantly to secretion of gluconic acid produced by oxidation of glucose by the glucose dehydrogenase enzyme and its cofactor, pyrroloquinoline quinone. This study analyzes pqqE gene expression and pqq promoter activity in the native phosphate-solubilizing bacterium Serratia sp S119 growing under P-limitation, and in the presence of root exudates obtained from peanut plants, also growing under P-limitation. Results indicated that Serratia sp. S119 contains a pqq operon composed of six genes (pqqA,B,C,D,E,F) and two promoters, one upstream of pqqA and other between pqqA and pqqB. PqqE gene expression and pqq promoter activity increased under P-limiting growth conditions and not under N-deficient conditions. In the plant-bacteria interaction assay, the activity of the bacterial pqq promoter region varied depending on the concentration and type of root exudates and on the bacterial growth phase. Root exudates from peanut plants growing under P-available and P-limiting conditions showed differences in their composition. It is concluded from this study that the response of Serratia sp. S119 to phosphorus limitation involves an increase in expression of pqq genes, and that molecules exuded by peanut roots modify expression of these phosphate-solubilizing bacterial genes during plant-bacteria interactions. Copyright © 2017 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Transcriptomic analysis of fruit stored under cold conditions using controlled atmosphere in Prunus persica cv. "Red Pearl".

PubMed

Sanhueza, Dayan; Vizoso, Paula; Balic, Iván; Campos-Vargas, Reinaldo; Meneses, Claudio

2015-01-01

Cold storage (CS) can induce a physiological disorder known as chilling injury (CI) in nectarine fruits. The main symptom is mealiness that is perceived as non-juicy fruit by consumers. Postharvest treatments such as controlled atmosphere (CA; a high CO2 concentration and low O2) have been used under cold conditions to avoid this disorder. With the objective of exploring the mechanisms involved in the CA effect on mealiness prevention, we analyzed transcriptomic changes under six conditions of "Red Pearl" nectarines by RNA-Seq. Our analysis included just harvested nectarines, juicy non-stored fruits, fruits affected for CI after CS and fruits stored in a combination of CA plus CS without CI phenotype. Nectarines stored in cold conditions combined with CA treatment resulted in less mealiness; we obtained 21.6% of juice content compared with just CS fruits (7.7%; mealy flesh). RNA-Seq data analyses were carried out to study the gene expression for different conditions assayed. During ripening, we detected that nectarines exposed to CA treatment expressed a similar number of genes compared with fruits that were not exposed to cold conditions. Firm fruits have more differentially expressed genes than soft fruits, which suggest that most important changes occur during CS. On the other hand, gene ontology analysis revealed enrichment mainly in metabolic and cellular processes. Differentially expressed genes analysis showed that low O2 concentrations combined with cold conditions slows the metabolic processes more than just the cold storage, resulting mainly in the suppression of primary metabolism and cold stress response. This is a significant step toward unraveling the molecular mechanism that explains the effectiveness of CA as a tool to prevent CI development on fruits.

Transcriptomic analysis of fruit stored under cold conditions using controlled atmosphere in Prunus persica cv. “Red Pearl”

PubMed Central

Sanhueza, Dayan; Vizoso, Paula; Balic, Iván; Campos-Vargas, Reinaldo; Meneses, Claudio

2015-01-01

Cold storage (CS) can induce a physiological disorder known as chilling injury (CI) in nectarine fruits. The main symptom is mealiness that is perceived as non-juicy fruit by consumers. Postharvest treatments such as controlled atmosphere (CA; a high CO2 concentration and low O2) have been used under cold conditions to avoid this disorder. With the objective of exploring the mechanisms involved in the CA effect on mealiness prevention, we analyzed transcriptomic changes under six conditions of “Red Pearl” nectarines by RNA-Seq. Our analysis included just harvested nectarines, juicy non-stored fruits, fruits affected for CI after CS and fruits stored in a combination of CA plus CS without CI phenotype. Nectarines stored in cold conditions combined with CA treatment resulted in less mealiness; we obtained 21.6% of juice content compared with just CS fruits (7.7%; mealy flesh). RNA-Seq data analyses were carried out to study the gene expression for different conditions assayed. During ripening, we detected that nectarines exposed to CA treatment expressed a similar number of genes compared with fruits that were not exposed to cold conditions. Firm fruits have more differentially expressed genes than soft fruits, which suggest that most important changes occur during CS. On the other hand, gene ontology analysis revealed enrichment mainly in metabolic and cellular processes. Differentially expressed genes analysis showed that low O2 concentrations combined with cold conditions slows the metabolic processes more than just the cold storage, resulting mainly in the suppression of primary metabolism and cold stress response. This is a significant step toward unraveling the molecular mechanism that explains the effectiveness of CA as a tool to prevent CI development on fruits. PMID:26483806

Identification and evaluation of reference genes for qRT-PCR studies in Lentinula edodes

PubMed Central

Qin, Peng; He, Maolan; Yu, Xiumei; Zhao, Ke; Zhang, Xiaoping; Ma, Menggen; Chen, Qiang; Chen, Xiaoqiong; Zeng, Xianfu; Gu, Yunfu

2018-01-01

Lentinula edodes (shiitake mushroom) is a common edible mushroom with a number of potential therapeutic and nutritional applications. It contains various medically important molecules, such as polysaccharides, terpenoids, sterols, and lipids, were contained in this mushroom. Quantitative real-time polymerase chain reaction (qRT-PCR) is a powerful tool to analyze the mechanisms underlying the biosynthetic pathways of these substances. qRT-PCR is used for accurate analyses of transcript levels owing to its rapidity, sensitivity, and reliability. However, its accuracy and reliability for the quantification of transcripts rely on the expression stability of the reference genes used for data normalization. To ensure the reliability of gene expression analyses using qRT-PCR in L. edodes molecular biology research, it is necessary to systematically evaluate reference genes. In the current study, ten potential reference genes were selected from L. edodes genomic data and their expression levels were measured by qRT-PCR using various samples. The expression stability of each candidate gene was analyzed by three commonly used software packages: geNorm, NormFinder, and BestKeeper. Base on the results, Rpl4 was the most stable reference gene across all experimental conditions, and Atu was the most stable gene among strains. 18S was found to be the best reference gene for different development stages, and Rpl4 was the most stably expressed gene under various nutrient conditions. The present work will contribute to qRT-PCR studies in L. edodes. PMID:29293626

Identification and evaluation of reference genes for qRT-PCR studies in Lentinula edodes.

PubMed

Xiang, Quanju; Li, Jin; Qin, Peng; He, Maolan; Yu, Xiumei; Zhao, Ke; Zhang, Xiaoping; Ma, Menggen; Chen, Qiang; Chen, Xiaoqiong; Zeng, Xianfu; Gu, Yunfu

2018-01-01

Lentinula edodes (shiitake mushroom) is a common edible mushroom with a number of potential therapeutic and nutritional applications. It contains various medically important molecules, such as polysaccharides, terpenoids, sterols, and lipids, were contained in this mushroom. Quantitative real-time polymerase chain reaction (qRT-PCR) is a powerful tool to analyze the mechanisms underlying the biosynthetic pathways of these substances. qRT-PCR is used for accurate analyses of transcript levels owing to its rapidity, sensitivity, and reliability. However, its accuracy and reliability for the quantification of transcripts rely on the expression stability of the reference genes used for data normalization. To ensure the reliability of gene expression analyses using qRT-PCR in L. edodes molecular biology research, it is necessary to systematically evaluate reference genes. In the current study, ten potential reference genes were selected from L. edodes genomic data and their expression levels were measured by qRT-PCR using various samples. The expression stability of each candidate gene was analyzed by three commonly used software packages: geNorm, NormFinder, and BestKeeper. Base on the results, Rpl4 was the most stable reference gene across all experimental conditions, and Atu was the most stable gene among strains. 18S was found to be the best reference gene for different development stages, and Rpl4 was the most stably expressed gene under various nutrient conditions. The present work will contribute to qRT-PCR studies in L. edodes.

Gene Expression Dynamics Accompanying the Sponge Thermal Stress Response.

PubMed

Guzman, Christine; Conaco, Cecilia

2016-01-01

Marine sponges are important members of coral reef ecosystems. Thus, their responses to changes in ocean chemistry and environmental conditions, particularly to higher seawater temperatures, will have potential impacts on the future of these reefs. To better understand the sponge thermal stress response, we investigated gene expression dynamics in the shallow water sponge, Haliclona tubifera (order Haplosclerida, class Demospongiae), subjected to elevated temperature. Using high-throughput transcriptome sequencing, we show that these conditions result in the activation of various processes that interact to maintain cellular homeostasis. Short-term thermal stress resulted in the induction of heat shock proteins, antioxidants, and genes involved in signal transduction and innate immunity pathways. Prolonged exposure to thermal stress affected the expression of genes involved in cellular damage repair, apoptosis, signaling and transcription. Interestingly, exposure to sublethal temperatures may improve the ability of the sponge to mitigate cellular damage under more extreme stress conditions. These insights into the potential mechanisms of adaptation and resilience of sponges contribute to a better understanding of sponge conservation status and the prediction of ecosystem trajectories under future climate conditions.

The nature of electron acceptor (MnIV/NO3) triggers differential expression of genes associated with stress and ammonium limitation responses in Shewanella algae C6G3.

PubMed

Aigle, Axel; Bonin, Patricia; -Nunez, Nicolas Fernandez; Loriod, Béatrice; Guasco, Sophie; Bergon, Aurélie; Armougom, Fabrice; Iobbi-Nivol, Chantal; Imbert, Jean; Michotey, Valérie

2018-03-16

Shewanella algae C6G3 can reduce dissimilatively nitrate into ammonium and manganese-oxide (MnIV) into MnII. It has the unusual ability to produce anaerobically nitrite from ammonium in the presence of MnIV. To gain insight into their metabolic capabilities, global mRNA expression patterns were investigated by RNA-seq and qRT-PCR in cells growing with lactate and ammonium as carbon and nitrogen sources and with either MnIV or nitrate as electron acceptors. Gene exhibiting higher expression levels in the presence of MnIV belonged to functional categories of carbohydrate, coenzyme, lipid metabolisms and inorganic ion transport. Comparative transcriptomic pattern between MnIV and NO3 revealed that the strain presented an ammonium limitation status with MnIV, despite the presence of non-limiting concentration of ammonium under both culture conditions. In addition, in presence of MnIV, ntrB/nrtC regulators, ammonium channel, nitrogen regulatory protein P-II, glutamine synthetase and asparagine synthetase glutamine dependent genes were over-represented. Under nitrate condition, the expression of genes involved in the synthesis of several amino acids was increased. Finally, expression level of genes associated with the general stress response was also amplified and among them, katE, a putative catalase/peroxidase present on several Shewanella genomes, was highly expressed with a relative median value higher in MnIV condition.

Versatile control of Plasmodium falciparum gene expression with an inducible protein-RNA interaction

PubMed Central

Goldfless, Stephen J.; Wagner, Jeffrey C.; Niles, Jacquin C.

2014-01-01

The available tools for conditional gene expression in Plasmodium falciparum are limited. Here, to enable reliable control of target gene expression, we build a system to efficiently modulate translation. We overcame several problems associated with other approaches for regulating gene expression in P. falciparum. Specifically, our system functions predictably across several native and engineered promoter contexts, and affords control over reporter and native parasite proteins irrespective of their subcellular compartmentalization. Induction and repression of gene expression are rapid, homogeneous, and stable over prolonged periods. To demonstrate practical application of our system, we used it to reveal direct links between antimalarial drugs and their native parasite molecular target. This is an important out come given the rapid spread of resistance, and intensified efforts to efficiently discover and optimize new antimalarial drugs. Overall, the studies presented highlight the utility of our system for broadly controlling gene expression and performing functional genetics in P. falciparum. PMID:25370483

Utilization of urea and expression profiles of related genes in the dinoflagellate Prorocentrum donghaiense

PubMed Central

Jing, Xiaoli; Lin, Senjie; Zhang, Huan; Koerting, Claudia; Yu, Zhigang

2017-01-01

Urea has been shown to contribute more than half of total nitrogen (N) required by phytoplankton in some estuaries and coastal waters and to provide a substantial portion of the N demand for many harmful algal blooms (HABs) of dinoflagellates. In this study, we investigated the physiological and transcriptional responses in Prorocentrum donghaiense to changes in nitrate and urea availability. We found that this species could efficiently utilize urea as sole N source and achieve comparable growth rate and photosynthesis capability as it did under nitrate. These physiological parameters were markedly lower in cultures grown under nitrate- or urea-limited conditions. P. donghaiense N content was similarly low under nitrate- or urea-limited culture condition, but was markedly higher under urea-replete condition than under nitrate-replete condition. Carbon (C) content was consistently elevated under N-limited condition. Consequently, the C:N ratio was as high as 21:1 under nitrate- or urea-limitation, but 7:1 under urea-replete condition and 9:1 to 10:1 under nitrate-replete condition. Using quantitative reverse transcription PCR, we investigated the expression pattern for four genes involved in N transport and assimilation. The results indicated that genes encoding nitrate transport, urea hydrolysis, and nickel transporter gene were sensitive to changes in general N nutrient availability whereas the urea transporter gene responded much more strongly to changes in urea concentration. Taken together, our study shows the high bioavailability of urea, its impact on C:N stoichiometry, and the sensitivity of urea transporter gene expression to urea availability. PMID:29117255

Utilization of urea and expression profiles of related genes in the dinoflagellate Prorocentrum donghaiense.

PubMed

Jing, Xiaoli; Lin, Senjie; Zhang, Huan; Koerting, Claudia; Yu, Zhigang

2017-01-01

Urea has been shown to contribute more than half of total nitrogen (N) required by phytoplankton in some estuaries and coastal waters and to provide a substantial portion of the N demand for many harmful algal blooms (HABs) of dinoflagellates. In this study, we investigated the physiological and transcriptional responses in Prorocentrum donghaiense to changes in nitrate and urea availability. We found that this species could efficiently utilize urea as sole N source and achieve comparable growth rate and photosynthesis capability as it did under nitrate. These physiological parameters were markedly lower in cultures grown under nitrate- or urea-limited conditions. P. donghaiense N content was similarly low under nitrate- or urea-limited culture condition, but was markedly higher under urea-replete condition than under nitrate-replete condition. Carbon (C) content was consistently elevated under N-limited condition. Consequently, the C:N ratio was as high as 21:1 under nitrate- or urea-limitation, but 7:1 under urea-replete condition and 9:1 to 10:1 under nitrate-replete condition. Using quantitative reverse transcription PCR, we investigated the expression pattern for four genes involved in N transport and assimilation. The results indicated that genes encoding nitrate transport, urea hydrolysis, and nickel transporter gene were sensitive to changes in general N nutrient availability whereas the urea transporter gene responded much more strongly to changes in urea concentration. Taken together, our study shows the high bioavailability of urea, its impact on C:N stoichiometry, and the sensitivity of urea transporter gene expression to urea availability.

Transcriptome-wide analysis of differential gene expression in response to light:dark cycles in a model cnidarian.

PubMed

Leach, W B; Macrander, J; Peres, R; Reitzel, A M

2018-06-01

Animals respond to diurnal shifts in their environment with a combination of behavioral, physiological, and molecular changes to synchronize with regularly-timed external cues. Reproduction, movement, and metabolism in cnidarians have all been shown to be regulated by diurnal lighting, but the molecular mechanisms that may be responsible for these phenotypes remain largely unknown. The starlet sea anemone, Nematostella vectensis, has oscillating patterns of locomotion and respiration, as well as the molecular components of a putative circadian clock that may provide a mechanism for these light-induced responses. Here, we compare transcriptomic responses of N. vectensis when cultured under a diurnal lighting condition (12 h light: 12 h dark) with sea anemones cultured under constant darkness for 20 days. More than 3,000 genes (~13% of transcripts) had significant differences in expression between light and dark, with most genes having higher expression in the photoperiod. Following removal of the light cue 678 genes lost differential expression, suggesting that light-entrained gene expression by the circadian clock has temporal limits. Grouping of genes differentially expressed in light:dark conditions showed that cell cycle and transcription maintained diel expression in the absence of light, while many of the genes related to metabolism, antioxidants, immunity, and signal transduction lost differential expression without a light cue. Our data highlight the importance of diel light cycles on circadian mechanisms in this species, prompting new hypotheses for the role of photoreception in major biological processes, e.g., metabolism, immunity. Copyright © 2018 Elsevier Inc. All rights reserved.

Fungal Gene Expression on Demand: an Inducible, Tunable, and Metabolism-Independent Expression System for Aspergillus niger▿†

PubMed Central

Meyer, Vera; Wanka, Franziska; van Gent, Janneke; Arentshorst, Mark; van den Hondel, Cees A. M. J. J.; Ram, Arthur F. J.

2011-01-01

Filamentous fungi are the cause of serious human and plant diseases but are also exploited in biotechnology as production platforms. Comparative genomics has documented their genetic diversity, and functional genomics and systems biology approaches are under way to understand the functions and interaction of fungal genes and proteins. In these approaches, gene functions are usually inferred from deletion or overexpression mutants. However, studies at these extreme points give only limited information. Moreover, many overexpression studies use metabolism-dependent promoters, often causing pleiotropic effects and thus limitations in their significance. We therefore established and systematically evaluated a tunable expression system for Aspergillus niger that is independent of carbon and nitrogen metabolism and silent under noninduced conditions. The system consists of two expression modules jointly targeted to a defined genomic locus. One module ensures constitutive expression of the tetracycline-dependent transactivator rtTA2S-M2, and one module harbors the rtTA2S-M2-dependent promoter that controls expression of the gene of interest (the Tet-on system). We show here that the system is tight, responds within minutes after inducer addition, and allows fine-tuning based on the inducer concentration or gene copy number up to expression levels higher than the expression levels of the gpdA promoter. We also validate the Tet-on system for the generation of conditional overexpression mutants and demonstrate its power when combined with a gene deletion approach. Finally, we show that the system is especially suitable when the functions of essential genes must be examined. PMID:21378046

Steady-state and dynamic gene expression programs in Saccharomyces cerevisiae in response to variation in environmental nitrogen

PubMed Central

Airoldi, Edoardo M.; Miller, Darach; Athanasiadou, Rodoniki; Brandt, Nathan; Abdul-Rahman, Farah; Neymotin, Benjamin; Hashimoto, Tatsu; Bahmani, Tayebeh; Gresham, David

2016-01-01

Cell growth rate is regulated in response to the abundance and molecular form of essential nutrients. In Saccharomyces cerevisiae (budding yeast), the molecular form of environmental nitrogen is a major determinant of cell growth rate, supporting growth rates that vary at least threefold. Transcriptional control of nitrogen use is mediated in large part by nitrogen catabolite repression (NCR), which results in the repression of specific transcripts in the presence of a preferred nitrogen source that supports a fast growth rate, such as glutamine, that are otherwise expressed in the presence of a nonpreferred nitrogen source, such as proline, which supports a slower growth rate. Differential expression of the NCR regulon and additional nitrogen-responsive genes results in >500 transcripts that are differentially expressed in cells growing in the presence of different nitrogen sources in batch cultures. Here we find that in growth rate–controlled cultures using nitrogen-limited chemostats, gene expression programs are strikingly similar regardless of nitrogen source. NCR expression is derepressed in all nitrogen-limiting chemostat conditions regardless of nitrogen source, and in these conditions, only 34 transcripts exhibit nitrogen source–specific differential gene expression. Addition of either the preferred nitrogen source, glutamine, or the nonpreferred nitrogen source, proline, to cells growing in nitrogen-limited chemostats results in rapid, dose-dependent repression of the NCR regulon. Using a novel means of computational normalization to compare global gene expression programs in steady-state and dynamic conditions, we find evidence that the addition of nitrogen to nitrogen-limited cells results in the transient overproduction of transcripts required for protein translation. Simultaneously, we find that that accelerated mRNA degradation underlies the rapid clearing of a subset of transcripts, which is most pronounced for the highly expressed NCR-regulated permease genes GAP1, MEP2, DAL5, PUT4, and DIP5. Our results reveal novel aspects of nitrogen-regulated gene expression and highlight the need for a quantitative approach to study how the cell coordinates protein translation and nitrogen assimilation to optimize cell growth in different environments. PMID:26941329

Validation of endogenous internal real-time PCR controls in renal tissues.

PubMed

Cui, Xiangqin; Zhou, Juling; Qiu, Jing; Johnson, Martin R; Mrug, Michal

2009-01-01

Endogenous internal controls ('reference' or 'housekeeping' genes) are widely used in real-time PCR (RT-PCR) analyses. Their use relies on the premise of consistently stable expression across studied experimental conditions. Unfortunately, none of these controls fulfills this premise across a wide range of experimental conditions; consequently, none of them can be recommended for universal use. To determine which endogenous RT-PCR controls are suitable for analyses of renal tissues altered by kidney disease, we studied the expression of 16 commonly used 'reference genes' in 7 mildly and 7 severely affected whole kidney tissues from a well-characterized cystic kidney disease model. Expression levels of these 16 genes, determined by TaqMan RT-PCR analyses and Affymetrix GeneChip arrays, were normalized and tested for overall variance and equivalence of the means. Both statistical approaches and both TaqMan- and GeneChip-based methods converged on 3 out of the 4 top-ranked genes (Ppia, Gapdh and Pgk1) that had the most constant expression levels across the studied phenotypes. A combination of the top-ranked genes will provide a suitable endogenous internal control for similar studies of kidney tissues across a wide range of disease severity. Copyright 2009 S. Karger AG, Basel.

Transcriptome Analysis of CD4+ T Cells in Coeliac Disease Reveals Imprint of BACH2 and IFNγ Regulation

PubMed Central

Molloy, Ben; Dominguez Castro, Patricia; Cormican, Paul; Trimble, Valerie; Mahmud, Nasir; McManus, Ross

2015-01-01

Genetic studies have to date identified 43 genome wide significant coeliac disease susceptibility (CD) loci comprising over 70 candidate genes. However, how altered regulation of such disease associated genes contributes to CD pathogenesis remains to be elucidated. Recently there has been considerable emphasis on characterising cell type specific and stimulus dependent genetic variants. Therefore in this study we used RNA sequencing to profile over 70 transcriptomes of CD4+ T cells, a cell type crucial for CD pathogenesis, in both stimulated and resting samples from individuals with CD and unaffected controls. We identified extensive transcriptional changes across all conditions, with the previously established CD gene IFNy the most strongly up-regulated gene (log2 fold change 4.6; Padjusted = 2.40x10-11) in CD4+ T cells from CD patients compared to controls. We show a significant correlation of differentially expressed genes with genetic studies of the disease to date (Padjusted = 0.002), and 21 CD candidate susceptibility genes are differentially expressed under one or more of the conditions used in this study. Pathway analysis revealed significant enrichment of immune related processes. Co-expression network analysis identified several modules of coordinately expressed CD genes. Two modules were particularly highly enriched for differentially expressed genes (P<2.2x10-16) and highlighted IFNy and the genetically associated transcription factor BACH2 which showed significantly reduced expression in coeliac samples (log2FC -1.75; Padjusted = 3.6x10-3) as key regulatory genes in CD. Genes regulated by BACH2 were very significantly over-represented among our differentially expressed genes (P<2.2x10-16) indicating that reduced expression of this master regulator of T cell differentiation promotes a pro-inflammatory response and strongly corroborates genetic evidence that BACH2 plays an important role in CD pathogenesis. PMID:26444573

Turning Bone Morphogenetic Protein 2 (BMP2) On and Off in Mesenchymal Cells†

PubMed Central

Rogers, Melissa B.; Shah, Tapan A.; Shaikh, Nadia N.

2016-01-01

The concentration, location, and timing of bone morphogenetic protein 2 (BMP2, HGNC:1069, GeneID: 650) gene expression must be precisely regulated. Abnormal BMP2 levels cause congenital anomalies and diseases involving the mesenchymal cells that differentiate into muscle, fat, cartilage, and bone. The molecules and conditions that influence BMP2 synthesis are diverse. Understandably, complex mechanisms control Bmp2 gene expression. This review includes a compilation of agents and conditions that can induce Bmp2. The currently known trans-regulatory factors and cis-regulatory elements that modulate Bmp2 expression are summarized and discussed. This article is protected by copyright. All rights reserved PMID:25776852

Stability of gene expression in human T cells in different gravity environments is clustered in chromosomal region 11p15.4.

PubMed

Thiel, Cora S; Huge, Andreas; Hauschild, Swantje; Tauber, Svantje; Lauber, Beatrice A; Polzer, Jennifer; Paulsen, Katrin; Lier, Hartwin; Engelmann, Frank; Schmitz, Burkhard; Schütte, Andreas; Layer, Liliana E; Ullrich, Oliver

2017-01-01

In the last decades, a plethora of in vitro studies with living human cells contributed a vast amount of knowledge about cellular and molecular effects of microgravity. Previous studies focused mostly on the identification of gravity-responsive genes, whereas a multi-platform analysis at an integrative level, which specifically evaluates the extent and robustness of transcriptional response to an altered gravity environment was not performed so far. Therefore, we investigated the stability of gene expression response in non-activated human Jurkat T lymphocytic cells in different gravity environments through the combination of parabolic flights with a suborbital ballistic rocket and 2D clinostat and centrifuge experiments, using strict controls for excluding all possible other factors of influence. We revealed an overall high stability of gene expression in microgravity and identified olfactory gene expression in the chromosomal region 11p15.4 as particularly robust to altered gravity. We identified that classical reference genes ABCA5 , GAPDH , HPRT1 , PLA2G4A , and RPL13A were stably expressed in all tested gravity conditions and platforms, while ABCA5 and GAPDH were also known to be stably expressed in U937 cells in all gravity conditions. In summary, 10-20% of all transcripts remained totally unchanged in any gravitational environment tested (between 10 -4 and 9 g), 20-40% remained unchanged in microgravity (between 10 -4 and 10 -2  g) and 97-99% were not significantly altered in microgravity if strict exclusion criteria were applied. Therefore, we suppose a high stability of gene expression in microgravity. Comparison with other stressors suggests that microgravity alters gene expression homeostasis not stronger than other environmental factors.

Genetic accommodation in the wild: evolution of gene expression plasticity during character displacement.

PubMed

Levis, N A; Serrato-Capuchina, A; Pfennig, D W

2017-09-01

Ecological character displacement is considered crucial in promoting diversification, yet relatively little is known of its underlying mechanisms. We examined whether evolutionary shifts in gene expression plasticity ('genetic accommodation') mediate character displacement in spadefoot toads. Where Spea bombifrons and S. multiplicata occur separately in allopatry (the ancestral condition), each produces alternative, diet-induced, larval ecomorphs: omnivores, which eat detritus, and carnivores, which specialize on shrimp. By contrast, where these two species occur together in sympatry (the derived condition), selection to minimize competition for detritus has caused S. bombifrons to become nearly fixed for producing only carnivores, suggesting that character displacement might have arisen through an extreme form of genetic accommodation ('genetic assimilation') in which plasticity is lost. Here, we asked whether we could infer a signature of this process in regulatory changes of specific genes. In particular, we investigated whether genes that are normally expressed more highly in one morph ('biased' genes) have evolved reduced plasticity in expression levels among S. bombifrons from sympatry compared to S. bombifrons from allopatry. We reared individuals from sympatry vs. allopatry on detritus or shrimp and measured the reaction norms of nine biased genes. Although different genes displayed different patterns of gene regulatory evolution, the combined gene expression profiles revealed that sympatric individuals had indeed lost the diet-induced gene expression plasticity present in allopatric individuals. Our data therefore provide one of the few examples from natural populations in which genetic accommodation/assimilation can be traced to regulatory changes of specific genes. Such genetic accommodation might mediate character displacement in many systems. © 2017 European Society For Evolutionary Biology. Journal of Evolutionary Biology © 2017 European Society For Evolutionary Biology.

Gene expression changes in response to aging compared to heat stress, oxidative stress and ionizing radiation in Drosophila melanogaster.

PubMed

Landis, Gary; Shen, Jie; Tower, John

2012-11-01

Gene expression changes in response to aging, heat stress, hyperoxia, hydrogen peroxide, and ionizing radiation were compared using microarrays. A set of 18 genes were up-regulated across all conditions, indicating a general stress response shared with aging, including the heat shock protein (Hsp) genes Hsp70, Hsp83 and l(2)efl, the glutathione-S-transferase gene GstD2, and the mitochondrial unfolded protein response (mUPR) gene ref(2)P. Selected gene expression changes were confirmed using quantitative PCR, Northern analysis and GstD-GFP reporter constructs. Certain genes were altered in only a subset of the conditions, for example, up-regulation of numerous developmental pathway and signaling genes in response to hydrogen peroxide. While aging shared features with each stress, aging was more similar to the stresses most associated with oxidative stress (hyperoxia, hydrogen peroxide, ionizing radiation) than to heat stress. Aging is associated with down-regulation of numerous mitochondrial genes, including electron-transport-chain (ETC) genes and mitochondrial metabolism genes, and a sub-set of these changes was also observed upon hydrogen peroxide stress and ionizing radiation stress. Aging shared the largest number of gene expression changes with hyperoxia. The extensive down-regulation of mitochondrial and ETC genes during aging is consistent with an aging-associated failure in mitochondrial maintenance, which may underlie the oxidative stress-like and proteotoxic stress-like responses observed during aging.

Gene expression changes in response to aging compared to heat stress, oxidative stress and ionizing radiation in Drosophila melanogaster

PubMed Central

Landis, Gary; Shen, Jie; Tower, John

2012-01-01

Gene expression changes in response to aging, heat stress, hyperoxia, hydrogen peroxide, and ionizing radiation were compared using microarrays. A set of 18 genes were up-regulated across all conditions, indicating a general stress response shared with aging, including the heat shock protein (Hsp) genes Hsp70, Hsp83 and l(2)efl, the glutathione-S-transferase gene GstD2, and the mitochondrial unfolded protein response (mUPR) gene ref(2)P. Selected gene expression changes were confirmed using quantitative PCR, Northern analysis and GstD-GFP reporter constructs. Certain genes were altered in only a subset of the conditions, for example, up-regulation of numerous developmental pathway and signaling genes in response to hydrogen peroxide. While aging shared features with each stress, aging was more similar to the stresses most associated with oxidative stress (hyperoxia, hydrogen peroxide, ionizing radiation) than to heat stress. Aging is associated with down-regulation of numerous mitochondrial genes, including electron-transport-chain (ETC) genes and mitochondrial metabolism genes, and a sub-set of these changes was also observed upon hydrogen peroxide stress and ionizing radiation stress. Aging shared the largest number of gene expression changes with hyperoxia. The extensive down-regulation of mitochondrial and ETC genes during aging is consistent with an aging-associated failure in mitochondrial maintenance, which may underlie the oxidative stress-like and proteotoxic stress-like responses observed during aging. PMID:23211361

Diversity and expression of different forms of RubisCO genes in polluted groundwater under different redox conditions.

PubMed

Alfreider, Albin; Schirmer, Mario; Vogt, Carsten

2012-03-01

Groundwater polluted with methyl-tert-butyl ether (MTBE) and ammonium was investigated for chemolithoautotrophic CO(2) fixation capabilities based on detailed analyses of ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) large subunit genes. Samples retrieved from a groundwater conditioning unit, characterized by different redox conditions, were examined for the presence of form IA, form IC (cbbL) and form II (cbbM) RubisCO genes and transcripts obtained from DNA- and RNA-extracts. Form IA RubisCO sequences, which revealed a complex and distinct variety in different sampling stations, were expressed in the original groundwater and in samples amended with oxygen, but not in the aquifer groundwater enriched with nitrate. Form IC RubisCO genes were exclusively detected in groundwater supplied with oxygen and sequences were affiliated with cbbL genes in nitrifying bacteria. cbbM genes were not expressed in the oxygen-amended groundwater, probably due to the low CO(2) /O(2) substrate specificity of this enzyme. Most form II RubisCO transcripts were affiliated with RubisCO genes of denitrifiers, which are important residents in the groundwater supplied with nitrate. The distinct distribution pattern and diversity of RubisCO genes and transcripts obtained in this study suggest that the induction of different RubisCO enzymes is highly regulated and closely linked to the actual environmental conditions. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Time-Course Gene Set Analysis for Longitudinal Gene Expression Data

PubMed Central

Hejblum, Boris P.; Skinner, Jason; Thiébaut, Rodolphe

2015-01-01

Gene set analysis methods, which consider predefined groups of genes in the analysis of genomic data, have been successfully applied for analyzing gene expression data in cross-sectional studies. The time-course gene set analysis (TcGSA) introduced here is an extension of gene set analysis to longitudinal data. The proposed method relies on random effects modeling with maximum likelihood estimates. It allows to use all available repeated measurements while dealing with unbalanced data due to missing at random (MAR) measurements. TcGSA is a hypothesis driven method that identifies a priori defined gene sets with significant expression variations over time, taking into account the potential heterogeneity of expression within gene sets. When biological conditions are compared, the method indicates if the time patterns of gene sets significantly differ according to these conditions. The interest of the method is illustrated by its application to two real life datasets: an HIV therapeutic vaccine trial (DALIA-1 trial), and data from a recent study on influenza and pneumococcal vaccines. In the DALIA-1 trial TcGSA revealed a significant change in gene expression over time within 69 gene sets during vaccination, while a standard univariate individual gene analysis corrected for multiple testing as well as a standard a Gene Set Enrichment Analysis (GSEA) for time series both failed to detect any significant pattern change over time. When applied to the second illustrative data set, TcGSA allowed the identification of 4 gene sets finally found to be linked with the influenza vaccine too although they were found to be associated to the pneumococcal vaccine only in previous analyses. In our simulation study TcGSA exhibits good statistical properties, and an increased power compared to other approaches for analyzing time-course expression patterns of gene sets. The method is made available for the community through an R package. PMID:26111374

Identification of Suitable Reference Genes for Gene Expression Normalization in qRT-PCR Analysis in Watermelon

PubMed Central

Gao, Lingyun; Zhao, Shuang; Jiang, Wei; Huang, Yuan; Bie, Zhilong

2014-01-01

Watermelon is one of the major Cucurbitaceae crops and the recent availability of genome sequence greatly facilitates the fundamental researches on it. Quantitative real-time reverse transcriptase PCR (qRT–PCR) is the preferred method for gene expression analyses, and using validated reference genes for normalization is crucial to ensure the accuracy of this method. However, a systematic validation of reference genes has not been conducted on watermelon. In this study, transcripts of 15 candidate reference genes were quantified in watermelon using qRT–PCR, and the stability of these genes was compared using geNorm and NormFinder. geNorm identified ClTUA and ClACT, ClEF1α and ClACT, and ClCAC and ClTUA as the best pairs of reference genes in watermelon organs and tissues under normal growth conditions, abiotic stress, and biotic stress, respectively. NormFinder identified ClYLS8, ClUBCP, and ClCAC as the best single reference genes under the above experimental conditions, respectively. ClYLS8 and ClPP2A were identified as the best reference genes across all samples. Two to nine reference genes were required for more reliable normalization depending on the experimental conditions. The widely used watermelon reference gene 18SrRNA was less stable than the other reference genes under the experimental conditions. Catalase family genes were identified in watermelon genome, and used to validate the reliability of the identified reference genes. ClCAT1and ClCAT2 were induced and upregulated in the first 24 h, whereas ClCAT3 was downregulated in the leaves under low temperature stress. However, the expression levels of these genes were significantly overestimated and misinterpreted when 18SrRNA was used as a reference gene. These results provide a good starting point for reference gene selection in qRT–PCR analyses involving watermelon. PMID:24587403

Identification of suitable reference genes for gene expression normalization in qRT-PCR analysis in watermelon.

PubMed

Kong, Qiusheng; Yuan, Jingxian; Gao, Lingyun; Zhao, Shuang; Jiang, Wei; Huang, Yuan; Bie, Zhilong

2014-01-01

Watermelon is one of the major Cucurbitaceae crops and the recent availability of genome sequence greatly facilitates the fundamental researches on it. Quantitative real-time reverse transcriptase PCR (qRT-PCR) is the preferred method for gene expression analyses, and using validated reference genes for normalization is crucial to ensure the accuracy of this method. However, a systematic validation of reference genes has not been conducted on watermelon. In this study, transcripts of 15 candidate reference genes were quantified in watermelon using qRT-PCR, and the stability of these genes was compared using geNorm and NormFinder. geNorm identified ClTUA and ClACT, ClEF1α and ClACT, and ClCAC and ClTUA as the best pairs of reference genes in watermelon organs and tissues under normal growth conditions, abiotic stress, and biotic stress, respectively. NormFinder identified ClYLS8, ClUBCP, and ClCAC as the best single reference genes under the above experimental conditions, respectively. ClYLS8 and ClPP2A were identified as the best reference genes across all samples. Two to nine reference genes were required for more reliable normalization depending on the experimental conditions. The widely used watermelon reference gene 18SrRNA was less stable than the other reference genes under the experimental conditions. Catalase family genes were identified in watermelon genome, and used to validate the reliability of the identified reference genes. ClCAT1and ClCAT2 were induced and upregulated in the first 24 h, whereas ClCAT3 was downregulated in the leaves under low temperature stress. However, the expression levels of these genes were significantly overestimated and misinterpreted when 18SrRNA was used as a reference gene. These results provide a good starting point for reference gene selection in qRT-PCR analyses involving watermelon.

Genome-Wide Identification and Testing of Superior Reference Genes for Transcript Normalization in Arabidopsis1[w

PubMed Central

Czechowski, Tomasz; Stitt, Mark; Altmann, Thomas; Udvardi, Michael K.; Scheible, Wolf-Rüdiger

2005-01-01

Gene transcripts with invariant abundance during development and in the face of environmental stimuli are essential reference points for accurate gene expression analyses, such as RNA gel-blot analysis or quantitative reverse transcription-polymerase chain reaction (PCR). An exceptionally large set of data from Affymetrix ATH1 whole-genome GeneChip studies provided the means to identify a new generation of reference genes with very stable expression levels in the model plant species Arabidopsis (Arabidopsis thaliana). Hundreds of Arabidopsis genes were found that outperform traditional reference genes in terms of expression stability throughout development and under a range of environmental conditions. Most of these were expressed at much lower levels than traditional reference genes, making them very suitable for normalization of gene expression over a wide range of transcript levels. Specific and efficient primers were developed for 22 genes and tested on a diverse set of 20 cDNA samples. Quantitative reverse transcription-PCR confirmed superior expression stability and lower absolute expression levels for many of these genes, including genes encoding a protein phosphatase 2A subunit, a coatomer subunit, and an ubiquitin-conjugating enzyme. The developed PCR primers or hybridization probes for the novel reference genes will enable better normalization and quantification of transcript levels in Arabidopsis in the future. PMID:16166256

Transcriptome database resource and gene expression atlas for the rose

PubMed Central

2012-01-01

Background For centuries roses have been selected based on a number of traits. Little information exists on the genetic and molecular basis that contributes to these traits, mainly because information on expressed genes for this economically important ornamental plant is scarce. Results Here, we used a combination of Illumina and 454 sequencing technologies to generate information on Rosa sp. transcripts using RNA from various tissues and in response to biotic and abiotic stresses. A total of 80714 transcript clusters were identified and 76611 peptides have been predicted among which 20997 have been clustered into 13900 protein families. BLASTp hits in closely related Rosaceae species revealed that about half of the predicted peptides in the strawberry and peach genomes have orthologs in Rosa dataset. Digital expression was obtained using RNA samples from organs at different development stages and under different stress conditions. qPCR validated the digital expression data for a selection of 23 genes with high or low expression levels. Comparative gene expression analyses between the different tissues and organs allowed the identification of clusters that are highly enriched in given tissues or under particular conditions, demonstrating the usefulness of the digital gene expression analysis. A web interface ROSAseq was created that allows data interrogation by BLAST, subsequent analysis of DNA clusters and access to thorough transcript annotation including best BLAST matches on Fragaria vesca, Prunus persica and Arabidopsis. The rose peptides dataset was used to create the ROSAcyc resource pathway database that allows access to the putative genes and enzymatic pathways. Conclusions The study provides useful information on Rosa expressed genes, with thorough annotation and an overview of expression patterns for transcripts with good accuracy. PMID:23164410

Emiliania huxleyi increases calcification but not expression of calcification-related genes in long-term exposure to elevated temperature and pCO2.

PubMed

Benner, Ina; Diner, Rachel E; Lefebvre, Stephane C; Li, Dian; Komada, Tomoko; Carpenter, Edward J; Stillman, Jonathon H

2013-01-01

Increased atmospheric pCO2 is expected to render future oceans warmer and more acidic than they are at present. Calcifying organisms such as coccolithophores that fix and export carbon into the deep sea provide feedbacks to increasing atmospheric pCO2. Acclimation experiments suggest negative effects of warming and acidification on coccolithophore calcification, but the ability of these organisms to adapt to future environmental conditions is not well understood. Here, we tested the combined effect of pCO2 and temperature on the coccolithophore Emiliania huxleyi over more than 700 generations. Cells increased inorganic carbon content and calcification rate under warm and acidified conditions compared with ambient conditions, whereas organic carbon content and primary production did not show any change. In contrast to findings from short-term experiments, our results suggest that long-term acclimation or adaptation could change, or even reverse, negative calcification responses in E. huxleyi and its feedback to the global carbon cycle. Genome-wide profiles of gene expression using RNA-seq revealed that genes thought to be essential for calcification are not those that are most strongly differentially expressed under long-term exposure to future ocean conditions. Rather, differentially expressed genes observed here represent new targets to study responses to ocean acidification and warming.

Selection of suitable soybean EF1α genes as internal controls for real-time PCR analyses of tissues during plant development and under stress conditions.

PubMed

Saraiva, Kátia D C; Fernandes de Melo, Dirce; Morais, Vanessa D; Vasconcelos, Ilka M; Costa, José H

2014-09-01

The EF1α genes were stable in the large majority of soybean tissues during development and in specific tissues/conditions under stress. Quantitative real-time PCR (qPCR) analysis strongly depends on transcript normalization using stable reference genes. Reference genes are generally encoded by multigene families and are used in qPCR normalization; however, little effort has been made to verify the stability of different gene members within a family. Here, the expression stability of members of the soybean EF1α gene family (named EF1α 1a1, 1a2, 1b, 2a, 2b and 3) was evaluated in different tissues during plant development and stress exposure (SA and PEG). Four genes (UKN1, SKIP 16, EF1β and MTP) already established as stably expressed were also used in the comparative analysis. GeNorm analyses revealed different combinations of reference genes as stable in soybean tissues during development. The EF1α genes were the most stable in cotyledons (EF1α 3 and EF1α 1b), epicotyls (EF1α 1a2, EF1α 2b and EF1α 1a1), hypocotyls (EF1α 1a1 and EF1β), pods (EF1α 2a and EF1α 2b) and roots (EF1α 2a and UKN1) and less stable in tissues such as trifoliate and unifoliate leaves and germinating seeds. Under stress conditions, no suitable combination including only EF1α genes was found; however, some genes were relatively stable in leaves (EF1α 1a2) and roots (EF1α 1a1) treated with SA as well as in roots treated with PEG (EF1α 2b). EF1α 2a was the most stably expressed EF1α gene in all soybean tissues under stress. Taken together, our data provide guidelines for the selection of EF1α genes for use as reference genes in qPCR expression analyses during plant development and under stress conditions.

Temperature and Oxidative Stress as Triggers for Virulence Gene Expression in Pathogenic Leptospira spp.

PubMed Central

Fraser, Tricia; Brown, Paul D.

2017-01-01

Leptospirosis is a zooanthroponosis aetiologically caused by pathogenic bacteria belonging to the genus, Leptospira. Environmental signals such as increases in temperatures or oxidative stress can trigger response regulatory modes of virulence genes during infection. This study sought to determine the effect of temperature and oxidative stress on virulence associated genes in highly passaged Leptospira borgpeterseneii Jules and L. interrogans Portlandvere. Bacteria were grown in EMJH at 30°C, 37°C, or at 30°C before being transferred to 37°C. A total of 14 virulence-associated genes (fliY, invA, lenA, ligB, lipL32, lipL36, lipL41, lipL45, loa22, lsa21, mce, ompL1, sph2, and tlyC) were assessed using endpoint PCR. Transcriptional analyses of lenA, lipL32, lipL41, loa22, sph2 were assessed by quantitative real-time RT-PCR at the temperature conditions. To assess oxidative stress, bacteria were exposed to H2O2 for 30 and 60 min with or without the temperature stress. All genes except ligB (for Portlandvere) and ligB and mce (for Jules) were detectable in the strains. Quantitatively, temperature stress resulted in significant changes in gene expression within species or between species. Temperature changes were more influential in gene expression for Jules, particularly at 30°C and upshift conditions; at 37°C, expression levels were higher for Portlandvere. However, compared to Jules, where temperature was influential in two of five genes, temperature was an essential element in four of five genes in Portlandvere exposed to oxidative stress. At both low and high oxidative stress levels, the interplay between genetic predisposition (larger genome size) and temperature was biased towards Portlandvere particularly at 30°C and upshift conditions. While it is clear that expression of many virulence genes in highly passaged strains of Leptospira are attenuated or lost, genetic predisposition, changes in growth temperature and/or oxidative intensity and/or duration were factors which acted in isolation or together with other regulatory cues to contribute to the variable gene expression observed in this study. Overall, differential gene expression in serovar Portlandvere was more responsive to temperature and oxidative stress. PMID:28536558

Plasticity in the Rat Prefrontal Cortex: Linking Gene Expression and an Operant Learning with a Computational Theory

PubMed Central

Rapanelli, Maximiliano; Lew, Sergio Eduardo; Frick, Luciana Romina; Zanutto, Bonifacio Silvano

2010-01-01

The plasticity in the medial Prefrontal Cortex (mPFC) of rodents or lateral prefrontal cortex in non human primates (lPFC), plays a key role neural circuits involved in learning and memory. Several genes, like brain-derived neurotrophic factor (BDNF), cAMP response element binding (CREB), Synapsin I, Calcium/calmodulin-dependent protein kinase II (CamKII), activity-regulated cytoskeleton-associated protein (Arc), c-jun and c-fos have been related to plasticity processes. We analysed differential expression of related plasticity genes and immediate early genes in the mPFC of rats during learning an operant conditioning task. Incompletely and completely trained animals were studied because of the distinct events predicted by our computational model at different learning stages. During learning an operant conditioning task, we measured changes in the mRNA levels by Real-Time RT-PCR during learning; expression of these markers associated to plasticity was incremented while learning and such increments began to decline when the task was learned. The plasticity changes in the lPFC during learning predicted by the model matched up with those of the representative gene BDNF. Herein, we showed for the first time that plasticity in the mPFC in rats during learning of an operant conditioning is higher while learning than when the task is learned, using an integrative approach of a computational model and gene expression. PMID:20111591

Production of human interferon alfa 2b in plants of Nicotiana excelsior by Agrobacterium-mediated transient expression.

PubMed

Sindarovska, Y R; Gerasymenko, I M; Sheludko, Y V; Olevinskaya, Z M; Spivak, N Y; Kuchuk, N V

2010-01-01

Human interferon alpha2b gene was transiently expressed in Nicotiana excelsior plants. Fusion with N. plumbaginifolia calreticulin signal peptide for improved apoplast targeting and carrying out the expression under optimized conditions resulted in maximal interferon activity of 3.2 x 10(3) IU/g fresh weight (FW) with an average of 2.1 +/- 0.8 x 10(3) IU/g FW. It proves that N. excelsior is a suitable host for Agrobacterium-mediated transient expression of genes encoding physiologically active human proteins. The transient expression conditions optimized for GFP marker protein were confirmed to be preferable for hIFN alpha2b.

Determination of internal controls for quantitative real time RT-PCR analysis of the effect of Edwardsiella tarda infection on gene expression in turbot (Scophthalmus maximus).

PubMed

Dang, Wei; Sun, Li

2011-02-01

In recent years, quantitative real time reverse transcriptase-PCR (qRT-PCR) has been used frequently in the study of gene expression in turbot (Scophthalmus maximus) in relation to bacterial infection. However, no investigations on appropriate qRT-PCR reference genes have been documented. In this report, we determined the potential of eight housekeeping genes, i.e. β-actin (ACTB), ribosomal protein L17 (RPL17), α-tubulin (TUBA), elongation factor-1-α(EF1A), β-2-Microglobulin (B2M), RNA polymerase II subunit D (RPSD), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and 18S ribosomal RNA (18S rRNA), as internal standards for qRT-PCR analysis of gene expression in turbot as a function of bacterial infection. For this purpose, the expression of the eight housekeeping genes in seven turbot tissues was determined by qRT-PCR before and after bacterial challenge, and the data were analyzed with the geNorm and NormFinder algorisms. The results showed that the expression of all the examined genes exhibited tissue-dependent variations both before and after bacterial challenge. Before bacterial challenge, geNorm and NormFinder identified RPSD as the gene that showed least tissue specific expression. At 12 h post-bacterial infection, geNorm ranked ACTB/GAPDH, 18S rRNA/ACTB, ACTB/GAPDH, 18S rRNA/ACTB, RPL17/TUBA, RPSD/GAPDH, and RPSD/B2M, respectively, as the most stably expressed genes in liver, spleen, kidney, gill, heart, muscle, and brain. Comparable ranking orders were produced by NormFinder. Similar results were obtained at 24 h post-bacterial infection. Taken together, these results indicate that RPSD is the most stable gene across tissue types under normal physiological conditions and that, during bacterial infection, ACTB might be used as an internal standard for the normalization of gene expression in immune relevant organs; however, no single gene or single pair of genes in the examined set of housekeeping genes can serve as a universal reference across all tissue types under the condition of bacterial infection. Copyright © 2011 Elsevier Ltd. All rights reserved.

Cafeteria diet overfeeding in young male rats impairs the adaptive response to fed/fasted conditions and increases adiposity independent of body weight.

PubMed

Castro, H; Pomar, C A; Picó, C; Sánchez, J; Palou, A

2015-03-01

We analyzed the effects of a short exposure to a cafeteria diet during early infancy in rats on their metabolic response to fed/fasting conditions in key tissues involved in energy homeostasis. Ten-day-old male pups were fed a control or a cafeteria diet for 12 days and then killed under ad libitum feeding conditions or 12 h fasting. The expression of key genes related to energy metabolism in liver, retroperitoneal white adipose tissue (WAT) and hypothalamus were analyzed. Despite no differences in body weight, cafeteria-fed animals had almost double the fat mass of control rats. They also showed higher food intake, higher leptinemia and altered hypothalamic expression of Neuropetide Y, suggesting a dysfunction in the control of food intake. Unlike controls, cafeteria-fed animals did not decrease WAT expression of Pparg, sterol regulatory element binding transcription factor 1 or Cidea under fasting conditions, and displayed lower Pnpla2 expression than controls. In liver, compared with controls, cafeteria animals presented: (i) lower expression of genes related with fatty acid uptake and lipogenesis under ad libitum-fed conditions; (ii) higher expression of fatty acid oxidation-related genes and glucokinase under fasting conditions; (iii) greater expression of leptin and insulin receptors; and higher protein levels of insulin receptor and the pAMPK/AMPK ratio. A short period of exposure to a cafeteria diet in early infancy in rat pups is enough to disturb the metabolic response to fed/fasting conditions in key tissues involved in energy homeostasis, particularly in WAT, and hence induces an exacerbated body fat accumulation and increased metabolic risk, with no apparent effects on body weight.

Transcriptome analysis of Haloquadratum walsbyi: vanity is but the surface.

PubMed

Bolhuis, Henk; Martín-Cuadrado, Ana Belén; Rosselli, Riccardo; Pašić, Lejla; Rodriguez-Valera, Francisco

2017-07-03

Haloquadratum walsbyi dominates saturated thalassic lakes worldwide where they can constitute up to 80-90% of the total prokaryotic community. Despite the abundance of the enigmatic square-flattened cells, only 7 isolates are currently known with 2 genomes fully sequenced and annotated due to difficulties to grow them under laboratory conditions. We have performed a transcriptomic analysis of one of these isolates, the Spanish strain HBSQ001 in order to investigate gene transcription under light and dark conditions. Despite a potential advantage for light as additional source of energy, no significant differences were found between light and dark expressed genes. Constitutive high gene expression was observed in genes encoding surface glycoproteins, light mediated proton pumping by bacteriorhodopsin, several nutrient uptake systems, buoyancy and storage of excess carbon. Two low expressed regions of the genome were characterized by a lower codon adaptation index, low GC content and high incidence of hypothetical genes. Under the extant cultivation conditions, the square hyperhalophile devoted most of its transcriptome towards processes maintaining cell integrity and exploiting solar energy. Surface glycoproteins are essential for maintaining the large surface to volume ratio that facilitates light and organic nutrient harvesting whereas constitutive expression of bacteriorhodopsin warrants an immediate source of energy when light becomes available.

Detection of changes in gene regulatory patterns, elicited by perturbations of the Hsp90 molecular chaperone complex, by visualizing multiple experiments with an animation

PubMed Central

2011-01-01

Background To make sense out of gene expression profiles, such analyses must be pushed beyond the mere listing of affected genes. For example, if a group of genes persistently display similar changes in expression levels under particular experimental conditions, and the proteins encoded by these genes interact and function in the same cellular compartments, this could be taken as very strong indicators for co-regulated protein complexes. One of the key requirements is having appropriate tools to detect such regulatory patterns. Results We have analyzed the global adaptations in gene expression patterns in the budding yeast when the Hsp90 molecular chaperone complex is perturbed either pharmacologically or genetically. We integrated these results with publicly accessible expression, protein-protein interaction and intracellular localization data. But most importantly, all experimental conditions were simultaneously and dynamically visualized with an animation. This critically facilitated the detection of patterns of gene expression changes that suggested underlying regulatory networks that a standard analysis by pairwise comparison and clustering could not have revealed. Conclusions The results of the animation-assisted detection of changes in gene regulatory patterns make predictions about the potential roles of Hsp90 and its co-chaperone p23 in regulating whole sets of genes. The simultaneous dynamic visualization of microarray experiments, represented in networks built by integrating one's own experimental with publicly accessible data, represents a powerful discovery tool that allows the generation of new interpretations and hypotheses. PMID:21672238

Short-term exposure of Arabidopsis cell culures to hyper-G: Short-term changes in transcription regulation expression

NASA Astrophysics Data System (ADS)

Babbick, Maren; Hampp, Rudiger

2005-08-01

Callus cultures of Arabidopsis thaliana (cv. Columbia) were used to screen for early changes in gene expression in response to altered gravitational fields. In a recent microarray study we found hyper- g dependent changes in gene expression which indicated the involvement of WRKY genes [Martzivanou M. and Hampp R., Physiol. Plant., 118, 221-231,2003]. WRKY genes code for a family of plant-specific regulators of gene expression. In this study we report on the exposure of Arabidopsis callus cultures to 8g for up to 30 min. Quantitative analysis by real time RT-PCR of the amount of transcripts of WRKYs 3, 6, 22, 46, 65 and 70 showed individual changes in expression. As far as their function is known, these WRKY proteins are mainly involved in stress responses. As most alterations in transcript amount occurred within 10 min of treatment, such genes can be used for the investigation of microgravity-related effects on gene expression under sounding rocket conditions (TEXUS, MAXUS).

Identification and Evaluation of Reliable Reference Genes in the Medicinal Fungus Shiraia bambusicola.

PubMed

Song, Liang; Li, Tong; Fan, Li; Shen, Xiao-Ye; Hou, Cheng-Lin

2016-04-01

The stability of reference genes plays a vital role in real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis, which is generally regarded as a convenient and sensitive tool for the analysis of gene expression. A well-known medicinal fungus, Shiraia bambusicola, has great potential in the pharmaceutical, agricultural and food industries, but its suitable reference genes have not yet been determined. In the present study, 11 candidate reference genes in S. bambusicola were first evaluated and validated comprehensively. To identify the suitable reference genes for qRT-PCR analysis, three software-based algorithms, geNorm, NormFinder and Best Keeper, were applied to rank the tested genes. RNA samples were collected from seven fermentation stages using different media (potato dextrose or Czapek medium) and under different light conditions (12-h light/12-h dark and all-dark). The three most appropriate reference genes, ubi, tfc and ags, were able to normalize the qRT-PCR results under the culturing conditions of 12-h light/12-h dark, whereas the other three genes, vac, gke and acyl, performed better in the culturing conditions of all-dark growth. Therefore, under different light conditions, at least two reference genes (ubi and vac) could be employed to assure the reliability of qRT-PCR results. For both the natural culture medium (the most appropriate genes of this group: ubi, tfc and ags) and the chemically defined synthetic medium (the most stable genes of this group: tfc, vac and ef), the tfc gene remained the best gene used for normalizing the gene expression found with qRT-PCR. It is anticipated that these results would improve the selection of suitable reference genes for qRT-PCR assays and lay the foundation for an accurate analysis of gene expression in S. bambusicola.

Promoter sequence of 3-phosphoglycerate kinase gene 1 of lactic acid-producing fungus rhizopus oryzae and a method of expressing a gene of interest in fungal species

DOEpatents

Gao, Johnway [Richland, WA; Skeen, Rodney S [Pendleton, OR

2002-10-15

The present invention provides the promoter clone discovery of phosphoglycerate kinase gene 1 of a lactic acid-producing filamentous fungal strain, Rhizopus oryzae. The isolated promoter can constitutively regulate gene expression under various carbohydrate conditions. In addition, the present invention also provides a design of an integration vector for the transformation of a foreign gene in Rhizopus oryzae.

Promoter sequence of 3-phosphoglycerate kinase gene 2 of lactic acid-producing fungus rhizopus oryzae and a method of expressing a gene of interest in fungal species

DOEpatents

Gao, Johnway [Richland, WA; Skeen, Rodney S [Pendleton, OR

2003-03-04

The present invention provides the promoter clone discovery of phosphoglycerate kinase gene 2 of a lactic acid-producing filamentous fungal strain, Rhizopus oryzae. The isolated promoter can constitutively regulate gene expression under various carbohydrate conditions. In addition, the present invention also provides a design of an integration vector for the transformation of a foreign gene in Rhizopus oryzae.

Characterizing differential gene expression in polyploid grasses lacking a reference transcriptome

USDA-ARS?s Scientific Manuscript database

Basal transcriptome characterization and differential gene expression in response to varying conditions are often addressed through next generation sequencing (NGS) and data analysis techniques. While these strategies are commonly used, there are countless tools, pipelines, data analysis methods an...

Microarray evaluation of gene expression profiles in inflamed and healthy human dental pulp: the role of IL1beta and CD40 in pulp inflammation.

PubMed

Gatta, V; Zizzari, V L; Dd ' Amico, V; Salini, L; D' Aurora, M; Franchi, S; Antonucci, I; Sberna, M T; Gherlone, E; Stuppia, L; Tetè, S

2012-01-01

Dental pulp undergoes a number of changes passing from healthy status to inflammation due to deep decay. These changes are regulated by several genes resulting differently expressed in inflamed and healthy dental pulp, and the knowledge of the processes underlying this differential expression is of great relevance in the identification of the pathogenesis of the disease. In this study, the gene expression profile of inflamed and healthy dental pulps were compared by microarray analysis, and data obtained were analyzed by Ingenuity Pathway Analysis (IPA) software. This analysis allows to focus on a variety of genes, typically expressed in inflamed tissues. The comparison analysis showed an increased expression of several genes in inflamed pulp, among which IL1β and CD40 resulted of particular interest. These results indicate that gene expression profile of human dental pulp in different physiological and pathological conditions may become an useful tool for improving our knowledge about processes regulating pulp inflammation.

Transcriptome dynamics during the transition from anaerobic photosynthesis to aerobic respiration in Rhodobacter sphaeroides 2.4.1.

PubMed

Arai, Hiroyuki; Roh, Jung Hyeob; Kaplan, Samuel

2008-01-01

Rhodobacter sphaeroides 2.4.1 is a facultative photosynthetic anaerobe that grows by anoxygenic photosynthesis under anaerobic-light conditions. Changes in energy generation pathways under photosynthetic and aerobic respiratory conditions are primarily controlled by oxygen tensions. In this study, we performed time series microarray analyses to investigate transcriptome dynamics during the transition from anaerobic photosynthesis to aerobic respiration. Major changes in gene expression profiles occurred in the initial 15 min after the shift from anaerobic-light to aerobic-dark conditions, with changes continuing to occur up to 4 hours postshift. Those genes whose expression levels changed significantly during the time series were grouped into three major classes by clustering analysis. Class I contained genes, such as that for the aa3 cytochrome oxidase, whose expression levels increased after the shift. Class II contained genes, such as those for the photosynthetic apparatus and Calvin cycle enzymes, whose expression levels decreased after the shift. Class III contained genes whose expression levels temporarily increased during the time series. Many genes for metabolism and transport of carbohydrates or lipids were significantly induced early during the transition, suggesting that those endogenous compounds were initially utilized as carbon sources. Oxidation of those compounds might also be required for maintenance of redox homeostasis after exposure to oxygen. Genes for the repair of protein and sulfur groups and uptake of ferric iron were temporarily upregulated soon after the shift, suggesting they were involved in a response to oxidative stress. The flagellar-biosynthesis genes were expressed in a hierarchical manner at 15 to 60 min after the shift. Numerous transporters were induced at various time points, suggesting that the cellular composition went through significant changes during the transition from anaerobic photosynthesis to aerobic respiration. Analyses of these data make it clear that numerous regulatory activities come into play during the transition from one homeostatic state to another.

IRE1 inhibition affects the expression of insulin-like growth factor binding protein genes and modifies its sensitivity to glucose deprivation in U87 glioma cells.

PubMed

Minchenko, D O; Kharkova, A P; Tsymbal, D O; Karbovskyi, L L; Minchenko, O H

2015-10-01

The aim of the present study was to investigate the effect of inhibition of endoplasmic reticulum stress signaling mediated by IRE1/ERN1 (inositol-requiring enzyme 1/endoplasmic reticulum to nucleus signaling 1) on the expression of genes encoding different groups of insulin-like growth binding proteins (IGFBP6 and IGFBP7) and CCN family (IGFBP8/CTGF/CCN2, IGFBP9/NOV/CCN3, IGFBP10/CYR61/CCN1, WISP1/CCN4, and WISP2/CCN5) and its sensitivity to glucose deprivation in U87 glioma cells. The expression of IGFBP6, IGFBP7, IGFBP8, IGFBP9, IGFBP10, WISP1, and WISP2 genes was studied by qPCR in control U87 glioma cells (wild-type) and its subline with IRE1 signaling enzyme loss of function upon glucose deprivation. The expression of IGFBP8, IGFBP9, and WISP2 genes was up-regulated in control glioma cells upon glucose deprivation with most significant changes for IGFBP9 gene. At the same time, the expression of IGFBP6, IGFBP10, and WISP1 genes was resistant to glucose deprivation in these glioma cells, but the IGFBP7 gene expression was down-regulated. The inhibition of both enzymatic activities (kinase and endoribonuclease) of IRE1 in glioma cells modified the sensitivity of most studied gene expressions to glucose deprivation condition: introduced sensitivity of IGFBP10 and WISP1 genes to glucose deprivation, enhanced the effect of this deprivation on IGFBP7 and IGFBP9 gene expressions, and reduced this effect on WISP2 gene and induced suppressive effect of glucose deprivation on the expression of IGFBP8 gene. Furthermore, the inhibition of IRE1 strongly affected the expression of all studied genes in glioma cells upon regular growing condition in gene specific manner: up-regulated the expression levels of IGFBP7, IGFBP8, IGFBP10, WISP1, and WISP2 genes and down-regulated the IGFBP6 and IGFBP9 genes. The data of this investigation demonstrate that the expression of IGFBP7, IGFBP8, IGFBP9, and WISP2 genes are sensitive to glucose deprivation in U87 glioma cells and that inhibition of IRE1 signaling enzyme function may significantly affect the expression of all studied genes in the presence of glucose as well as modify the effect of glucose deprivation on the expression of most studied genes. These data also show that proteins encoded by these genes may participate in the regulation of metabolic and proliferative processes via IGF/INS receptors and possibly other signaling pathways as well, via IRE1 signaling, which is a central mediator of the unfolded protein response and an important component of the tumor growth and metabolic diseases.

Extensive Analysis of GmFTL and GmCOL Expression in Northern Soybean Cultivars in Field Conditions.

PubMed

Guo, Guangyu; Xu, Kun; Zhang, Xiaomei; Zhu, Jinlong; Lu, Mingyang; Chen, Fulu; Liu, Linpo; Xi, Zhang-Ying; Bachmair, Andreas; Chen, Qingshan; Fu, Yong-Fu

2015-01-01

The FLOWERING LOCUS T (FT) gene is a highly conserved florigen gene among flowering plants. Soybean genome encodes six homologs of FT, which display flowering activity in Arabidopsis thaliana. However, their contributions to flowering time in different soybean cultivars, especially in field conditions, are unclear. We employed six soybean cultivars with different maturities to extensively investigate expression patterns of GmFTLs (Glycine max FT-like) and GmCOLs (Glycine max CO-like) in the field conditions. The results show that GmFTL3 is an FT homolog with the highest transcript abundance in soybean, but other GmFTLs may also contribute to flower induction with different extents, because they have more or less similar expression patterns in developmental-, leaf-, and circadian-specific modes. And four GmCOL genes (GmCOL1/2/5/13) may confer to the expression of GmFTL genes. Artificial manipulation of GmFTL expression by transgenic strategy (overexpression and RNAi) results in a distinct change in soybean flowering time, indicating that GmFTLs not only impact on the control of flowering time, but have potential applications in the manipulation of photoperiodic adaptation in soybean. Additionally, transgenic plants show that GmFTLs play a role in formation of the first flowers and in vegetative growth.

Extensive Analysis of GmFTL and GmCOL Expression in Northern Soybean Cultivars in Field Conditions

PubMed Central

Zhu, Jinlong; Lu, Mingyang; Chen, Fulu; Liu, Linpo; Xi, Zhang-Ying; Bachmair, Andreas; Chen, Qingshan; Fu, Yong-Fu

2015-01-01

The FLOWERING LOCUS T (FT) gene is a highly conserved florigen gene among flowering plants. Soybean genome encodes six homologs of FT, which display flowering activity in Arabidopsis thaliana. However, their contributions to flowering time in different soybean cultivars, especially in field conditions, are unclear. We employed six soybean cultivars with different maturities to extensively investigate expression patterns of GmFTLs (Glycine max FT-like) and GmCOLs (Glycine max CO-like) in the field conditions. The results show that GmFTL3 is an FT homolog with the highest transcript abundance in soybean, but other GmFTLs may also contribute to flower induction with different extents, because they have more or less similar expression patterns in developmental-, leaf-, and circadian-specific modes. And four GmCOL genes (GmCOL1/2/5/13) may confer to the expression of GmFTL genes. Artificial manipulation of GmFTL expression by transgenic strategy (overexpression and RNAi) results in a distinct change in soybean flowering time, indicating that GmFTLs not only impact on the control of flowering time, but have potential applications in the manipulation of photoperiodic adaptation in soybean. Additionally, transgenic plants show that GmFTLs play a role in formation of the first flowers and in vegetative growth. PMID:26371882

Global Transcriptional Responses to Osmotic, Oxidative, and Imipenem Stress Conditions in Pseudomonas putida.

PubMed

Bojanovič, Klara; D'Arrigo, Isotta; Long, Katherine S

2017-04-01

Bacteria cope with and adapt to stress by modulating gene expression in response to specific environmental cues. In this study, the transcriptional response of Pseudomonas putida KT2440 to osmotic, oxidative, and imipenem stress conditions at two time points was investigated via identification of differentially expressed mRNAs and small RNAs (sRNAs). A total of 440 sRNA transcripts were detected, of which 10% correspond to previously annotated sRNAs, 40% to novel intergenic transcripts, and 50% to novel transcripts antisense to annotated genes. Each stress elicits a unique response as far as the extent and dynamics of the transcriptional changes. Nearly 200 protein-encoding genes exhibited significant changes in all stress types, implicating their participation in a general stress response. Almost half of the sRNA transcripts were differentially expressed under at least one condition, suggesting possible functional roles in the cellular response to stress conditions. The data show a larger fraction of differentially expressed sRNAs than of mRNAs with >5-fold expression changes. The work provides detailed insights into the mechanisms through which P. putida responds to different stress conditions and increases understanding of bacterial adaptation in natural and industrial settings. IMPORTANCE This study maps the complete transcriptional response of P. putida KT2440 to osmotic, oxidative, and imipenem stress conditions at short and long exposure times. Over 400 sRNA transcripts, consisting of both intergenic and antisense transcripts, were detected, increasing the number of identified sRNA transcripts in the strain by a factor of 10. Unique responses to each type of stress are documented, including both the extent and dynamics of the gene expression changes. The work adds rich detail to previous knowledge of stress response mechanisms due to the depth of the RNA sequencing data. Almost half of the sRNAs exhibit significant expression changes under at least one condition, suggesting their involvement in adaptation to stress conditions and identifying interesting candidates for further functional characterization. Copyright © 2017 American Society for Microbiology.

The in vitro maintenance of clock genes expression within the rat pineal gland under standard and norepinephrine-synchronized stimulation.

PubMed

Andrade-Silva, Jéssica; Cipolla-Neto, José; Peliciari-Garcia, Rodrigo A

2014-01-01

Although the norepinephrine (NE) synchronization protocol was proved to be an important procedure for further modulating in vitro pineal melatonin synthesis, the maintenance of clock genes under the same conditions remained to be investigated. The aim of this study was to investigate the maintenance of the clock genes expression in pineal gland cultures under standard and NE-synchronized stimulation. The glands were separated into three experimental groups: Control, Standard (acute NE-stimulation), and NE-synchronized. The expression of Bmal1, Per2, Cry2, Rev-erbα, the clock controlled gene Dbp and Arylalkylamine-N-acetyltransferase were investigated, as well as melatonin content. No oscillations were observed in the expression of the investigated genes from the control group. Under Standard NE stimulation, the clock genes did not exhibit a rhythmic pattern of expression. However, in the NE-synchronized condition, a rhythmic expression pattern was observed in all cases. An enhancement in pineal gland responsiveness to NE stimulation, reflected in an advanced synthesis of melatonin was also observed. Our results reinforce our previous hypothesis that NE synchronization of pineal gland culture mimics the natural rhythmic release of NE in the gland, increasing melatonin synthesis and keeping the pineal circadian clock synchronized, ensuring the fine adjustments that are relied in the clockwork machinery. Copyright © 2014 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

Monitoring the Single-Cell Stress Response of the Diatom Thalassiosira pseudonana by Quantitative Real-Time Reverse Transcription-PCR

PubMed Central

Shi, Xu; Gao, Weimin; Chao, Shih-hui

2013-01-01

Directly monitoring the stress response of microbes to their environments could be one way to inspect the health of microorganisms themselves, as well as the environments in which the microorganisms live. The ultimate resolution for such an endeavor could be down to a single-cell level. In this study, using the diatom Thalassiosira pseudonana as a model species, we aimed to measure gene expression responses of this organism to various stresses at a single-cell level. We developed a single-cell quantitative real-time reverse transcription-PCR (RT-qPCR) protocol and applied it to determine the expression levels of multiple selected genes under nitrogen, phosphate, and iron depletion stress conditions. The results, for the first time, provided a quantitative measurement of gene expression at single-cell levels in T. pseudonana and demonstrated that significant gene expression heterogeneity was present within the cell population. In addition, different expression patterns between single-cell- and bulk-cell-based analyses were also observed for all genes assayed in this study, suggesting that cell response heterogeneity needs to be taken into consideration in order to obtain accurate information that indicates the environmental stress condition. PMID:23315741

Monitoring the single-cell stress response of the diatom Thalassiosira pseudonana by quantitative real-time reverse transcription-PCR.

PubMed

Shi, Xu; Gao, Weimin; Chao, Shih-hui; Zhang, Weiwen; Meldrum, Deirdre R

2013-03-01

Directly monitoring the stress response of microbes to their environments could be one way to inspect the health of microorganisms themselves, as well as the environments in which the microorganisms live. The ultimate resolution for such an endeavor could be down to a single-cell level. In this study, using the diatom Thalassiosira pseudonana as a model species, we aimed to measure gene expression responses of this organism to various stresses at a single-cell level. We developed a single-cell quantitative real-time reverse transcription-PCR (RT-qPCR) protocol and applied it to determine the expression levels of multiple selected genes under nitrogen, phosphate, and iron depletion stress conditions. The results, for the first time, provided a quantitative measurement of gene expression at single-cell levels in T. pseudonana and demonstrated that significant gene expression heterogeneity was present within the cell population. In addition, different expression patterns between single-cell- and bulk-cell-based analyses were also observed for all genes assayed in this study, suggesting that cell response heterogeneity needs to be taken into consideration in order to obtain accurate information that indicates the environmental stress condition.

Differentially regulated gene expression in quiescence versus senescence and identification of ARID5A as a quiescence associated marker.

PubMed

Anwar, Tarique; Sen, Bijoya; Aggarwal, Savera; Nath, Rhisita; Pathak, Niteen; Katoch, Ajay; Aiyaz, Mohamed; Trehanpati, Nirupma; Khosla, Sanjeev; Ramakrishna, Gayatri

2018-05-01

In multicellular organisms majority of the cells remain in a non-dividing states of either quiescence (reversible) or senescence (irreversible). In the present study, gene expression signatures unique to quiescence and senescence were identified using microarray in osteosarcoma cell line, U2OS. It was noted that certain genes and pathways like NOD pathway was shared by both the growth arrest conditions. A major highlight of the present study was increased expression of number of chemokines and cytokines in both quiescence and senescence. While senescence-associated secretory phenotype (SASP) is well known, the quiescence-associated secretory phenotype (QASP) is relatively unknown and appeared novel in this study. ARID5A, a subunit of SWI/SNF complex was identified as a quiescence associated gene. The endogenous expression of ARID5A increased during serum starved condition of quiescence. Overexpression of ARID5A resulted in more number of cells in G0/G1 phase of cell cycle. Further ARID5A overexpressing cells when subjected to serum starvation showed a pronounced secretory phenotype. Overall, the present work has identified gene expression signatures which can distinguish quiescence from senescence. © 2017 Wiley Periodicals, Inc.

Transcriptomic profiling of genes in matured dimorphic seeds of euhalophyte Suaeda salsa.

PubMed

Xu, Yange; Zhao, Yuanqin; Duan, Huimin; Sui, Na; Yuan, Fang; Song, Jie

2017-09-13

Suaeda salsa (S. salsa) is a euhalophyte with high economic value. S. salsa can produce dimorphic seeds. Brown seeds are more salt tolerant, can germinate quickly and maintain the fitness of the species under high saline conditions. Black seeds are less salt tolerant, may become part of the seed bank and germinate when soil salinity is reduced. Previous reports have mainly focused on the ecophysiological traits of seed germination and production under saline conditions in this species. However, there is no information available on the molecular characteristics of S. salsa dimorphic seeds. In the present study, a total of 5825 differentially expressed genes were obtained; and 4648 differentially expressed genes were annotated based on a sequence similarity search, utilizing five public databases by transcriptome analysis. The different expression of these genes may be associated with embryo development, fatty acid, osmotic regulation substances and plant hormones in brown and black seeds. Compared to black seeds, most genes may relate to embryo development, and various genes that encode fatty acid desaturase and are involved in osmotic regulation substance synthesis or transport are upregulated in brown seeds. A large number of differentially expressed genes related to plant hormones were found in brown and black seeds, and their possible roles in regulating seed dormancy/germination were discussed. Upregulated genes involved in seed development and osmotic regulation substance accumulation may relate to bigger seed size and rapid seed germination in brown seeds, compared to black seeds. Differentially expressed genes of hormones may relate to seed dormancy/germination and the development of brown and black seeds. The transcriptome dataset will serve as a valuable resource to further understand gene expression and functional genomics in S. salsa dimorphic seeds.

[Influence of tissue-specific superoxide dismutase genes expression in brain cells on Drosophila melanogaster sensitivity to oxidative stress and viability].

PubMed

Vitushynska, M V; Matiytsiv, N P; Chernyk, Y

2015-01-01

The study has shown that both functional gene knockout Sodl and Sod2 and their overexpression in neurons and glial tissue increase the sensitivity of Drosophila melanogaster to oxidative stress (OS) conditions. The lowest survival rate was only 20.5% in insects with Sod2 knockout in neurons. Comparative analysis of the survival curves showed that adults with altered tissue-specific expression of the studied genes had reduced average and maximum life span. Under OS conditions induced by 5% hydrogen peroxide the life spans of wild type Oregon R and transgenic insects were significantly reduced. Altered Sod gene expression in glial tissue leads to degenerative changes in Drosophila brain at the young age. During the aging of insects and the action of pro-oxidants increasing of neurodegenerative phenotype is observed.

Integrated Analysis of the Effects of Cold and Dehydration on Rice Metabolites, Phytohormones, and Gene Transcripts1[W][OPEN

PubMed Central

Maruyama, Kyonoshin; Urano, Kaoru; Yoshiwara, Kyouko; Morishita, Yoshihiko; Sakurai, Nozomu; Suzuki, Hideyuki; Kojima, Mikiko; Sakakibara, Hitoshi; Shibata, Daisuke; Saito, Kazuki; Shinozaki, Kazuo; Yamaguchi-Shinozaki, Kazuko

2014-01-01

Correlations between gene expression and metabolite/phytohormone levels under abiotic stress conditions have been reported for Arabidopsis (Arabidopsis thaliana). However, little is known about these correlations in rice (Oryza sativa ‘Nipponbare’), despite its importance as a model monocot. We performed an integrated analysis to clarify the relationships among cold- and dehydration-responsive metabolites, phytohormones, and gene transcription in rice. An integrated analysis of metabolites and gene expression indicated that several genes encoding enzymes involved in starch degradation, sucrose metabolism, and the glyoxylate cycle are up-regulated in rice plants exposed to cold or dehydration and that these changes are correlated with the accumulation of glucose (Glc), fructose, and sucrose. In particular, high expression levels of genes encoding isocitrate lyase and malate synthase in the glyoxylate cycle correlate with increased Glc levels in rice, but not in Arabidopsis, under dehydration conditions, indicating that the regulation of the glyoxylate cycle may be involved in Glc accumulation under dehydration conditions in rice but not Arabidopsis. An integrated analysis of phytohormones and gene transcripts revealed an inverse relationship between abscisic acid (ABA) signaling and cytokinin (CK) signaling under cold and dehydration stresses; these stresses increase ABA signaling and decrease CK signaling. High levels of Oryza sativa 9-cis-epoxycarotenoid dioxygenase transcripts correlate with ABA accumulation, and low levels of Cytochrome P450 (CYP) 735A transcripts correlate with decreased levels of a CK precursor in rice. This reduced expression of CYP735As occurs in rice but not Arabidopsis. Therefore, transcriptional regulation of CYP735As might be involved in regulating CK levels under cold and dehydration conditions in rice but not Arabidopsis. PMID:24515831

Rotating wall vessel exposure alters protein secretion and global gene expression in Staphylococcus aureus

NASA Astrophysics Data System (ADS)

Rosado, Helena; O'Neill, Alex J.; Blake, Katy L.; Walther, Meik; Long, Paul F.; Hinds, Jason; Taylor, Peter W.

2012-04-01

Staphylococcus aureus is routinely recovered from air and surface samples taken aboard the International Space Station (ISS) and poses a health threat to crew. As bacteria respond to the low shear forces engendered by continuous rotation conditions in a Rotating Wall Vessel (RWV) and the reduced gravitational field of near-Earth flight by altering gene expression, we examined the effect of low-shear RWV growth on protein secretion and gene expression by three S. aureus isolates. When cultured under 1 g, the total amount of protein secreted by these strains varied up to fourfold; under continuous rotation conditions, protein secretion by all three strains was significantly reduced. Concentrations of individual proteins were differentially reduced and no evidence was found for increased lysis. These data suggest that growth under continuous rotation conditions reduces synthesis or secretion of proteins. A limited number of changes in gene expression under continuous rotation conditions were noted: in all isolates vraX, a gene encoding a polypeptide associated with cell wall stress, was down-regulated. A vraX deletion mutant of S. aureus SH1000 was constructed: no differences were found between SH1000 and ΔvraX with respect to colony phenotype, viability, protein export, antibiotic susceptibility, vancomycin kill kinetics, susceptibility to cold or heat and gene modulation. An ab initio protein-ligand docking simulation suggests a major binding site for β-lactam drugs such as imipenem. If such changes to the bacterial phenotype occur during spaceflight, they will compromise the capacity of staphylococci to cause systemic infection and to circumvent antibacterial chemotherapy.

The expression of the clock gene cycle has rhythmic pattern and is affected by photoperiod in the moth Sesamia nonagrioides.

PubMed

Kontogiannatos, Dimitrios; Gkouvitsas, Theodoros; Kourti, Anna

2017-06-01

To obtain clues to the link between the molecular mechanism of circadian and photoperiod clocks, we have cloned the circadian clock gene cycle (Sncyc) in the corn stalk borer, Sesamia nonagrioides, which undergoes facultative diapause controlled by photoperiod. Sequence analysis revealed a high degree of conservation among insects for this gene. SnCYC consists of 667 amino acids and structural analysis showed that it contains a BCTR domain in its C-terminal in addition to the common domains found in Drosophila CYC, i.e. bHLH, PAS-A, PAS-B domains. The results revealed that the sequence of Sncyc showed a similarity to that of its mammalian orthologue, Bmal1. We also investigated the expression patterns of Sncyc in the brain of larvae growing under long-day 16L: 8D (LD), constant darkness (DD) and short-day 10L: 14D (SD) conditions using qRT-PCR assays. The mRNAs of Sncyc expression was rhythmic in LD, DD and SD cycles. Also, it is remarkable that the photoperiodic conditions affect the expression patterns and/or amplitudes of circadian clock gene Sncyc. This gene is associated with diapause in S. nonagrioides, because under SD (diapause conditions) the photoperiodic signal altered mRNA accumulation. Sequence and expression analysis of cyc in S. nonagrioides shows interesting differences compared to Drosophila where this gene does not oscillate or change in expression patterns in response to photoperiod, suggesting that this species is an interesting new model to study the molecular control of insect circadian and photoperiodic clocks. Copyright © 2017 Elsevier Inc. All rights reserved.

Subset of heat-shock transcription factors required for the early response of Arabidopsis to excess light

PubMed Central

Jung, Hou-Sung; Crisp, Peter A.; Estavillo, Gonzalo M.; Cole, Benjamin; Hong, Fangxin; Mockler, Todd C.; Pogson, Barry J.; Chory, Joanne

2013-01-01

Sunlight provides energy for photosynthesis and is essential for nearly all life on earth. However, too much or too little light or rapidly fluctuating light conditions cause stress to plants. Rapid changes in the amount of light are perceived as a change in the reduced/oxidized (redox) state of photosynthetic electron transport components in chloroplasts. However, how this generates a signal that is relayed to changes in nuclear gene expression is not well understood. We modified redox state in the reference plant, Arabidopsis thaliana, using either excess light or low light plus the herbicide DBMIB (2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone), a well-known inhibitor of photosynthetic electron transport. Modification of redox state caused a change in expression of a common set of about 750 genes, many of which are known stress-responsive genes. Among the most highly enriched promoter elements in the induced gene set were heat-shock elements (HSEs), known motifs that change gene expression in response to high temperature in many systems. We show that HSEs from the promoter of the ASCORBATE PEROXIDASE 2 (APX2) gene were necessary and sufficient for APX2 expression in conditions of excess light, or under low light plus the herbicide. We tested APX2 expression phenotypes in overexpression and loss-of-function mutants of 15 Arabidopsis A-type heat-shock transcription factors (HSFs), and identified HSFA1D, HSFA2, and HSFA3 as key factors regulating APX2 expression in diverse stress conditions. Excess light regulates both the subcellular location of HSFA1D and its biochemical properties, making it a key early component of the excess light stress network of plants. PMID:23918368

Evaluation of reference genes for insect olfaction studies.

PubMed

Omondi, Bonaventure Aman; Latorre-Estivalis, Jose Manuel; Rocha Oliveira, Ivana Helena; Ignell, Rickard; Lorenzo, Marcelo Gustavo

2015-04-22

Quantitative reverse transcription PCR (qRT-PCR) is a robust and accessible method to assay gene expression and to infer gene regulation. Being a chain of procedures, this technique is subject to systematic error due to biological and technical limitations mainly set by the starting material and downstream procedures. Thus, rigorous data normalization is critical to grant reliability and repeatability of gene expression quantification by qRT-PCR. A number of 'housekeeping genes', involved in basic cellular functions, have been commonly used as internal controls for this normalization process. However, these genes could themselves be regulated and must therefore be tested a priori. We evaluated eight potential reference genes for their stability as internal controls for RT-qPCR studies of olfactory gene expression in the antennae of Rhodnius prolixus, a Chagas disease vector. The set of genes included were: α-tubulin; β-actin; Glyceraldehyde-3-phosphate dehydrogenase; Eukaryotic initiation factor 1A; Glutathione-S-transferase; Serine protease; Succinate dehydrogenase; and Glucose-6-phosphate dehydrogenase. Five experimental conditions, including changes in age,developmental stage and feeding status were tested in both sexes. We show that the evaluation of candidate reference genes is necessary for each combination of sex, tissue and physiological condition analyzed in order to avoid inconsistent results and conclusions. Although, Normfinder and geNorm software yielded different results between males and females, five genes (SDH, Tub, GAPDH, Act and G6PDH) appeared in the first positions in all rankings obtained. By using gene expression data of a single olfactory coreceptor gene as an example, we demonstrated the extent of changes expected using different internal standards. This work underlines the need for a rigorous selection of internal standards to grant the reliability of normalization processes in qRT-PCR studies. Furthermore, we show that particular physiological or developmental conditions require independent evaluation of a diverse set of potential reference genes.

AUCTSP: an improved biomarker gene pair class predictor.

PubMed

Kagaris, Dimitri; Khamesipour, Alireza; Yiannoutsos, Constantin T

2018-06-26

The Top Scoring Pair (TSP) classifier, based on the concept of relative ranking reversals in the expressions of pairs of genes, has been proposed as a simple, accurate, and easily interpretable decision rule for classification and class prediction of gene expression profiles. The idea that differences in gene expression ranking are associated with presence or absence of disease is compelling and has strong biological plausibility. Nevertheless, the TSP formulation ignores significant available information which can improve classification accuracy and is vulnerable to selecting genes which do not have differential expression in the two conditions ("pivot" genes). We introduce the AUCTSP classifier as an alternative rank-based estimator of the magnitude of the ranking reversals involved in the original TSP. The proposed estimator is based on the Area Under the Receiver Operating Characteristic (ROC) Curve (AUC) and as such, takes into account the separation of the entire distribution of gene expression levels in gene pairs under the conditions considered, as opposed to comparing gene rankings within individual subjects as in the original TSP formulation. Through extensive simulations and case studies involving classification in ovarian, leukemia, colon, breast and prostate cancers and diffuse large b-cell lymphoma, we show the superiority of the proposed approach in terms of improving classification accuracy, avoiding overfitting and being less prone to selecting non-informative (pivot) genes. The proposed AUCTSP is a simple yet reliable and robust rank-based classifier for gene expression classification. While the AUCTSP works by the same principle as TSP, its ability to determine the top scoring gene pair based on the relative rankings of two marker genes across all subjects as opposed to each individual subject results in significant performance gains in classification accuracy. In addition, the proposed method tends to avoid selection of non-informative (pivot) genes as members of the top-scoring pair.

Update of the Diatom EST Database: a new tool for digital transcriptomics

PubMed Central

Maheswari, Uma; Mock, Thomas; Armbrust, E. Virginia; Bowler, Chris

2009-01-01

The Diatom Expressed Sequence Tag (EST) Database was constructed to provide integral access to ESTs from these ecologically and evolutionarily interesting microalgae. It has now been updated with 130 000 Phaeodactylum tricornutum ESTs from 16 cDNA libraries and 77 000 Thalassiosira pseudonana ESTs from seven libraries, derived from cells grown in different nutrient and stress regimes. The updated relational database incorporates results from statistical analyses such as log-likelihood ratios and hierarchical clustering, which help to identify differentially expressed genes under different conditions, and allow similarities in gene expression in different libraries to be investigated in a functional context. The database also incorporates links to the recently sequenced genomes of P. tricornutum and T. pseudonana, enabling an easy cross-talk between the expression pattern of diatom orthologs and the genome browsers. These improvements will facilitate exploration of diatom responses to conditions of ecological relevance and will aid gene function identification of diatom-specific genes and in silico gene prediction in this largely unexplored class of eukaryotes. The updated Diatom EST Database is available at http://www.biologie.ens.fr/diatomics/EST3. PMID:19029140

Transcriptome Profiling of Shewanella oneidensis Gene Expression following Exposure to Acidic and Alkaline pH†

PubMed Central

Leaphart, Adam B.; Thompson, Dorothea K.; Huang, Katherine; Alm, Eric; Wan, Xiu-Feng; Arkin, Adam; Brown, Steven D.; Wu, Liyou; Yan, Tingfen; Liu, Xueduan; Wickham, Gene S.; Zhou, Jizhong

2006-01-01

The molecular response of Shewanella oneidensis MR-1 to variations in extracellular pH was investigated based on genomewide gene expression profiling. Microarray analysis revealed that cells elicited both general and specific transcriptome responses when challenged with environmental acid (pH 4) or base (pH 10) conditions over a 60-min period. Global responses included the differential expression of genes functionally linked to amino acid metabolism, transcriptional regulation and signal transduction, transport, cell membrane structure, and oxidative stress protection. Response to acid stress included the elevated expression of genes encoding glycogen biosynthetic enzymes, phosphate transporters, and the RNA polymerase sigma-38 factor (rpoS), whereas the molecular response to alkaline pH was characterized by upregulation of nhaA and nhaR, which are predicted to encode an Na+/H+ antiporter and transcriptional activator, respectively, as well as sulfate transport and sulfur metabolism genes. Collectively, these results suggest that S. oneidensis modulates multiple transporters, cell envelope components, and pathways of amino acid consumption and central intermediary metabolism as part of its transcriptome response to changing external pH conditions. PMID:16452448

Coordinate changes in gene expression and triacylglycerol composition in the developing seeds of oilseed rape (Brassica napus) and turnip rape (Brassica rapa).

PubMed

Vuorinen, Anssi L; Kalpio, Marika; Linderborg, Kaisa M; Kortesniemi, Maaria; Lehto, Kirsi; Niemi, Jarmo; Yang, Baoru; Kallio, Heikki P

2014-02-15

Crop production for vegetable oil in the northern latitudes utilises oilseed rape (Brassica napus subsp. oleifera) and turnip rape (B. rapa subsp. oleifera), having similar oil compositions. The oil consists mostly of triacylglycerols, which are synthesised during seed development. In this study, we characterised the oil composition and the expression levels of genes involved in triacylglycerol biosynthesis in the developing seeds in optimal, low temperature (15 °C) and short day (12-h day length) conditions. Gene expression levels of several genes were altered during seed development. Low temperature and short day treatments increased the level of 9,12,15-octadecatrienoic acid (18:3n-3) in turnip rape and short day treatment decreased the total oil content in both species. This study gives a novel view on seed oil biosynthesis under different growth conditions, bringing together gene expression levels of the triacylglycerol biosynthesis pathway and oil composition over a time series in two related oilseed species. Copyright © 2013 Elsevier Ltd. All rights reserved.

Abiotic stresses affect differently the intron splicing and expression of chloroplast genes in coffee plants (Coffea arabica) and rice (Oryza sativa).

PubMed

Nguyen Dinh, Sy; Sai, Than Zaw Tun; Nawaz, Ghazala; Lee, Kwanuk; Kang, Hunseung

2016-08-20

Despite the increasing understanding of the regulation of chloroplast gene expression in plants, the importance of intron splicing and processing of chloroplast RNA transcripts under stress conditions is largely unknown. Here, to understand how abiotic stresses affect the intron splicing and expression patterns of chloroplast genes in dicots and monocots, we carried out a comprehensive analysis of the intron splicing and expression patterns of chloroplast genes in the coffee plant (Coffea arabica) as a dicot and rice (Oryza sativa) as a monocot under abiotic stresses, including drought, cold, or combined drought and heat stresses. The photosynthetic activity of both coffee plants and rice seedlings was significantly reduced under all stress conditions tested. Analysis of the transcript levels of chloroplast genes revealed that the splicing of tRNAs and mRNAs in coffee plants and rice seedlings were significantly affected by abiotic stresses. Notably, abiotic stresses affected differently the splicing of chloroplast tRNAs and mRNAs in coffee plants and rice seedlings. The transcript levels of most chloroplast genes were markedly downregulated in both coffee plants and rice seedlings upon stress treatment. Taken together, these results suggest that coffee and rice plants respond to abiotic stresses via regulating the intron splicing and expression of different sets of chloroplast genes. Copyright © 2016 Elsevier GmbH. All rights reserved.

Metatranscriptomic analysis of lignocellulolytic microbial communities involved in high-solids decomposition of rice straw

DOE PAGES

Simmons, Christopher W.; Reddy, Amitha P.; D’haeseleer, Patrik; ...

2014-12-31

New lignocellulolytic enzymes are needed that maintain optimal activity under the harsh conditions present during industrial enzymatic deconstruction of biomass, including high temperatures, the absence of free water, and the presence of inhibitors from the biomass. Enriching lignocellulolytic microbial communities under these conditions provides a source of microorganisms that may yield robust lignocellulolytic enzymes tolerant to the extreme conditions needed to improve the throughput and efficiency of biomass enzymatic deconstruction. Identification of promising enzymes from these systems is challenging due to complex substrate-enzyme interactions and requirements to assay for activity. In this study, metatranscriptomes from compost-derived microbial communities enriched onmore » rice straw under thermophilic and mesophilic conditions were sequenced and analyzed to identify lignocellulolytic enzymes overexpressed under thermophilic conditions. To determine differential gene expression across mesophilic and thermophilic treatments, a method was developed which pooled gene expression by functional category, as indicated by Pfam annotations, since microbial communities performing similar tasks are likely to have overlapping functions even if they share no specific genes. Differential expression analysis identified enzymes from glycoside hydrolase family 48, carbohydrate binding module family 2, and carbohydrate binding module family 33 domains as significantly overexpressed in the thermophilic community. Overexpression of these protein families in the thermophilic community resulted from expression of a small number of genes not currently represented in any protein database. Genes in overexpressed protein families were predominantly expressed by a single Actinobacteria genus, Micromonospora. In conclusion, coupling measurements of deconstructive activity with comparative analyses to identify overexpressed enzymes in lignocellulolytic communities provides a targeted approach for discovery of candidate enzymes for more efficient biomass deconstruction. Furthermore, glycoside hydrolase family 48 cellulases and carbohydrate binding module family 33 polysaccharide monooxygenases with carbohydrate binding module family 2 domains may improve saccharification of lignocellulosic biomass under high-temperature and low moisture conditions relevant to industrial biofuel production.« less

Metatranscriptomic analysis of lignocellulolytic microbial communities involved in high-solids decomposition of rice straw

DOE Office of Scientific and Technical Information (OSTI.GOV)

Simmons, Christopher W.; Reddy, Amitha P.; D’haeseleer, Patrik

New lignocellulolytic enzymes are needed that maintain optimal activity under the harsh conditions present during industrial enzymatic deconstruction of biomass, including high temperatures, the absence of free water, and the presence of inhibitors from the biomass. Enriching lignocellulolytic microbial communities under these conditions provides a source of microorganisms that may yield robust lignocellulolytic enzymes tolerant to the extreme conditions needed to improve the throughput and efficiency of biomass enzymatic deconstruction. Identification of promising enzymes from these systems is challenging due to complex substrate-enzyme interactions and requirements to assay for activity. In this study, metatranscriptomes from compost-derived microbial communities enriched onmore » rice straw under thermophilic and mesophilic conditions were sequenced and analyzed to identify lignocellulolytic enzymes overexpressed under thermophilic conditions. To determine differential gene expression across mesophilic and thermophilic treatments, a method was developed which pooled gene expression by functional category, as indicated by Pfam annotations, since microbial communities performing similar tasks are likely to have overlapping functions even if they share no specific genes. Differential expression analysis identified enzymes from glycoside hydrolase family 48, carbohydrate binding module family 2, and carbohydrate binding module family 33 domains as significantly overexpressed in the thermophilic community. Overexpression of these protein families in the thermophilic community resulted from expression of a small number of genes not currently represented in any protein database. Genes in overexpressed protein families were predominantly expressed by a single Actinobacteria genus, Micromonospora. In conclusion, coupling measurements of deconstructive activity with comparative analyses to identify overexpressed enzymes in lignocellulolytic communities provides a targeted approach for discovery of candidate enzymes for more efficient biomass deconstruction. Furthermore, glycoside hydrolase family 48 cellulases and carbohydrate binding module family 33 polysaccharide monooxygenases with carbohydrate binding module family 2 domains may improve saccharification of lignocellulosic biomass under high-temperature and low moisture conditions relevant to industrial biofuel production.« less

Selection and evaluation of novel reference genes for quantitative reverse transcription PCR (qRT-PCR) based on genome and transcriptome data in Brassica napus L.

PubMed

Yang, Hongli; Liu, Jing; Huang, Shunmou; Guo, Tingting; Deng, Linbin; Hua, Wei

2014-03-15

Selection of reference genes in Brassica napus, a tetraploid (4×) species, is a very difficult task without information on genome and transcriptome. By now, only several traditional reference genes which show significant expression differentiation under different conditions are used in B. napus. In the present study, based on genome and transcriptome data of the rapeseed Zhongshuang-11 cultivar, 14 candidate reference genes were screened for investigation in different tissues, cultivars, and treated conditions of B. napus. These genes were as follows: ELF5, ENTH, F-BOX7, F-BOX2, FYPP1, GDI1, GYF, MCP2d, OTP80, PPR, SPOC, Unknown1, Unknown2 and UBA. Among them, excluding GYF and FYPP1, another 12 genes, were identified to perform better than traditional reference genes ACTIN7 and GAPDH. To further validate the accuracy of the newly developed reference genes in normalization, expression levels of BnCAT1 (B. napus catalase 1) in different rapeseed tissues and seedlings under stress conditions were normalized by the three most stable reference genes PPR, GDI1, and ENTH and little difference existed in normalization results. To the best of our knowledge, this is the first time B. napus reference genes have been provided with the help of complete genome and transcriptome information. The new reference genes provided in this study are more accurate than previously reported reference genes in quantifying expression levels of B. napus genes. Crown Copyright © 2014. Published by Elsevier B.V. All rights reserved.

Influence of elevated atmospheric carbon dioxide on transcriptional responses of Bradyrhizobium japonicum in the soybean rhizoplane.

PubMed

Sugawara, Masayuki; Sadowsky, Michael J

2013-01-01

Elevated atmospheric CO2 can influence the structure and function of rhizoplane and rhizosphere microorganisms by altering root growth and the quality and quantity of compounds released into the rhizoplane and rhizosphere via root exudation. In these studies we investigated the transcriptional responses of Bradyrhizobium japonicum cells growing in the rhizoplane of soybean plants exposed to elevated atmospheric CO2. The results of microarray analyses indicated that elevated atmospheric CO2 concentration indirectly influenced the expression of a large number of genes in Bradyrhizobium attached to soybean roots. In addition, relative to plants and bacteria grown under ambient CO2 growth conditions, genes involved in C1 metabolism, denitrification and FixK2-associated genes, including those involved in nitrogen fixation, microaerobic respiration, respiratory nitrite reductase, and heme biosynthesis, were significantly up-regulated under conditions of elevated CO2 in the rhizosphere. The expression profile of genes involved in lipochitooligosaccharide Nod factor biosynthesis and negative transcriptional regulators of nodulation genes, nolA and nodD2, were also influenced by plant growth under conditions of elevated CO2. Taken together, the results of these studies indicate that the growth of soybeans under conditions of elevated atmospheric CO2 influences gene expressions in B. japonicum in the soybean rhizoplane, resulting in changes to carbon/nitrogen metabolism, respiration, and nodulation efficiency.

Influence of Elevated Atmospheric Carbon Dioxide on Transcriptional Responses of Bradyrhizobium japonicum in the Soybean Rhizoplane

PubMed Central

Sugawara, Masayuki; Sadowsky, Michael J.

2013-01-01

Elevated atmospheric CO2 can influence the structure and function of rhizoplane and rhizosphere microorganisms by altering root growth and the quality and quantity of compounds released into the rhizoplane and rhizosphere via root exudation. In these studies we investigated the transcriptional responses of Bradyrhizobium japonicum cells growing in the rhizoplane of soybean plants exposed to elevated atmospheric CO2. The results of microarray analyses indicated that elevated atmospheric CO2 concentration indirectly influenced the expression of a large number of genes in Bradyrhizobium attached to soybean roots. In addition, relative to plants and bacteria grown under ambient CO2 growth conditions, genes involved in C1 metabolism, denitrification and FixK2-associated genes, including those involved in nitrogen fixation, microaerobic respiration, respiratory nitrite reductase, and heme biosynthesis, were significantly up-regulated under conditions of elevated CO2 in the rhizosphere. The expression profile of genes involved in lipochitooligosaccharide Nod factor biosynthesis and negative transcriptional regulators of nodulation genes, nolA and nodD2, were also influenced by plant growth under conditions of elevated CO2. Taken together, the results of these studies indicate that the growth of soybeans under conditions of elevated atmospheric CO2 influences gene expressions in B. japonicum in the soybean rhizoplane, resulting in changes to carbon/nitrogen metabolism, respiration, and nodulation efficiency. PMID:23666536

Transcriptomic differences between day and night in Acropora millepora provide new insights into metabolite exchange and light-enhanced calcification in corals.

PubMed

Bertucci, A; Forêt, S; Ball, E E; Miller, D J

2015-09-01

The evolutionary success of reef-building corals is often attributed to their symbiotic relationship with photosynthetic dinoflagellates of the genus Symbiodinium, but metabolic interactions between the partners and the molecular bases of light-enhanced calcification (LEC) are not well understood. Here, the metabolic bases of the interaction between the coral Acropora millepora and its dinoflagellate symbiont were investigated by comparing gene expression levels under light and dark conditions at the whole transcriptome level. Among the 497 differentially expressed genes identified, a suite of genes involved in cholesterol transport was found to be upregulated under light conditions, confirming the significance of this compound in the coral symbiosis. Although ion transporters likely to have roles in calcification were not differentially expressed in this study, expression levels of many genes associated with skeletal organic matrix composition and organization were higher in light conditions. This implies that the rate of organic matrix synthesis is one factor limiting calcification at night. Thus, LEC during the day is likely to be a consequence of increases in both matrix synthesis and the supply of precursor molecules as a result of photosynthetic activity. © 2015 John Wiley & Sons Ltd.

Transcriptome-wide analysis supports environmental adaptations of two Pinus pinaster populations from contrasting habitats.

PubMed

Cañas, Rafael A; Feito, Isabel; Fuente-Maqueda, José Francisco; Ávila, Concepción; Majada, Juan; Cánovas, Francisco M

2015-11-06

Maritime pine (Pinus pinaster Aiton) grows in a range of different climates in the southwestern Mediterranean region and the existence of a variety of latitudinal ecotypes or provenances is well established. In this study, we have conducted a deep analysis of the transcriptome in needles from two P. pinaster provenances, Leiria (Portugal) and Tamrabta (Morocco), which were grown in northern Spain under the same conditions. An oligonucleotide microarray (PINARRAY3) and RNA-Seq were used for whole-transcriptome analyses, and we found that 90.95% of the data were concordant between the two platforms. Furthermore, the two methods identified very similar percentages of differentially expressed genes with values of 5.5% for PINARRAY3 and 5.7% for RNA-Seq. In total, 6,023 transcripts were shared and 88 differentially expressed genes overlapped in the two platforms. Among the differentially expressed genes, all transport related genes except aquaporins were expressed at higher levels in Tamrabta than in Leiria. In contrast, genes involved in secondary metabolism were expressed at higher levels in Tamrabta, and photosynthesis-related genes were expressed more highly in Leiria. The genes involved in light sensing in plants were well represented in the differentially expressed groups of genes. In addition, increased levels of hormones such as abscisic acid, gibberellins, jasmonic and salicylic acid were observed in Leiria. Both transcriptome platforms have proven to be useful resources, showing complementary and reliable results. The results presented here highlight the different abilities of the two maritime pine populations to sense environmental conditions and reveal one type of regulation that can be ascribed to different genetic and epigenetic backgrounds.

Transcriptomic analysis reveals metabolic switches and surface remodeling as key processes for stage transition in Trypanosoma cruzi

PubMed Central

Greif, Gonzalo; Rodriguez, Matias; Alvarez-Valin, Fernando

2017-01-01

American trypanosomiasis is a chronic and endemic disease which affects millions of people. Trypanosoma cruzi, its causative agent, has a life cycle that involves complex morphological and functional transitions, as well as a variety of environmental conditions. This requires a tight regulation of gene expression, which is achieved mainly by post-transcriptional regulation. In this work we conducted an RNAseq analysis of the three major life cycle stages of T. cruzi: amastigotes, epimastigotes and trypomastigotes. This analysis allowed us to delineate specific transcriptomic profiling for each stage, and also to identify those biological processes of major relevance in each state. Stage specific expression profiling evidenced the plasticity of T. cruzi to adapt quickly to different conditions, with particular focus on membrane remodeling and metabolic shifts along the life cycle. Epimastigotes, which replicate in the gut of insect vectors, showed higher expression of genes related to energy metabolism, mainly Krebs cycle, respiratory chain and oxidative phosphorylation related genes, and anabolism related genes associated to nucleotide and steroid biosynthesis; also, a general down-regulation of surface glycoprotein coding genes was seen at this stage. Trypomastigotes, living extracellularly in the bloodstream of mammals, express a plethora of surface proteins and signaling genes involved in invasion and evasion of immune response. Amastigotes mostly express membrane transporters and genes involved in regulation of cell cycle, and also express a specific subset of surface glycoprotein coding genes. In addition, these results allowed us to improve the annotation of the Dm28c genome, identifying new ORFs and set the stage for construction of networks of co-expression, which can give clues about coded proteins of unknown functions. PMID:28286708

Coral thermal tolerance: tuning gene expression to resist thermal stress.

PubMed

Bellantuono, Anthony J; Granados-Cifuentes, Camila; Miller, David J; Hoegh-Guldberg, Ove; Rodriguez-Lanetty, Mauricio

2012-01-01

The acclimatization capacity of corals is a critical consideration in the persistence of coral reefs under stresses imposed by global climate change. The stress history of corals plays a role in subsequent response to heat stress, but the transcriptomic changes associated with these plastic changes have not been previously explored. In order to identify host transcriptomic changes associated with acquired thermal tolerance in the scleractinian coral Acropora millepora, corals preconditioned to a sub-lethal temperature of 3°C below bleaching threshold temperature were compared to both non-preconditioned corals and untreated controls using a cDNA microarray platform. After eight days of hyperthermal challenge, conditions under which non-preconditioned corals bleached and preconditioned corals (thermal-tolerant) maintained Symbiodinium density, a clear differentiation in the transcriptional profiles was revealed among the condition examined. Among these changes, nine differentially expressed genes separated preconditioned corals from non-preconditioned corals, with 42 genes differentially expressed between control and preconditioned treatments, and 70 genes between non-preconditioned corals and controls. Differentially expressed genes included components of an apoptotic signaling cascade, which suggest the inhibition of apoptosis in preconditioned corals. Additionally, lectins and genes involved in response to oxidative stress were also detected. One dominant pattern was the apparent tuning of gene expression observed between preconditioned and non-preconditioned treatments; that is, differences in expression magnitude were more apparent than differences in the identity of genes differentially expressed. Our work revealed a transcriptomic signature underlying the tolerance associated with coral thermal history, and suggests that understanding the molecular mechanisms behind physiological acclimatization would be critical for the modeling of reefs in impending climate change scenarios.

Single administration of recombinant IL-6 restores the gene expression of lipogenic enzymes in liver of fasting IL-6-deficient mice.

PubMed

Gavito, A L; Cabello, R; Suarez, J; Serrano, A; Pavón, F J; Vida, M; Romero, M; Pardo, V; Bautista, D; Arrabal, S; Decara, J; Cuesta, A L; Valverde, A M; Rodríguez de Fonseca, F; Baixeras, E

2016-03-01

Lipogenesis is intimately controlled by hormones and cytokines as well as nutritional conditions. IL-6 participates in the regulation of fatty acid metabolism in the liver. We investigated the role of IL-6 in mediating fasting/re-feeding changes in the expression of hepatic lipogenic enzymes. Gene and protein expression of lipogenic enzymes were examined in livers of wild-type (WT) and IL-6-deficient (IL-6(-/-) ) mice during fasting and re-feeding conditions. Effects of exogenous IL-6 administration on gene expression of these enzymes were evaluated in vivo. The involvement of STAT3 in mediating these IL-6 responses was investigated by using siRNA in human HepG2 cells. During feeding, the up-regulation in the hepatic expression of lipogenic genes presented similar time kinetics in WT and IL-6(-/-) mice. During fasting, expression of lipogenic genes decreased gradually over time in both strains, although the initial drop was more marked in IL-6(-/-) mice. Protein levels of hepatic lipogenic enzymes were lower in IL-6(-/-) than in WT mice at the end of the fasting period. In WT, circulating IL-6 levels paralleled gene expression of hepatic lipogenic enzymes. IL-6 administration in vivo and in vitro showed that IL-6-mediated signalling was associated with the up-regulation of hepatic lipogenic enzyme genes. Moreover, silencing STAT3 in HepG2 cells attenuated IL-6 mediated up-regulation of lipogenic gene transcription levels. IL-6 sustains levels of hepatic lipogenic enzymes during fasting through activation of STAT3. Our findings indicate that clinical use of STAT3-associated signalling cytokines, particularly against steatosis, should be undertaken with caution. © 2016 The British Pharmacological Society.

Coral Thermal Tolerance: Tuning Gene Expression to Resist Thermal Stress

PubMed Central

Bellantuono, Anthony J.; Granados-Cifuentes, Camila; Miller, David J.; Hoegh-Guldberg, Ove; Rodriguez-Lanetty, Mauricio

2012-01-01

The acclimatization capacity of corals is a critical consideration in the persistence of coral reefs under stresses imposed by global climate change. The stress history of corals plays a role in subsequent response to heat stress, but the transcriptomic changes associated with these plastic changes have not been previously explored. In order to identify host transcriptomic changes associated with acquired thermal tolerance in the scleractinian coral Acropora millepora, corals preconditioned to a sub-lethal temperature of 3°C below bleaching threshold temperature were compared to both non-preconditioned corals and untreated controls using a cDNA microarray platform. After eight days of hyperthermal challenge, conditions under which non-preconditioned corals bleached and preconditioned corals (thermal-tolerant) maintained Symbiodinium density, a clear differentiation in the transcriptional profiles was revealed among the condition examined. Among these changes, nine differentially expressed genes separated preconditioned corals from non-preconditioned corals, with 42 genes differentially expressed between control and preconditioned treatments, and 70 genes between non-preconditioned corals and controls. Differentially expressed genes included components of an apoptotic signaling cascade, which suggest the inhibition of apoptosis in preconditioned corals. Additionally, lectins and genes involved in response to oxidative stress were also detected. One dominant pattern was the apparent tuning of gene expression observed between preconditioned and non-preconditioned treatments; that is, differences in expression magnitude were more apparent than differences in the identity of genes differentially expressed. Our work revealed a transcriptomic signature underlying the tolerance associated with coral thermal history, and suggests that understanding the molecular mechanisms behind physiological acclimatization would be critical for the modeling of reefs in impending climate change scenarios. PMID:23226355

The role of transcriptome resilience in resistance of corals to bleaching.

PubMed

Seneca, Francois O; Palumbi, Stephen R

2015-04-01

Wild populations increasingly experience extreme conditions as climate change amplifies environmental variability. How individuals respond to environmental extremes determines the impact of climate change overall. The variability of response from individual to individual can represent the opportunity for natural selection to occur as a result of extreme conditions. Here, we experimentally replicated the natural exposure to extreme temperatures of the reef lagoon at Ofu Island (American Samoa), where corals can experience severe heat stress during midday low tide. We investigated the bleaching and transcriptome response of 20 Acropora hyacinthus colonies 5 and 20 h after exposure to control (29 °C) or heated (35 °C) conditions. We found a highly dynamic transcriptome response: 27% of the coral transcriptome was significantly regulated 1 h postheat exposure. Yet 15 h later, when heat-induced coral bleaching became apparent, only 12% of the transcriptome was differentially regulated. A large proportion of responsive genes at the first time point returned to control levels, others remained differentially expressed over time, while an entirely different subset of genes was successively regulated at the second time point. However, a noteworthy variability in gene expression was observed among individual coral colonies. Among the genes of which expression lingered over time, fast return to normal levels was associated with low bleaching. Colonies that maintained higher expression levels of these genes bleached severely. Return to normal levels of gene expression after stress has been termed transcriptome resilience, and in the case of some specific genes may signal the physiological health and response ability of individuals to environmental stress. © 2015 John Wiley & Sons Ltd.

Selecting and validating reference genes for quantitative real-time PCR in Plutella xylostella (L.).

PubMed

You, Yanchun; Xie, Miao; Vasseur, Liette; You, Minsheng

2018-05-01

Gene expression analysis provides important clues regarding gene functions, and quantitative real-time PCR (qRT-PCR) is a widely used method in gene expression studies. Reference genes are essential for normalizing and accurately assessing gene expression. In the present study, 16 candidate reference genes (ACTB, CyPA, EF1-α, GAPDH, HSP90, NDPk, RPL13a, RPL18, RPL19, RPL32, RPL4, RPL8, RPS13, RPS4, α-TUB, and β-TUB) from Plutella xylostella were selected to evaluate gene expression stability across different experimental conditions using five statistical algorithms (geNorm, NormFinder, Delta Ct, BestKeeper, and RefFinder). The results suggest that different reference genes or combinations of reference genes are suitable for normalization in gene expression studies of P. xylostella according to the different developmental stages, strains, tissues, and insecticide treatments. Based on the given experimental sets, the most stable reference genes were RPS4 across different developmental stages, RPL8 across different strains and tissues, and EF1-α across different insecticide treatments. A comprehensive and systematic assessment of potential reference genes for gene expression normalization is essential for post-genomic functional research in P. xylostella, a notorious pest with worldwide distribution and a high capacity to adapt and develop resistance to insecticides.

Gene expression profiling--Opening the black box of plant ecosystem responses to global change

DOE Office of Scientific and Technical Information (OSTI.GOV)

Leakey, A.D.B.; Ainsworth, E.A.; Bernard, S.M.

The use of genomic techniques to address ecological questions is emerging as the field of genomic ecology. Experimentation under environmentally realistic conditions to investigate the molecular response of plants to meaningful changes in growth conditions and ecological interactions is the defining feature of genomic ecology. Since the impact of global change factors on plant performance are mediated by direct effects at the molecular, biochemical and physiological scales, gene expression analysis promises important advances in understanding factors that have previously been consigned to the 'black box' of unknown mechanism. Various tools and approaches are available for assessing gene expression in modelmore » and non-model species as part of global change biology studies. Each approach has its own unique advantages and constraints. A first generation of genomic ecology studies in managed ecosystems and mesocosms have provided a testbed for the approach and have begun to reveal how the experimental design and data analysis of gene expression studies can be tailored for use in an ecological context.« less

Gene Expression Profiling in Fish Toxicology: A Review.

PubMed

Kumar, Girish; Denslow, Nancy D

In this review, we present an overview of transcriptomic responses to chemical exposures in a variety of fish species. We have discussed the use of several molecular approaches such as northern blotting, differential display reverse transcription-polymerase chain reaction (DDRT-PCR), suppression subtractive hybridization (SSH), real time quantitative PCR (RT-qPCR), microarrays, and next-generation sequencing (NGS) for measuring gene expression. These techniques have been mainly used to measure the toxic effects of single compounds or simple mixtures in laboratory conditions. In addition, only few studies have been conducted to examine the biological significance of differentially expressed gene sets following chemical exposure. Therefore, future studies should focus more under field conditions using a multidisciplinary approach (genomics, proteomics and metabolomics) to understand the synergetic effects of multiple environmental stressors and to determine the functional significance of differentially expressed genes. Nevertheless, recent developments in NGS technologies and decreasing costs of sequencing holds the promise to uncover the complexity of anthropogenic impacts and biological effects in wild fish populations.

Identifying Growth Conditions for Nicotiana benthimiana Resulting in Predictable Gene Expression of Promoter-Gus Fusion

NASA Astrophysics Data System (ADS)

Sandoval, V.; Barton, K.; Longhurst, A.

2012-12-01

Revoluta (Rev) is a transcription factor that establishes leaf polarity inArabidopsis thaliana. Through previous work in Dr. Barton's Lab, it is known that Revoluta binds to the ZPR3 promoter, thus activating the ZPR3 gene product inArabidopsis thaliana. Using this knowledge, two separate DNA constructs were made, one carrying revgene and in the other, the ZPR3 promoter fussed with the GUS gene. When inoculated in Nicotiana benthimiana (tobacco), the pMDC32 plasmid produces the Rev protein. Rev binds to the ZPR3 promoter thereby activating the transcription of the GUS gene, which can only be expressed in the presence of Rev. When GUS protein comes in contact with X-Gluc it produce the blue stain seen (See Figure 1). In the past, variability has been seen of GUS expression on tobacco therefore we hypothesized that changing the growing conditions and leaf age might improve how well it's expressed.

A systems approach to model the relationship between aflatoxin gene cluster expression, environmental factors, growth and toxin production by Aspergillus flavus

PubMed Central

Abdel-Hadi, Ahmed; Schmidt-Heydt, Markus; Parra, Roberto; Geisen, Rolf; Magan, Naresh

2012-01-01

A microarray analysis was used to examine the effect of combinations of water activity (aw, 0.995–0.90) and temperature (20–42°C) on the activation of aflatoxin biosynthetic genes (30 genes) in Aspergillus flavus grown on a conducive YES (20 g yeast extract, 150 g sucrose, 1 g MgSO4·7H2O) medium. The relative expression of 10 key genes (aflF, aflD, aflE, aflM, aflO, aflP, aflQ, aflX, aflR and aflS) in the biosynthetic pathway was examined in relation to different environmental factors and phenotypic aflatoxin B1 (AFB1) production. These data, plus data on relative growth rates and AFB1 production under different aw × temperature conditions were used to develop a mixed-growth-associated product formation model. The gene expression data were normalized and then used as a linear combination of the data for all 10 genes and combined with the physical model. This was used to relate gene expression to aw and temperature conditions to predict AFB1 production. The relationship between the observed AFB1 production provided a good linear regression fit to the predicted production based in the model. The model was then validated by examining datasets outside the model fitting conditions used (37°C, 40°C and different aw levels). The relationship between structural genes (aflD, aflM) in the biosynthetic pathway and the regulatory genes (aflS, aflJ) was examined in relation to aw and temperature by developing ternary diagrams of relative expression. These findings are important in developing a more integrated systems approach by combining gene expression, ecophysiological influences and growth data to predict mycotoxin production. This could help in developing a more targeted approach to develop prevention strategies to control such carcinogenic natural metabolites that are prevalent in many staple food products. The model could also be used to predict the impact of climate change on toxin production. PMID:21880616

Genomic and transcriptomic analysis of aroma synthesis in two hybrids between Saccharomyces cerevisiae and S. kudriavzevii in winemaking conditions.

PubMed

Gamero, Amparo; Belloch, Carmela; Querol, Amparo

2015-09-04

Aroma is one of the most important attributes defining wine quality in which yeasts play a crucial role, synthesizing aromatic compounds or releasing odourless conjugates. A present-day trend in winemaking consists of lowering fermentation temperature to achieve higher aroma production and retention. S. cerevisiae × S. kudriavzevii hybrids seem to have inherited beneficial traits from their parental species, like fermenting efficiently at low temperature or producing higher amounts of certain aromatic compounds. In this study, allelic composition and gene expression of the genes related to aroma synthesis in two genetically and phenotypically different S. cerevisiae × S. kudriavzevii hybrids, Lalvin W27 and VIN7, were compared and related to aroma production in microvinifications at 12 and 28 °C. In addition, the contribution of the allele coming from each parental to the overall expression was explored by RT-PCR. The results indicated large differences in allele composition, gene expression and the contribution of each parental to the overall expression at the fermentation temperatures tested. Results obtained by RT-PCR showed that in ARO1 and ATF2 genes the S. kudriavzevii allele was more expressed than that of S. cerevisiae particularly at 12 °C. This study revealed high differences regarding allele composition and gene expression in two S. cerevisiae × S. kudriavzevii hybrids, which may have led to different aroma profiles in winemaking conditions. The contribution of the alleles coming from each parental to the overall expression has proved to differently influence aroma synthesis. Besides, the quantitative contribution to the overall gene expression of the alleles coming from one parental strain or the other was clearly determined by the fermentation temperature for some genes.

Selection of Reference Genes for Quantitative Gene Expression in Porcine Mesenchymal Stem Cells Derived from Various Sources along with Differentiation into Multilineages

PubMed Central

Lee, Won-Jae; Jeon, Ryoung-Hoon; Jang, Si-Jung; Park, Ji-Sung; Lee, Seung-Chan; Baregundi Subbarao, Raghavendra; Lee, Sung-Lim; Park, Bong-Wook; King, William Allan; Rho, Gyu-Jin

2015-01-01

The identification of stable reference genes is a prerequisite for ensuring accurate validation of gene expression, yet too little is known about stable reference genes of porcine MSCs. The present study was, therefore, conducted to assess the stability of reference genes in porcine MSCs derived from bone marrow (BMSCs), adipose (AMSCs), and skin (SMSCs) with their in vitro differentiated cells into mesenchymal lineages such as adipocytes, osteocytes, and chondrocytes. Twelve commonly used reference genes were investigated for their threshold cycle (Ct) values by qRT-PCR. The Ct values of candidate reference genes were analyzed by geNorm software to clarify stable expression regardless of experimental conditions. Thus, Pearson's correlation was applied to determine correlation between the three most stable reference genes (NF3) and optimal number of reference genes (NFopt). In assessment of stability of reference gene across experimental conditions by geNorm analysis, undifferentiated MSCs and each differentiated status into mesenchymal lineages showed slightly different results but similar patterns about more or less stable rankings. Furthermore, Pearson's correlation revealed high correlation (r > 0.9) between NF3 and NFopt. Overall, the present study showed that HMBS, YWHAZ, SDHA, and TBP are suitable reference genes for qRT-PCR in porcine MSCs. PMID:25972899

Octopaminergic gene expression and flexible social behaviour in the subsocial burying beetle Nicrophorus vespilloides

PubMed Central

Cunningham, C B; Douthit, M K; Moore, A J

2014-01-01

Flexible behaviour allows organisms to respond appropriately to changing environmental and social conditions. In the subsocial beetle Nicrophorus vespilloides, females tolerate conspecifics when mating, become aggressive when defending resources, and return to social tolerance when transitioning to parenting. Given the association between octopamine and aggression in insects, we hypothesized that genes in the octopaminergic system would be differentially expressed across different social and reproductive contexts. To test this in N. vespilloides, we first obtained the sequences of orthologues of the synthetic enzymes and receptors of the octopaminergic system. We next compared relative gene expression from virgin females, mated females, mated females alone on a resource required for reproduction and mated females on a resource with a male. Expression varied for five receptor genes. The expression of octopamine β receptor 1 and octopamine β receptor 2 was relatively higher in mated females than in other social conditions. Octopamine β receptor 3 was influenced by the presence or absence of a resource and less by social environment. Octopamine α receptor and octopamine/tyramine receptor 1 gene expression was relatively lower in the mated females with a resource and a male. We suggest that in N. vespilloides the octopaminergic system is associated with the expression of resource defence, alternative mating tactics, social tolerance and indirect parental care. PMID:24646461

In-depth analysis of internal control genes for quantitative real-time PCR in Brassica oleracea var. botrytis.

PubMed

Sheng, X G; Zhao, Z Q; Yu, H F; Wang, J S; Zheng, C F; Gu, H H

2016-07-15

Quantitative reverse-transcription PCR (qRT-PCR) is a versatile technique for the analysis of gene expression. The selection of stable reference genes is essential for the application of this technique. Cauliflower (Brassica oleracea L. var. botrytis) is a commonly consumed vegetable that is rich in vitamin, calcium, and iron. Thus far, to our knowledge, there have been no reports on the validation of suitable reference genes for the data normalization of qRT-PCR in cauliflower. In the present study, we analyzed 12 candidate housekeeping genes in cauliflower subjected to different abiotic stresses, hormone treatment conditions, and accessions. geNorm and NormFinder algorithms were used to assess the expression stability of these genes. ACT2 and TIP41 were selected as suitable reference genes across all experimental samples in this study. When different accessions were compared, ACT2 and UNK3 were found to be the most suitable reference genes. In the hormone and abiotic stress treatments, ACT2, TIP41, and UNK2 were the most stably expressed. Our study also provided guidelines for selecting the best reference genes under various experimental conditions.

Transcriptomics of environmental acclimatization and survival in wild adult Pacific sockeye salmon (Oncorhynchus nerka) during spawning migration.

PubMed

Evans, Tyler G; Hammill, Edd; Kaukinen, Karia; Schulze, Angela D; Patterson, David A; English, Karl K; Curtis, Janelle M R; Miller, Kristina M

2011-11-01

Environmental shifts accompanying salmon spawning migrations from ocean feeding grounds to natal freshwater streams can be severe, with the underlying stress often cited as a cause of increased mortality. Here, a salmonid microarray was used to characterize changes in gene expression occurring between ocean and river habitats in gill and liver tissues of wild migrating sockeye salmon (Oncorhynchus nerka Walbaum) returning to spawn in the Fraser River, British Columbia, Canada. Expression profiles indicate that the transcriptome of migrating salmon is strongly affected by shifting abiotic and biotic conditions encountered along migration routes. Conspicuous shifts in gene expression associated with changing salinity, temperature, pathogen exposure and dissolved oxygen indicate that these environmental variables most strongly impact physiology during spawning migrations. Notably, transcriptional changes related to osmoregulation were largely preparatory and occurred well before salmon encountered freshwater. In the river environment, differential expression of genes linked with elevated temperatures indicated that thermal regimes within the Fraser River are approaching tolerance limits for adult salmon. To empirically correlate gene expression with survival, biopsy sampling of gill tissue and transcriptomic profiling were combined with telemetry. Many genes correlated with environmental variables were differentially expressed between premature mortalities and successful migrants. Parametric survival analyses demonstrated a broad-scale transcriptional regulator, cofactor required for Sp1 transcriptional activation (CRSP), to be significantly predictive of survival. As the environmental characteristics of salmon habitats continue to change, establishing how current environmental conditions influence salmon physiology under natural conditions is critical to conserving this ecologically and economically important fish species. © 2011 Blackwell Publishing Ltd.

Biofilm formation, phenotypic production of cellulose and gene expression in Salmonella enterica decrease under anaerobic conditions.

PubMed

Lamas, A; Miranda, J M; Vázquez, B; Cepeda, A; Franco, C M

2016-12-05

Salmonella enterica subsp. enterica is one of the main food-borne pathogens. This microorganism combines an aerobic life outside the host with an anaerobic life within the host. One of the main concerns related to S. enterica is biofilm formation and cellulose production. In this study, biofilm formation, morphotype, cellulose production and transcription of biofilm and quorum sensing-related genes of 11 S. enterica strains were tested under three different conditions: aerobiosis, microaerobiosis, and anaerobiosis. The results showed an influence of oxygen levels on biofilm production. Biofilm formation was significantly higher (P<0.05) in aerobiosis than in microaerobiosis and anaerobiosis. Cellulose production and RDAR (red, dry, and rough) were expressed only in aerobiosis. In microaerobiosis, the strains expressed the SAW (smooth and white) morphotype, while in anaerobiosis the colonies appeared small and red. The expression of genes involved in cellulose synthesis (csgD and adrA) and quorum sensing (sdiA and luxS) was reduced in microaerobiosis and anaerobiosis in all S. enterica strains tested. This gene expression levels were less reduced in S. Typhimurium and S. Enteritidis compared to the tested serotypes. There was a relationship between the expression of biofilm and quorum sensing-related genes. Thus, the results from this study indicate that biofilm formation and cellulose production are highly influenced by atmospheric conditions. This must be taken into account as contamination with these bacteria can occur during food processing under vacuum or modified atmospheres. Copyright © 2016 Elsevier B.V. All rights reserved.

Analytical workflow profiling gene expression in murine macrophages

PubMed Central

Nixon, Scott E.; González-Peña, Dianelys; Lawson, Marcus A.; McCusker, Robert H.; Hernandez, Alvaro G.; O’Connor, Jason C.; Dantzer, Robert; Kelley, Keith W.

2015-01-01

Comprehensive and simultaneous analysis of all genes in a biological sample is a capability of RNA-Seq technology. Analysis of the entire transcriptome benefits from summarization of genes at the functional level. As a cellular response of interest not previously explored with RNA-Seq, peritoneal macrophages from mice under two conditions (control and immunologically challenged) were analyzed for gene expression differences. Quantification of individual transcripts modeled RNA-Seq read distribution and uncertainty (using a Beta Negative Binomial distribution), then tested for differential transcript expression (False Discovery Rate-adjusted p-value < 0.05). Enrichment of functional categories utilized the list of differentially expressed genes. A total of 2079 differentially expressed transcripts representing 1884 genes were detected. Enrichment of 92 categories from Gene Ontology Biological Processes and Molecular Functions, and KEGG pathways were grouped into 6 clusters. Clusters included defense and inflammatory response (Enrichment Score = 11.24) and ribosomal activity (Enrichment Score = 17.89). Our work provides a context to the fine detail of individual gene expression differences in murine peritoneal macrophages during immunological challenge with high throughput RNA-Seq. PMID:25708305

Establishing references for gene expression analyses by RT-qPCR in Theobroma cacao tissues.

PubMed

Pinheiro, T T; Litholdo, C G; Sereno, M L; Leal, G A; Albuquerque, P S B; Figueira, A

2011-11-17

Lack of continuous progress in Theobroma cacao (Malvaceae) breeding, especially associated with seed quality traits, requires more efficient selection methods based on genomic information. Reverse transcript quantitative PCR (RT-qPCR) has become the method of choice for gene expression analysis, but relative expression analysis requires various reference genes, which must be stable across various biological conditions. We sought suitable reference genes for various tissues of cacao, especially developing seeds. Ten potential reference genes were analyzed for stability at various stages of embryo development, leaves, stems, roots, flowers, and pod epicarp; seven of them were also evaluated in shoot tips treated either with hormones (salicylate; ethefon; methyl-jasmonate) or after inoculation with the fungus Moniliophthora perniciosa (Marasmiaceae sensu lato). For developing embryos, the three most stable genes were actin (ACT), polyubiquitin (PUB), and ribosomal protein L35 (Rpl35). In the analyses of various tissues, the most stable genes were malate dehydrogenase (MDH), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and acyl-carrier protein B (ACP B). GAPDH, MDH and tubulin (TUB) were the most appropriate for normalization when shoot apexes were treated with hormones, while ACT, TUB and Rpl35 were the most appropriate after inoculation with M. perniciosa. We conclude that for each plant system and biological or ontogenetical condition, there is a need to define suitable reference genes. This is the first report to define reference genes for expression studies in cacao.

Parallel human genome analysis: microarray-based expression monitoring of 1000 genes.

PubMed Central

Schena, M; Shalon, D; Heller, R; Chai, A; Brown, P O; Davis, R W

1996-01-01

Microarrays containing 1046 human cDNAs of unknown sequence were printed on glass with high-speed robotics. These 1.0-cm2 DNA "chips" were used to quantitatively monitor differential expression of the cognate human genes using a highly sensitive two-color hybridization assay. Array elements that displayed differential expression patterns under given experimental conditions were characterized by sequencing. The identification of known and novel heat shock and phorbol ester-regulated genes in human T cells demonstrates the sensitivity of the assay. Parallel gene analysis with microarrays provides a rapid and efficient method for large-scale human gene discovery. Images Fig. 1 Fig. 2 Fig. 3 PMID:8855227

The Fur-Iron Complex Modulates Expression of the Quorum-Sensing Master Regulator, SmcR, To Control Expression of Virulence Factors in Vibrio vulnificus

PubMed Central

Kim, In Hwang; Wen, Yancheng; Son, Jee-Soo; Lee, Kyu-Ho

2013-01-01

The gene vvpE, encoding the virulence factor elastase, is a member of the quorum-sensing regulon in Vibrio vulnificus and displays enhanced expression at high cell density. We observed that this gene was repressed under iron-rich conditions and that the repression was due to a Fur (ferric uptake regulator)-dependent repression of smcR, a gene encoding a quorum-sensing master regulator with similarity to luxR in Vibrio harveyi. A gel mobility shift assay and a footprinting experiment demonstrated that the Fur-iron complex binds directly to two regions upstream of smcR (−82 to −36 and −2 to +27, with respect to the transcription start site) with differing affinities. However, binding of the Fur-iron complex is reversible enough to allow expression of smcR to be induced by quorum sensing at high cell density under iron-rich conditions. Under iron-limiting conditions, Fur fails to bind either region and the expression of smcR is regulated solely by quorum sensing. These results suggest that two biologically important environmental signals, iron and quorum sensing, converge to direct the expression of smcR, which then coordinates the expression of virulence factors. PMID:23716618

Transcriptome analysis of Schistosoma mansoni larval development using serial analysis of gene expression (SAGE).

PubMed

Taft, A S; Vermeire, J J; Bernier, J; Birkeland, S R; Cipriano, M J; Papa, A R; McArthur, A G; Yoshino, T P

2009-04-01

Infection of the snail, Biomphalaria glabrata, by the free-swimming miracidial stage of the human blood fluke, Schistosoma mansoni, and its subsequent development to the parasitic sporocyst stage is critical to establishment of viable infections and continued human transmission. We performed a genome-wide expression analysis of the S. mansoni miracidia and developing sporocyst using Long Serial Analysis of Gene Expression (LongSAGE). Five cDNA libraries were constructed from miracidia and in vitro cultured 6- and 20-day-old sporocysts maintained in sporocyst medium (SM) or in SM conditioned by previous cultivation with cells of the B. glabrata embryonic (Bge) cell line. We generated 21 440 SAGE tags and mapped 13 381 to the S. mansoni gene predictions (v4.0e) either by estimating theoretical 3' UTR lengths or using existing 3' EST sequence data. Overall, 432 transcripts were found to be differentially expressed amongst all 5 libraries. In total, 172 tags were differentially expressed between miracidia and 6-day conditioned sporocysts and 152 were differentially expressed between miracidia and 6-day unconditioned sporocysts. In addition, 53 and 45 tags, respectively, were differentially expressed in 6-day and 20-day cultured sporocysts, due to the effects of exposure to Bge cell-conditioned medium.

NF-κB mediates Gadd45β expression and DNA demethylation in the hippocampus during fear memory formation.

PubMed

Jarome, Timothy J; Butler, Anderson A; Nichols, Jessica N; Pacheco, Natasha L; Lubin, Farah D

2015-01-01

Gadd45-mediated DNA demethylation mechanisms have been implicated in the process of memory formation. However, the transcriptional mechanisms involved in the regulation of Gadd45 gene expression during memory formation remain unexplored. NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) controls transcription of genes in neurons and is a critical regulator of synaptic plasticity and memory formation. In silico analysis revealed several NF-κB (p65/RelA and cRel) consensus sequences within the Gadd45β gene promoter. Whether NF-κB activity regulates Gadd45 expression and associated DNA demethylation in neurons during memory formation is unknown. Here, we found that learning in a fear conditioning paradigm increased Gadd45β gene expression and brain-derivedneurotrophic factor (BDNF) DNA demethylation in area CA1 of the hippocampus, both of which were prevented with pharmacological inhibition of NF-κB activity. Further experiments found that conditional mutations in p65/RelA impaired fear memory formation but did not alter changes in Gadd45β expression. The learning-induced increases in Gadd45β mRNA levels, Gadd45β binding at the BDNF gene and BDNF DNA demethylation were blocked in area CA1 of the c-rel knockout mice. Additionally, local siRNA-mediated knockdown of c-rel in area CA1 prevented fear conditioning-induced increases in Gadd45β expression and BDNF DNA demethylation, suggesting that c-Rel containing NF-κB transcription factor complex is responsible for Gadd45β regulation during memory formation. Together, these results support a novel transcriptional role for NF-κB in regulation of Gadd45β expression and DNA demethylation in hippocampal neurons during fear memory.

Paired termini stabilize antisense RNAs and enhance conditional gene silencing in Escherichia coli

PubMed Central

Nakashima, Nobutaka; Tamura, Tomohiro; Good, Liam

2006-01-01

Reliable methods for conditional gene silencing in bacteria have been elusive. To improve silencing by expressed antisense RNAs (asRNAs), we systematically altered several design parameters and targeted multiple reporter and essential genes in Escherichia coli. A paired termini (PT) design, where flanking inverted repeats create paired dsRNA termini, proved effective. PTasRNAs targeted against the ackA gene within the acetate kinase-phosphotransacetylase operon (ackA-pta) triggered target mRNA decay and a 78% reduction in AckA activity with high genetic penetrance. PTasRNAs are abundant and stable and function through an RNase III independent mechanism that requires a large stoichiometric excess of asRNA. Conditional ackA silencing reduced carbon flux to acetate and increased heterologous gene expression. The PT design also improved silencing of the essential fabI gene. Full anti-fabI PTasRNA induction prevented growth and partial induction sensitized cells to a FabI inhibitor. PTasRNAs have potential for functional genomics, antimicrobial discovery and metabolic flux control. PMID:17062631

Paired termini stabilize antisense RNAs and enhance conditional gene silencing in Escherichia coli.

PubMed

Nakashima, Nobutaka; Tamura, Tomohiro; Good, Liam

2006-01-01

Reliable methods for conditional gene silencing in bacteria have been elusive. To improve silencing by expressed antisense RNAs (asRNAs), we systematically altered several design parameters and targeted multiple reporter and essential genes in Escherichia coli. A paired termini (PT) design, where flanking inverted repeats create paired dsRNA termini, proved effective. PTasRNAs targeted against the ackA gene within the acetate kinase-phosphotransacetylase operon (ackA-pta) triggered target mRNA decay and a 78% reduction in AckA activity with high genetic penetrance. PTasRNAs are abundant and stable and function through an RNase III independent mechanism that requires a large stoichiometric excess of asRNA. Conditional ackA silencing reduced carbon flux to acetate and increased heterologous gene expression. The PT design also improved silencing of the essential fabI gene. Full anti-fabI PTasRNA induction prevented growth and partial induction sensitized cells to a FabI inhibitor. PTasRNAs have potential for functional genomics, antimicrobial discovery and metabolic flux control.

Dynamics of the Streptococcus gordonii Transcriptome in Response to Medium, Salivary α-Amylase, and Starch

PubMed Central

Haase, Elaine M.; Feng, Xianghui; Pan, Jiachuan; Miecznikowski, Jeffrey C.

2015-01-01

Streptococcus gordonii, a primary colonizer of the tooth surface, interacts with salivary α-amylase via amylase-binding protein A (AbpA). This enzyme hydrolyzes starch to glucose, maltose, and maltodextrins that can be utilized by various oral bacteria for nutrition. Microarray studies demonstrated that AbpA modulates gene expression in response to amylase, suggesting that the amylase-streptococcal interaction may function in ways other than nutrition. The goal of this study was to explore the role of AbpA in gene regulation through comparative transcriptional profiling of wild-type KS1 and AbpA− mutant KS1ΩabpA under various environmental conditions. A portion of the total RNA isolated from mid-log-phase cells grown in 5% CO2 in (i) complex medium with or without amylase, (ii) defined medium (DM) containing 0.8% glucose with/without amylase, and (iii) DM containing 0.2% glucose and amylase with or without starch was reverse transcribed to cDNA and the rest used for RNA sequencing. Changes in the expression of selected genes were validated by quantitative reverse transcription-PCR. Maltodextrin-associated genes, fatty acid synthesis genes and competence genes were differentially expressed in a medium-dependent manner. Genes in another cluster containing a putative histidine kinase/response regulator, peptide methionine sulfoxide reductase, thioredoxin protein, lipoprotein, and cytochrome c-type protein were downregulated in KS1ΩabpA under all of the environmental conditions tested. Thus, AbpA appears to modulate genes associated with maltodextrin utilization/transport and fatty acid synthesis. Importantly, in all growth conditions AbpA was associated with increased expression of a potential two-component signaling system associated with genes involved in reducing oxidative stress, suggesting a role in signal transduction and stress tolerance. PMID:26025889

Evaluation of Reference Genes for RT qPCR Analyses of Structure-Specific and Hormone Regulated Gene Expression in Physcomitrella patens Gametophytes

PubMed Central

Le Bail, Aude; Scholz, Sebastian; Kost, Benedikt

2013-01-01

The use of the moss Physcomitrella patens as a model system to study plant development and physiology is rapidly expanding. The strategic position of P. patens within the green lineage between algae and vascular plants, the high efficiency with which transgenes are incorporated by homologous recombination, advantages associated with the haploid gametophyte representing the dominant phase of the P. patens life cycle, the simple structure of protonemata, leafy shoots and rhizoids that constitute the haploid gametophyte, as well as a readily accessible high-quality genome sequence make this moss a very attractive experimental system. The investigation of the genetic and hormonal control of P. patens development heavily depends on the analysis of gene expression patterns by real time quantitative PCR (RT qPCR). This technique requires well characterized sets of reference genes, which display minimal expression level variations under all analyzed conditions, for data normalization. Sets of suitable reference genes have been described for most widely used model systems including e.g. Arabidopsis thaliana, but not for P. patens. Here, we present a RT qPCR based comparison of transcript levels of 12 selected candidate reference genes in a range of gametophytic P. patens structures at different developmental stages, and in P. patens protonemata treated with hormones or hormone transport inhibitors. Analysis of these RT qPCR data using GeNorm and NormFinder software resulted in the identification of sets of P. patens reference genes suitable for gene expression analysis under all tested conditions, and suggested that the two best reference genes are sufficient for effective data normalization under each of these conditions. PMID:23951063

Thermal processing of bone: in vitro response of mesenchymal cells to bone-conditioned medium.

PubMed

Sawada, K; Caballé-Serrano, J; Schuldt Filho, G; Bosshardt, D D; Schaller, B; Buser, D; Gruber, R

2015-08-01

The autoclaving, pasteurization, and freezing of bone grafts to remove bacteria and viruses, and for preservation, respectively, is considered to alter biological properties during graft consolidation. Fresh bone grafts release paracrine-like signals that are considered to support tissue regeneration. However, the impact of the autoclaving, pasteurization, and freezing of bone grafts on paracrine signals remains unknown. Therefore, conditioned medium was prepared from porcine cortical bone chips that had undergone thermal processing. The biological properties of the bone-conditioned medium were assessed by examining the changes in expression of target genes in oral fibroblasts. The data showed that conditioned medium obtained from bone chips that had undergone pasteurization and freezing changed the expression of adrenomedullin, pentraxin 3, BTB/POZ domain-containing protein 11, interleukin 11, NADPH oxidase 4, and proteoglycan 4 by at least five-fold in oral fibroblasts. Bone-conditioned medium obtained from autoclaved bone chips, however, failed to change the expression of the respective genes. Also, when bone-conditioned medium was prepared from fresh bone chips, autoclaving blocked the capacity of bone-conditioned medium to modulate gene expression. These in vitro results suggest that pasteurization and freezing of bone grafts preserve the release of biologically active paracrine signals, but autoclaving does not. Copyright © 2015 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.

Gene expression inference with deep learning.

PubMed

Chen, Yifei; Li, Yi; Narayan, Rajiv; Subramanian, Aravind; Xie, Xiaohui

2016-06-15

Large-scale gene expression profiling has been widely used to characterize cellular states in response to various disease conditions, genetic perturbations, etc. Although the cost of whole-genome expression profiles has been dropping steadily, generating a compendium of expression profiling over thousands of samples is still very expensive. Recognizing that gene expressions are often highly correlated, researchers from the NIH LINCS program have developed a cost-effective strategy of profiling only ∼1000 carefully selected landmark genes and relying on computational methods to infer the expression of remaining target genes. However, the computational approach adopted by the LINCS program is currently based on linear regression (LR), limiting its accuracy since it does not capture complex nonlinear relationship between expressions of genes. We present a deep learning method (abbreviated as D-GEX) to infer the expression of target genes from the expression of landmark genes. We used the microarray-based Gene Expression Omnibus dataset, consisting of 111K expression profiles, to train our model and compare its performance to those from other methods. In terms of mean absolute error averaged across all genes, deep learning significantly outperforms LR with 15.33% relative improvement. A gene-wise comparative analysis shows that deep learning achieves lower error than LR in 99.97% of the target genes. We also tested the performance of our learned model on an independent RNA-Seq-based GTEx dataset, which consists of 2921 expression profiles. Deep learning still outperforms LR with 6.57% relative improvement, and achieves lower error in 81.31% of the target genes. D-GEX is available at https://github.com/uci-cbcl/D-GEX CONTACT: xhx@ics.uci.edu Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Gene expression inference with deep learning

PubMed Central

Chen, Yifei; Li, Yi; Narayan, Rajiv; Subramanian, Aravind; Xie, Xiaohui

2016-01-01

Motivation: Large-scale gene expression profiling has been widely used to characterize cellular states in response to various disease conditions, genetic perturbations, etc. Although the cost of whole-genome expression profiles has been dropping steadily, generating a compendium of expression profiling over thousands of samples is still very expensive. Recognizing that gene expressions are often highly correlated, researchers from the NIH LINCS program have developed a cost-effective strategy of profiling only ∼1000 carefully selected landmark genes and relying on computational methods to infer the expression of remaining target genes. However, the computational approach adopted by the LINCS program is currently based on linear regression (LR), limiting its accuracy since it does not capture complex nonlinear relationship between expressions of genes. Results: We present a deep learning method (abbreviated as D-GEX) to infer the expression of target genes from the expression of landmark genes. We used the microarray-based Gene Expression Omnibus dataset, consisting of 111K expression profiles, to train our model and compare its performance to those from other methods. In terms of mean absolute error averaged across all genes, deep learning significantly outperforms LR with 15.33% relative improvement. A gene-wise comparative analysis shows that deep learning achieves lower error than LR in 99.97% of the target genes. We also tested the performance of our learned model on an independent RNA-Seq-based GTEx dataset, which consists of 2921 expression profiles. Deep learning still outperforms LR with 6.57% relative improvement, and achieves lower error in 81.31% of the target genes. Availability and implementation: D-GEX is available at https://github.com/uci-cbcl/D-GEX. Contact: xhx@ics.uci.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:26873929

Selection and validation of reference genes for gene expression analysis in apomictic and sexual Cenchrus ciliaris

PubMed Central

2013-01-01

Background Apomixis is a naturally occurring asexual mode of seed reproduction resulting in offspring genetically identical to the maternal plant. Identifying differential gene expression patterns between apomictic and sexual plants is valuable to help deconstruct the trait. Quantitative RT-PCR (qRT-PCR) is a popular method for analyzing gene expression. Normalizing gene expression data using proper reference genes which show stable expression under investigated conditions is critical in qRT-PCR analysis. We used qRT-PCR to validate expression and stability of six potential reference genes (EF1alpha, EIF4A, UBCE, GAPDH, ACT2 and TUBA) in vegetative and reproductive tissues of B-2S and B-12-9 accessions of C. ciliaris. Findings Among tissue types evaluated, EF1alpha showed the highest level of expression while TUBA showed the lowest. When all tissue types were evaluated and compared between genotypes, EIF4A was the most stable reference gene. Gene expression stability for specific ovary stages of B-2S and B-12-9 was also determined. Except for TUBA, all other tested reference genes could be used for any stage-specific ovary tissue normalization, irrespective of the mode of reproduction. Conclusion Our gene expression stability assay using six reference genes, in sexual and apomictic accessions of C. ciliaris, suggests that EIF4A is the most stable gene across all tissue types analyzed. All other tested reference genes, with the exception of TUBA, could be used for gene expression comparison studies between sexual and apomictic ovaries over multiple developmental stages. This reference gene validation data in C. ciliaris will serve as an important base for future apomixis-related transcriptome data validation. PMID:24083672

Saccharomyces cerevisiae possesses a stress-inducible glycyl-tRNA synthetase gene.

PubMed

Chen, Shun-Jia; Wu, Yi-Hua; Huang, Hsiao-Yun; Wang, Chien-Chia

2012-01-01

Aminoacyl-tRNA synthetases are a large family of housekeeping enzymes that are pivotal in protein translation and other vital cellular processes. Saccharomyces cerevisiae possesses two distinct nuclear glycyl-tRNA synthetase (GlyRS) genes, GRS1 and GRS2. GRS1 encodes both cytoplasmic and mitochondrial activities, while GRS2 is essentially silent and dispensable under normal conditions. We herein present evidence that expression of GRS2 was drastically induced upon heat shock, ethanol or hydrogen peroxide addition, and high pH, while expression of GRS1 was somewhat repressed under those conditions. In addition, GlyRS2 (the enzyme encoded by GRS2) had a higher protein stability and a lower K(M) value for yeast tRNA(Gly) under heat shock conditions than under normal conditions. Moreover, GRS2 rescued the growth defect of a GRS1 knockout strain when highly expressed by a strong promoter at 37 °C, but not at the optimal temperature of 30 °C. These results suggest that GRS2 is actually an inducible gene that may function to rescue the activity of GRS1 under stress conditions.

Mining microarray datasets in nutrition: expression of the GPR120 (n-3 fatty acid receptor/sensor) gene is down-regulated in human adipocytes by macrophage secretions.

PubMed

Trayhurn, Paul; Denyer, Gareth

2012-01-01

Microarray datasets are a rich source of information in nutritional investigation. Targeted mining of microarray data following initial, non-biased bioinformatic analysis can provide key insight into specific genes and metabolic processes of interest. Microarrays from human adipocytes were examined to explore the effects of macrophage secretions on the expression of the G-protein-coupled receptor (GPR) genes that encode fatty acid receptors/sensors. Exposure of the adipocytes to macrophage-conditioned medium for 4 or 24 h had no effect on GPR40 and GPR43 expression, but there was a marked stimulation of GPR84 expression (receptor for medium-chain fatty acids), the mRNA level increasing 13·5-fold at 24 h relative to unconditioned medium. Importantly, expression of GPR120, which encodes an n-3 PUFA receptor/sensor, was strongly inhibited by the conditioned medium (15-fold decrease in mRNA at 24 h). Macrophage secretions have major effects on the expression of fatty acid receptor/sensor genes in human adipocytes, which may lead to an augmentation of the inflammatory response in adipose tissue in obesity.

Mining microarray datasets in nutrition: expression of the GPR120 (n-3 fatty acid receptor/sensor) gene is down-regulated in human adipocytes by macrophage secretions

PubMed Central

Trayhurn, Paul; Denyer, Gareth

2012-01-01

Microarray datasets are a rich source of information in nutritional investigation. Targeted mining of microarray data following initial, non-biased bioinformatic analysis can provide key insight into specific genes and metabolic processes of interest. Microarrays from human adipocytes were examined to explore the effects of macrophage secretions on the expression of the G-protein-coupled receptor (GPR) genes that encode fatty acid receptors/sensors. Exposure of the adipocytes to macrophage-conditioned medium for 4 or 24 h had no effect on GPR40 and GPR43 expression, but there was a marked stimulation of GPR84 expression (receptor for medium-chain fatty acids), the mRNA level increasing 13·5-fold at 24 h relative to unconditioned medium. Importantly, expression of GPR120, which encodes an n-3 PUFA receptor/sensor, was strongly inhibited by the conditioned medium (15-fold decrease in mRNA at 24 h). Macrophage secretions have major effects on the expression of fatty acid receptor/sensor genes in human adipocytes, which may lead to an augmentation of the inflammatory response in adipose tissue in obesity. PMID:25191551

A transcriptional approach to unravel the connection between phospholipases A₂ and D and ABA signal in citrus under water stress.

PubMed

Romero, Paco; Lafuente, M Teresa; Alférez, Fernando

2014-07-01

The effect of water stress on the interplay between phospholipases (PL) A2 and D and ABA signalling was investigated in fruit and leaves from the sweet orange Navelate and its fruit-specific ABA-deficient mutant Pinalate by studying simultaneously expression of 5 PLD and 3 PLA2-encoding genes. In general, expression levels of PLD-encoding genes were higher at harvest in the flavedo (coloured outer part of the peel) from Pinalate. Moreover, a higher and transient increase in expression of CsPLDα, CsPLDβ, CsPLDδ and CsPLDζ was observed in the mutant as compared to Navelate fruit under water stress, which may reflect a mechanism of acclimation to water stress influenced by ABA deficiency. An early induction in CsPLDγ gene expression, when increase in peel damage during fruit storage was most evident, suggested a role for this gene in membrane degradation processes during water stress. Exogenous ABA on mutant fruit modified the expression of all PLD genes and reduced the expression of CsPLDα and CsPLDβ by 1 week to levels similar to those of Navelate, suggesting a repressor role of ABA on these genes. In general, CssPLA2α and β transcript levels were lower in flavedo from Pinalate than from Navelate fruit during the first 3 weeks of storage, suggesting that expression of these genes also depends at least partially on ABA levels. Patterns of expression of PLD and PLA2-encoding genes were very similar in Navelate and Pinalate leaves, which have similar ABA levels, when comparing both RH conditions. Results comparison with other from previous works in the same experimental systems helped to decipher the effect of the stress severity on the differential response of some of these genes under dehydration conditions and pointed out the interplay between PLA2 and PLD families and their connection with ABA signalling in citrus. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Extreme Hypoxic Conditions Induce Selective Molecular Responses and Metabolic Reset in Detached Apple Fruit

PubMed Central

Cukrov, Dubravka; Zermiani, Monica; Brizzolara, Stefano; Cestaro, Alessandro; Licausi, Francesco; Luchinat, Claudio; Santucci, Claudio; Tenori, Leonardo; Van Veen, Hans; Zuccolo, Andrea; Ruperti, Benedetto; Tonutti, Pietro

2016-01-01

The ripening physiology of detached fruit is altered by low oxygen conditions with profound effects on quality parameters. To study hypoxia-related processes and regulatory mechanisms, apple (Malus domestica, cv Granny Smith) fruit, harvested at commercial ripening, were kept at 1°C under normoxic (control) and hypoxic (0.4 and 0.8 kPa oxygen) conditions for up to 60 days. NMR analyses of cortex tissue identified eight metabolites showing significantly different accumulations between samples, with ethanol and alanine displaying the most pronounced difference between hypoxic and normoxic treatments. A rapid up-regulation of alcohol dehydrogenase and pyruvate-related metabolism (lactate dehydrogenase, pyruvate decarboxylase, alanine aminotransferase) gene expression was detected under both hypoxic conditions with a more pronounced effect induced by the lowest (0.4 kPa) oxygen concentration. Both hypoxic conditions negatively affected ACC synthase and ACC oxidase transcript accumulation. Analysis of RNA-seq data of samples collected after 24 days of hypoxic treatment identified more than 1000 genes differentially expressed when comparing 0.4 vs. 0.8 kPa oxygen concentration samples. Genes involved in cell-wall, minor and major CHO, amino acid and secondary metabolisms, fermentation and glycolysis as well as genes involved in transport, defense responses, and oxidation-reduction appeared to be selectively affected by treatments. The lowest oxygen concentration induced a higher expression of transcription factors belonging to AUX/IAA, WRKY, HB, Zinc-finger families, while MADS box family genes were more expressed when apples were kept under 0.8 kPa oxygen. Out of the eight group VII ERF members present in apple genome, two genes showed a rapid up-regulation under hypoxia, and western blot analysis showed that apple MdRAP2.12 proteins were differentially accumulated in normoxic and hypoxic samples, with the highest level reached under 0.4 kPa oxygen. These data suggest that ripe apple tissues finely and specifically modulate sensing and regulatory mechanisms in response to different hypoxic stress conditions. PMID:26909091

[Endoplasmic reticulum stress in INS-1-3 cell associated with the expression changes of MODY gene pathway].

PubMed

Liu, Y T; Li, S R; Wang, Z; Xiao, J Z

2016-09-13

Objective: To profile the gene expression changes associated with endoplasmic reticulum stress in INS-1-3 cells induced by thapsigargin (TG) and tunicamycin (TM). Methods: Normal cultured INS-1-3 cells were used as a control. TG and TM were used to induce endoplasmic reticulum stress in INS-1-3 cells. Digital gene expression profiling technique was used to detect differentially expressed gene. The changes of gene expression were detected by expression pattern clustering analysis, gene ontology (GO) function and pathway enrichment analysis. Real time polymerase chain reaction (RT-PCR) was used to verify the key changes of gene expression. Results: Compared with the control group, there were 57 (45 up-regulated, 12 down-regulated) and 135 (99 up-regulated, 36 down-regulated) differentially expressed genes in TG and TM group, respectively. GO function enrichment analyses indicated that the main enrichment was in the endoplasmic reticulum. In signaling pathway analysis, the identified pathways were related with endoplasmic reticulum stress, antigen processing and presentation, protein export, and most of all, the maturity onset diabetes of the young (MODY) pathway. Conclusion: Under the condition of endoplasmic reticulum stress, the related expression changes of transcriptional factors in MODY signaling pathway may be related with the impaired function in islet beta cells.

With Reference to Reference Genes: A Systematic Review of Endogenous Controls in Gene Expression Studies.

PubMed

Chapman, Joanne R; Waldenström, Jonas

2015-01-01

The choice of reference genes that are stably expressed amongst treatment groups is a crucial step in real-time quantitative PCR gene expression studies. Recent guidelines have specified that a minimum of two validated reference genes should be used for normalisation. However, a quantitative review of the literature showed that the average number of reference genes used across all studies was 1.2. Thus, the vast majority of studies continue to use a single gene, with β-actin (ACTB) and/or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) being commonly selected in studies of vertebrate gene expression. Few studies (15%) tested a panel of potential reference genes for stability of expression before using them to normalise data. Amongst studies specifically testing reference gene stability, few found ACTB or GAPDH to be optimal, whereby these genes were significantly less likely to be chosen when larger panels of potential reference genes were screened. Fewer reference genes were tested for stability in non-model organisms, presumably owing to a dearth of available primers in less well characterised species. Furthermore, the experimental conditions under which real-time quantitative PCR analyses were conducted had a large influence on the choice of reference genes, whereby different studies of rat brain tissue showed different reference genes to be the most stable. These results highlight the importance of validating the choice of normalising reference genes before conducting gene expression studies.

Bone-related gene profiles in developing calvaria.

PubMed

Cho, Je-Yoel; Lee, Won-Bong; Kim, Hyun-Jung; Mi Woo, Kyung; Baek, Jeong-Hwa; Choi, Je-Yong; Hur, Cheol-Gu; Ryoo, Hyun-Mo

2006-05-10

Generating a comprehensive understanding of osteogenesis-related gene profiles is very important in the development of new treatments for osteopenic conditions. Developing calvaria undergoes a typical intramembranous bone-forming process. To identify genes associated with osteoblast differentiation, we isolated total RNAs from parietal bones, that represent active osteoblasts, and sutural mesenchyme, that represents osteoprogenitor cells, and comprehensively analyzed their gene expression profiles using an oligo-based Affymetrix microarray chip containing 22,690 probes. About 2100 genes with "Present" calls had more than 2-fold higher expression in bone compared to sutures while 73 of these genes had more than 8-fold expression. Some of these genes are already known to be bone-related biomarkers: VitD receptor, bone sialoprotein, osteocalcin, osteopontin, MMP13, etc. Eight genes were selected and subjected to confirmation by quantitative real-time RT-PCR analyses. All the genes tested showed higher expression in bones, ranging from 5- to 140-fold. Several of these genes are ESTs while others are already known but their functions in osteogenesis were not previously known. Most genes of the BMP and FGF families probed in the Genechip analysis were more highly expressed in bone tissues compared to suture. All differentially-expressed Runx and Dlx family genes also showed higher expression in bone. These results imply that our data is valid and can be used as a good standard for the mining of osteogenesis-related genes.

Long-range transcriptional interference in E. coli used to construct a dual positive selection system for genetic switches

PubMed Central

Hoffmann, Stefan A.; Kruse, Sabrina M.; Arndt, Katja M.

2016-01-01

Abstract We have investigated transcriptional interference between convergent genes in E. coli and demonstrate substantial interference for inter-promoter distances of as far as 3 kb. Interference can be elicited by both strong σ70 dependent and T7 promoters. In the presented design, a strong promoter driving gene expression of a ‘forward’ gene interferes with the expression of a ‘reverse’ gene by a weak promoter. This arrangement allows inversely correlated gene expression without requiring further regulatory components. Thus, modulation of the activity of the strong promoter alters expression of both the forward and the reverse gene. We used this design to develop a dual selection system for conditional operator site binding, allowing positive selection both for binding and for non-binding to DNA. This study demonstrates the utility of this novel system using the Lac repressor as a model protein for conditional DNA binding, and spectinomycin and chloramphenicol resistance genes as positive selection markers in liquid culture. Randomized LacI libraries were created and subjected to subsequent dual selection, but mispairing IPTG and selection cues in respect to the wild-type LacI response, allowing the isolation of a LacI variant with a reversed IPTG response within three rounds of library generation and dual selection. PMID:26932362

Effect of hypoxia on the expression of genes encoding insulin-like growth factors and some related proteins in U87 glioma cells without IRE1 function.

PubMed

Minchenko, Dmytro O; Kharkova, A P; Halkin, O V; Karbovskyi, L L; Minchenko, O H

2016-04-01

The aim of the present study was to investigate the effect of hypoxia on the expression of genes encoding insulin-like growth factors (IGF1 and IGF2), their receptor (IGF1R), binding protein-4 (IGFBP4), and stanniocalcin 2 (STC2) in U87 glioma cells in relation to inhibition of endoplasmic reticulum stress signaling mediated by IRE1 (inositol requiring enzyme 1) for evaluation of their possible significance in the control of tumor growth. The expression of IGF1, IGF2, IGF1R, IGFBP4, and STC2 genes in U87 glioma cells transfected by empty vector pcDNA3.1 (control) and cells without IRE1 signaling enzyme function (transfected by dnIRE1) upon hypoxia was studied by qPCR. The expression of IGF1 and IGF2 genes is down-regulated in glioma cells without IRE1 signaling enzyme function in comparison with the control cells. At the same time, the expression of IGF1R, IGFBP4, and STC2 genes was up-regulated in glioma cells upon inhibition of IRE1, with more significant changes for IGFBP4 and STC2 genes. We also showed that hypoxia does not change significantly the expression of IGF1, IGF2, and IGF1R genes but up-regulated IGFBP4 and STC2 genes expression in control glioma cells. Moreover, the inhibition of both enzymatic activities (kinase and endoribonuclease) of IRE1 in glioma cells does not change significantly the effect of hypoxia on the expression of IGF1, IGF1R, and IGFBP4 genes but introduces sensitivity of IGF2 gene to hypoxic condition. Thus, the expression of IGF2 gene is resistant to hypoxia only in control glioma cells and significantly down-regulated in cells without functional activity of IRE1 signaling enzyme, which is central mediator of the unfolded protein response and an important component of the tumor growth as well as metabolic diseases. Results of this study demonstrate that the expression of IGF1 and IGF1R genes is resistant to hypoxic condition both in control U87 glioma cells and cells without IRE1 signaling enzyme function. However, hypoxia significantly up-regulates the expression of IGFBP4 gene independently on the inhibition of IRE1 enzyme. These data show that proteins encoded by these genes are resistant to hypoxia except IGFBP4 and participate in the regulation of metabolic and proliferative processes through IRE1 signaling.

ATNT: an enhanced system for expression of polycistronic secondary metabolite gene clusters in Aspergillus niger.

PubMed

Geib, Elena; Brock, Matthias

2017-01-01

Fungi are treasure chests for yet unexplored natural products. However, exploitation of their real potential remains difficult as a significant proportion of biosynthetic gene clusters appears silent under standard laboratory conditions. Therefore, elucidation of novel products requires gene activation or heterologous expression. For heterologous gene expression, we previously developed an expression platform in Aspergillus niger that is based on the transcriptional regulator TerR and its target promoter P terA . In this study, we extended this system by regulating expression of terR  by the doxycycline inducible Tet-on system. Reporter genes cloned under the control of the target promoter P terA remained silent in the absence of doxycycline, but were strongly expressed when doxycycline was added. Reporter quantification revealed that the coupled system results in about five times higher expression rates compared to gene expression under direct control of the Tet-on system. As production of secondary metabolites generally requires the expression of several biosynthetic genes, the suitability of the self-cleaving viral peptide sequence P2A was tested in this optimised expression system. P2A allowed polycistronic expression of genes required for Asp-melanin formation in combination with the gene coding for the red fluorescent protein tdTomato. Gene expression and Asp-melanin formation was prevented in the absence of doxycycline and strongly induced by addition of doxycycline. Fluorescence studies confirmed the correct subcellular localisation of the respective enzymes. This tightly regulated but strongly inducible expression system enables high level production of secondary metabolites most likely even those with toxic potential. Furthermore, this system is compatible with polycistronic gene expression and, thus, suitable for the discovery of novel natural products.

Inhibition of N-Myc down regulated gene 1 in in vitro cultured human glioblastoma cells

PubMed Central

Said, Harun M; Polat, Buelent; Stein, Susanne; Guckenberger, Mathias; Hagemann, Carsten; Staab, Adrian; Katzer, Astrid; Anacker, Jelena; Flentje, Michael; Vordermark, Dirk

2012-01-01

AIM: To study short dsRNA oligonucleotides (siRNA) as a potent tool for artificially modulating gene expression of N-Myc down regulated gene 1 (NDRG1) gene induced under different physiological conditions (Normoxia and hypoxia) modulating NDRG1 transcription, mRNA stability and translation. METHODS: A cell line established from a patient with glioblastoma multiforme. Plasmid DNA for transfections was prepared with the Endofree Plasmid Maxi kit. From plates containing 5 × 107 cells, nuclear extracts were prepared according to previous protocols. The pSUPER-NDRG1 vectors were designed, two sequences were selected from the human NDRG1 cDNA (5’-GCATTATTGGCATGGGAAC-3’ and 5’-ATGCAGAGTAACGTGGAAG-3’. reverse transcription polymerase chain reaction was performed using primers designed using published information on β-actin and hypoxia-inducible factor (HIF)-1α mRNA sequences in GenBank. NDRG1 mRNA and protein level expression results under different conditions of hypoxia or reoxygenation were compared to aerobic control conditions using the Mann-Whitney U test. Reoxygenation values were also compared to the NDRG1 levels after 24 h of hypoxia (P < 0.05 was considered significant). RESULTS: siRNA- and iodoacetate (IAA)-mediated downregulation of NDRG1 mRNA and protein expression in vitro in human glioblastoma cell lines showed a nearly complete inhibition of NDRG1 expression when compared to the results obtained due to the inhibitory role of glycolysis inhibitor IAA. Hypoxia responsive elements bound by nuclear HIF-1 in human glioblastoma cells in vitro under different oxygenation conditions and the clearly enhanced binding of nuclear extracts from glioblastoma cell samples exposed to extreme hypoxic conditions confirmed the HIF-1 Western blotting results. CONCLUSION: NDRG1 represents an additional diagnostic marker for brain tumor detection, due to the role of hypoxia in regulating this gene, and it can represent a potential target for tumor treatment in human glioblastoma. The siRNA method can represent an elegant alternative to modulate the expression of the hypoxia induced NDRG1 gene and can help to monitor the development of the cancer disease treatment outcome through monitoring the expression of this gene in the patients undergoing the different therapeutic treatment alternatives available nowadays. PMID:22787578

Validation of Reference Genes for Gene Expression Studies in Virus-Infected Nicotiana benthamiana Using Quantitative Real-Time PCR

PubMed Central

Han, Chenggui; Yu, Jialin; Li, Dawei; Zhang, Yongliang

2012-01-01

Nicotiana benthamiana is the most widely-used experimental host in plant virology. The recent release of the draft genome sequence for N. benthamiana consolidates its role as a model for plant–pathogen interactions. Quantitative real-time PCR (qPCR) is commonly employed for quantitative gene expression analysis. For valid qPCR analysis, accurate normalisation of gene expression against an appropriate internal control is required. Yet there has been little systematic investigation of reference gene stability in N. benthamiana under conditions of viral infections. In this study, the expression profiles of 16 commonly used housekeeping genes (GAPDH, 18S, EF1α, SAMD, L23, UK, PP2A, APR, UBI3, SAND, ACT, TUB, GBP, F-BOX, PPR and TIP41) were determined in N. benthamiana and those with acceptable expression levels were further selected for transcript stability analysis by qPCR of complementary DNA prepared from N. benthamiana leaf tissue infected with one of five RNA plant viruses (Tobacco necrosis virus A, Beet black scorch virus, Beet necrotic yellow vein virus, Barley stripe mosaic virus and Potato virus X). Gene stability was analysed in parallel by three commonly-used dedicated algorithms: geNorm, NormFinder and BestKeeper. Statistical analysis revealed that the PP2A, F-BOX and L23 genes were the most stable overall, and that the combination of these three genes was sufficient for accurate normalisation. In addition, the suitability of PP2A, F-BOX and L23 as reference genes was illustrated by expression-level analysis of AGO2 and RdR6 in virus-infected N. benthamiana leaves. This is the first study to systematically examine and evaluate the stability of different reference genes in N. benthamiana. Our results not only provide researchers studying these viruses a shortlist of potential housekeeping genes to use as normalisers for qPCR experiments, but should also guide the selection of appropriate reference genes for gene expression studies of N. benthamiana under other biotic and abiotic stress conditions. PMID:23029521

Extraordinary diversity of visual opsin genes in dragonflies

PubMed Central

Futahashi, Ryo; Kawahara-Miki, Ryouka; Kinoshita, Michiyo; Yoshitake, Kazutoshi; Yajima, Shunsuke; Arikawa, Kentaro; Fukatsu, Takema

2015-01-01

Dragonflies are colorful and large-eyed animals strongly dependent on color vision. Here we report an extraordinary large number of opsin genes in dragonflies and their characteristic spatiotemporal expression patterns. Exhaustive transcriptomic and genomic surveys of three dragonflies of the family Libellulidae consistently identified 20 opsin genes, consisting of 4 nonvisual opsin genes and 16 visual opsin genes of 1 UV, 5 short-wavelength (SW), and 10 long-wavelength (LW) type. Comprehensive transcriptomic survey of the other dragonflies representing an additional 10 families also identified as many as 15–33 opsin genes. Molecular phylogenetic analysis revealed dynamic multiplications and losses of the opsin genes in the course of evolution. In contrast to many SW and LW genes expressed in adults, only one SW gene and several LW genes were expressed in larvae, reflecting less visual dependence and LW-skewed light conditions for their lifestyle under water. In this context, notably, the sand-burrowing or pit-dwelling species tended to lack SW gene expression in larvae. In adult visual organs: (i) many SW genes and a few LW genes were expressed in the dorsal region of compound eyes, presumably for processing SW-skewed light from the sky; (ii) a few SW genes and many LW genes were expressed in the ventral region of compound eyes, probably for perceiving terrestrial objects; and (iii) expression of a specific LW gene was associated with ocelli. Our findings suggest that the stage- and region-specific expressions of the diverse opsin genes underlie the behavior, ecology, and adaptation of dragonflies. PMID:25713365

Promises and pitfalls of a Pannexin1 transgenic mouse line.

PubMed

Hanstein, Regina; Negoro, Hiromitsu; Patel, Naman K; Charollais, Anne; Meda, Paolo; Spray, David C; Suadicani, Sylvia O; Scemes, Eliana

2013-01-01

Gene targeting strategies have become a powerful technology for elucidating mammalian gene function. The recently generated knockout (KO)-first strategy produces a KO at the RNA processing level and also allows for the generation of conditional KO alleles by combining FLP/FRT and Cre/loxP systems, thereby providing high flexibility in gene manipulation. However, this multipurpose KO-first cassette might produce hypomorphic rather than complete KOs if the RNA processing module is bypassed. Moreover, the generation of a conditional phenotype is also dependent on specific activity of Cre recombinase. Here, we report the use of an efficient molecular biological approach to test pannexin1 (Panx1) mRNA expression in global and conditional Panx1 KO mice derived from the KO-first mouse line, Panx1(tm1a(KOMP)Wtsi). Using qRT-PCR, we demonstrate that tissues from wild-type (WT) mice show a range of Panx1 mRNA expression levels, with highest expression in trigeminal ganglia, bladder and spleen. Unexpectedly, we found that in mice homozygous for the KO-first allele, Panx1 mRNA expression is not abolished but reduced by 70% compared to that of WT tissues. Thus, Panx1 KO-first mice present a hypomorphic phenotype. Crosses of Panx1 KO-first with FLP deleter mice generated Panx1(f/f) mice. Further crosses of the latter mice with mGFAP-Cre or NFH-Cre mice were used to generate astrocyte- and neuron-specific Panx1 deletions, respectively. A high incidence of ectopic Cre expression was found in offspring of both types of conditional Panx1 KO mice. Our study demonstrates that Panx1 expression levels in the global and conditional Panx1 KO mice derived from KO-first mouse lines must be carefully characterized to ensure modulation of Panx1 gene expression. The precise quantitation of Panx1 expression and its relation to function is expected to provide a foundation for future efforts aimed at deciphering the role of Panx1 under physiological and pathological conditions.

Iron-Regulated Expression of Alginate Production, Mucoid Phenotype, and Biofilm Formation by Pseudomonas aeruginosa

PubMed Central

Wiens, Jacinta R.; Vasil, Adriana I.; Schurr, Michael J.; Vasil, Michael L.

2014-01-01

ABSTRACT Pseudomonas aeruginosa strains of non-cystic fibrosis (non-CF) origin do not produce significant amounts of extracellular alginate and are nonmucoid. In CF, such isolates can become mucoid through mutation of one of the genes (mucA, mucB, mucC, or mucD) that produce regulatory factors that sequester AlgU, required for increased expression of alginate genes. Mutation of the muc genes in the nonmucoid PAO1, PA14, PAKS-1, and Ps388 strains led to increased levels of extracellular alginate and an obvious mucoid phenotype, but only under iron-limiting growth conditions (≤5 µM), not under iron-replete conditions (≥10 µM). In contrast, >50% of P. aeruginosa isolates from chronic CF pulmonary infections expressed increased levels of alginate and mucoidy both under iron-limiting and iron-replete conditions (i.e., iron-constitutive phenotype). No single iron regulatory factor (e.g., Fur, PvdS) was associated with this loss of iron-regulated alginate expression and mucoidy in these CF isolates. However, the loss of only pyoverdine production, or its uptake, abrogated the ability of P. aeruginosa to produce a robust biofilm that represents the Psl-type of biofilm. In contrast, we show that mutation of the pyoverdine and pyochelin biosynthesis genes and the pyoverdine receptor (FpvA) lead to iron-constitutive expression of the key alginate biosynthesis gene, algD, and an explicitly mucoid phenotype in both iron-limiting and iron-replete conditions. These data indicate that alginate production and mucoidy, in contrast to other types of biofilms produced by P. aeruginosa, are substantially enhanced under iron limitation. These results also have compelling implications in relation to the use of iron chelators in the treatment of P. aeruginosa CF infections. PMID:24496793

Identification of Reference Genes for RT-qPCR Data Normalization in Cannabis sativa Stem Tissues.

PubMed

Mangeot-Peter, Lauralie; Legay, Sylvain; Hausman, Jean-Francois; Esposito, Sergio; Guerriero, Gea

2016-09-15

Gene expression profiling via quantitative real-time PCR is a robust technique widely used in the life sciences to compare gene expression patterns in, e.g., different tissues, growth conditions, or after specific treatments. In the field of plant science, real-time PCR is the gold standard to study the dynamics of gene expression and is used to validate the results generated with high throughput techniques, e.g., RNA-Seq. An accurate relative quantification of gene expression relies on the identification of appropriate reference genes, that need to be determined for each experimental set-up used and plant tissue studied. Here, we identify suitable reference genes for expression profiling in stems of textile hemp (Cannabis sativa L.), whose tissues (isolated bast fibres and core) are characterized by remarkable differences in cell wall composition. We additionally validate the reference genes by analysing the expression of putative candidates involved in the non-oxidative phase of the pentose phosphate pathway and in the first step of the shikimate pathway. The goal is to describe the possible regulation pattern of some genes involved in the provision of the precursors needed for lignin biosynthesis in the different hemp stem tissues. The results here shown are useful to design future studies focused on gene expression analyses in hemp.

Understanding the impact of water distribution system conditions on the biodegradation of haloacetic acids and expression of bacterial dehalogenase genes.

PubMed

Behbahani, Mohsen; Lin, Boren; Phares, Tamara L; Seo, Youngwoo

2018-06-05

The objective of this study is to evaluate the influence of water distribution system conditions (pH, total organic carbon, residual chlorine, and phosphate) on haloacetic acids (HAAs) biodegradation. A series of batch microcosm tests were conducted to determine biodegradation kinetics and collected biomass was used for real time quantitative reverse transcription polymerase chain reaction analyses to monitor how these drinking water distribution system conditions affect the relative expression of bacterial dehalogenase genes. It was observed that tested water distribution system conditions affected HAA biodegradation with different removal efficiencies (0-100%). HAA biodegradation was improved in tested samples with TOC (3 mg/L) and pH 8.5 compared to those of TOC (0 mg/L) and pH 7, respectively. However, slight improvement was observed with the increased PO 4 concentration (3.5 mg/L), and the presence of residual chlorine even at low concentration prohibited biodegradation of HAAs. The observed trend in the relative expression of dehII genes was compatible with the HAA biodegradation trend. Overall relative expression ratio of dehII genes was lower at pH 7, phosphate (0.5 mg/L), and TOC (0 mg/L) in comparison with pH 8.5, phosphate (3.5 mg/L), and TOC (3 mg/L) in the same experimental conditions. Copyright © 2018 Elsevier B.V. All rights reserved.

Mediator phosphorylation prevents stress response transcription during non-stress conditions.

PubMed

Miller, Christian; Matic, Ivan; Maier, Kerstin C; Schwalb, Björn; Roether, Susanne; Strässer, Katja; Tresch, Achim; Mann, Matthias; Cramer, Patrick

2012-12-28

The multiprotein complex Mediator is a coactivator of RNA polymerase (Pol) II transcription that is required for the regulated expression of protein-coding genes. Mediator serves as an end point of signaling pathways and regulates Pol II transcription, but the mechanisms it uses are not well understood. Here, we used mass spectrometry and dynamic transcriptome analysis to investigate a functional role of Mediator phosphorylation in gene expression. Affinity purification and mass spectrometry revealed that Mediator from the yeast Saccharomyces cerevisiae is phosphorylated at multiple sites of 17 of its 25 subunits. Mediator phosphorylation levels change upon an external stimulus set by exposure of cells to high salt concentrations. Phosphorylated sites in the Mediator tail subunit Med15 are required for suppression of stress-induced changes in gene expression under non-stress conditions. Thus dynamic and differential Mediator phosphorylation contributes to gene regulation in eukaryotic cells.

Post-capture immune gene expression studies in the deep-sea hydrothermal vent mussel Bathymodiolus azoricus acclimatized to atmospheric pressure.

PubMed

Barros, Inês; Divya, Baby; Martins, Inês; Vandeperre, Frederic; Santos, Ricardo Serrão; Bettencourt, Raul

2015-01-01

Deep-sea hydrothermal vents are extreme habitats that are distributed worldwide in association with volcanic and tectonic events, resulting thus in the establishment of particular environmental conditions, in which high pressure, steep temperature gradients, and potentially toxic concentrations of sulfur, methane and heavy metals constitute driving factors for the foundation of chemosynthetic-based ecosystems. Of all the different macroorganisms found at deep-sea hydrothermal vents, the mussel Bathymodiolus azoricus is the most abundant species inhabiting the vent ecosystems from the Mid-Atlantic Ridge (MAR). In the present study, the effect of long term acclimatization at atmospheric pressure on host-symbiotic associations were studied in light of the ensuing physiological adaptations from which the immune and endosymbiont gene expressions were concomitantly quantified by means of real-time PCR. The expression of immune genes at 0 h, 12 h, 24 h, 36 h, 48 h, 72 h, 1 week and 3 weeks post-capture acclimatization was investigated and their profiles compared across the samples tested. The gene signal distribution for host immune and bacterial genes followed phasic changes in gene expression at 24 h, 1 week and 3 weeks acclimatization when compared to other time points tested during this temporal expression study. Analyses of the bacterial gene expression also suggested that both bacterial density and activity could contribute to shaping the intricate association between endosymbionts and host immune genes whose expression patterns seem to be concomitant at 1 week acclimatization. Fluorescence in situ hybridization was used to assess the distribution and prevalence of endosymbiont bacteria within gill tissues confirming the gradual loss of sulfur-oxidizing (SOX) and methane-oxidizing (MOX) bacteria during acclimatization. The present study addresses the deep-sea vent mussel B. azoricus as a model organism to study how acclimatization in aquaria and the prevalence of symbiotic bacteria are driving the expression of host immune genes. Tight associations, unseen thus far, suggest that host immune and bacterial gene expression patterns reflect distinct physiological responses over the course of acclimatization under aquarium conditions. Copyright © 2014 Elsevier Ltd. All rights reserved.

Identifying Stable Reference Genes for qRT-PCR Normalisation in Gene Expression Studies of Narrow-Leafed Lupin (Lupinus angustifolius L.).

PubMed

Taylor, Candy M; Jost, Ricarda; Erskine, William; Nelson, Matthew N

2016-01-01

Quantitative Reverse Transcription PCR (qRT-PCR) is currently one of the most popular, high-throughput and sensitive technologies available for quantifying gene expression. Its accurate application depends heavily upon normalisation of gene-of-interest data with reference genes that are uniformly expressed under experimental conditions. The aim of this study was to provide the first validation of reference genes for Lupinus angustifolius (narrow-leafed lupin, a significant grain legume crop) using a selection of seven genes previously trialed as reference genes for the model legume, Medicago truncatula. In a preliminary evaluation, the seven candidate reference genes were assessed on the basis of primer specificity for their respective targeted region, PCR amplification efficiency, and ability to discriminate between cDNA and gDNA. Following this assessment, expression of the three most promising candidates [Ubiquitin C (UBC), Helicase (HEL), and Polypyrimidine tract-binding protein (PTB)] was evaluated using the NormFinder and RefFinder statistical algorithms in two narrow-leafed lupin lines, both with and without vernalisation treatment, and across seven organ types (cotyledons, stem, leaves, shoot apical meristem, flowers, pods and roots) encompassing three developmental stages. UBC was consistently identified as the most stable candidate and has sufficiently uniform expression that it may be used as a sole reference gene under the experimental conditions tested here. However, as organ type and developmental stage were associated with greater variability in relative expression, it is recommended using UBC and HEL as a pair to achieve optimal normalisation. These results highlight the importance of rigorously assessing candidate reference genes for each species across a diverse range of organs and developmental stages. With emerging technologies, such as RNAseq, and the completion of valuable transcriptome data sets, it is possible that other potentially more suitable reference genes will be identified for this species in future.

Identifying Stable Reference Genes for qRT-PCR Normalisation in Gene Expression Studies of Narrow-Leafed Lupin (Lupinus angustifolius L.)

PubMed Central

Erskine, William; Nelson, Matthew N.

2016-01-01

Quantitative Reverse Transcription PCR (qRT-PCR) is currently one of the most popular, high-throughput and sensitive technologies available for quantifying gene expression. Its accurate application depends heavily upon normalisation of gene-of-interest data with reference genes that are uniformly expressed under experimental conditions. The aim of this study was to provide the first validation of reference genes for Lupinus angustifolius (narrow-leafed lupin, a significant grain legume crop) using a selection of seven genes previously trialed as reference genes for the model legume, Medicago truncatula. In a preliminary evaluation, the seven candidate reference genes were assessed on the basis of primer specificity for their respective targeted region, PCR amplification efficiency, and ability to discriminate between cDNA and gDNA. Following this assessment, expression of the three most promising candidates [Ubiquitin C (UBC), Helicase (HEL), and Polypyrimidine tract-binding protein (PTB)] was evaluated using the NormFinder and RefFinder statistical algorithms in two narrow-leafed lupin lines, both with and without vernalisation treatment, and across seven organ types (cotyledons, stem, leaves, shoot apical meristem, flowers, pods and roots) encompassing three developmental stages. UBC was consistently identified as the most stable candidate and has sufficiently uniform expression that it may be used as a sole reference gene under the experimental conditions tested here. However, as organ type and developmental stage were associated with greater variability in relative expression, it is recommended using UBC and HEL as a pair to achieve optimal normalisation. These results highlight the importance of rigorously assessing candidate reference genes for each species across a diverse range of organs and developmental stages. With emerging technologies, such as RNAseq, and the completion of valuable transcriptome data sets, it is possible that other potentially more suitable reference genes will be identified for this species in future. PMID:26872362

Comparison of the global gene expression of choroid plexus and meninges and associated vasculature under control conditions and after pronounced hyperthermia or amphetamine toxicity

PubMed Central

2013-01-01

Background The meninges (arachnoid and pial membranes) and associated vasculature (MAV) and choroid plexus are important in maintaining cerebrospinal fluid (CSF) generation and flow. MAV vasculature was previously observed to be adversely affected by environmentally-induced hyperthermia (EIH) and more so by a neurotoxic amphetamine (AMPH) exposure. Herein, microarray and RT-PCR analysis was used to compare the gene expression profiles between choroid plexus and MAV under control conditions and at 3 hours and 1 day after EIH or AMPH exposure. Since AMPH and EIH are so disruptive to vasculature, genes related to vasculature integrity and function were of interest. Results Our data shows that, under control conditions, many of the genes with relatively high expression in both the MAV and choroid plexus are also abundant in many epithelial tissues. These genes function in transport of water, ions, and solutes, and likely play a role in CSF regulation. Most genes that help form the blood–brain barrier (BBB) and tight junctions were also highly expressed in MAV but not in choroid plexus. In MAV, exposure to EIH and more so to AMPH decreased the expression of BBB-related genes such as Sox18, Ocln, and Cldn5, but they were much less affected in the choroid plexus. There was a correlation between the genes related to reactive oxidative stress and damage that were significantly altered in the MAV and choroid plexus after either EIH or AMPH. However, AMPH (at 3 hr) significantly affected about 5 times as many genes as EIH in the MAV, while in the choroid plexus EIH affected more genes than AMPH. Several unique genes that are not specifically related to vascular damage increased to a much greater extent after AMPH compared to EIH in the MAV (Lbp, Reg3a, Reg3b, Slc15a1, Sct and Fst) and choroid plexus (Bmp4, Dio2 and Lbp). Conclusions Our study indicates that the disruption of choroid plexus function and damage produced by AMPH and EIH is significant, but the changes may not be as pronounced as they are in the MAV, particularly for AMPH. Expression profiles in the MAV and choroid plexus differed to some extent and differences were not restricted to vascular related genes. PMID:23497014

Comparison of the global gene expression of choroid plexus and meninges and associated vasculature under control conditions and after pronounced hyperthermia or amphetamine toxicity.

PubMed

Bowyer, John F; Patterson, Tucker A; Saini, Upasana T; Hanig, Joseph P; Thomas, Monzy; Camacho, Luísa; George, Nysia I; Chen, James J

2013-03-05

The meninges (arachnoid and pial membranes) and associated vasculature (MAV) and choroid plexus are important in maintaining cerebrospinal fluid (CSF) generation and flow. MAV vasculature was previously observed to be adversely affected by environmentally-induced hyperthermia (EIH) and more so by a neurotoxic amphetamine (AMPH) exposure. Herein, microarray and RT-PCR analysis was used to compare the gene expression profiles between choroid plexus and MAV under control conditions and at 3 hours and 1 day after EIH or AMPH exposure. Since AMPH and EIH are so disruptive to vasculature, genes related to vasculature integrity and function were of interest. Our data shows that, under control conditions, many of the genes with relatively high expression in both the MAV and choroid plexus are also abundant in many epithelial tissues. These genes function in transport of water, ions, and solutes, and likely play a role in CSF regulation. Most genes that help form the blood-brain barrier (BBB) and tight junctions were also highly expressed in MAV but not in choroid plexus. In MAV, exposure to EIH and more so to AMPH decreased the expression of BBB-related genes such as Sox18, Ocln, and Cldn5, but they were much less affected in the choroid plexus. There was a correlation between the genes related to reactive oxidative stress and damage that were significantly altered in the MAV and choroid plexus after either EIH or AMPH. However, AMPH (at 3 hr) significantly affected about 5 times as many genes as EIH in the MAV, while in the choroid plexus EIH affected more genes than AMPH. Several unique genes that are not specifically related to vascular damage increased to a much greater extent after AMPH compared to EIH in the MAV (Lbp, Reg3a, Reg3b, Slc15a1, Sct and Fst) and choroid plexus (Bmp4, Dio2 and Lbp). Our study indicates that the disruption of choroid plexus function and damage produced by AMPH and EIH is significant, but the changes may not be as pronounced as they are in the MAV, particularly for AMPH. Expression profiles in the MAV and choroid plexus differed to some extent and differences were not restricted to vascular related genes.

Plasmid-Encoded Tetracycline Efflux Pump Protein Alters Bacterial Stress Responses and Ecological Fitness of Acinetobacter oleivorans

PubMed Central

Hong, Hyerim; Jung, Jaejoon; Park, Woojun

2014-01-01

Acquisition of the extracellular tetracycline (TC) resistance plasmid pAST2 affected host gene expression and phenotype in the oil-degrading soil bacterium, Acinetobacter oleivorans DR1. Whole-transcriptome profiling of DR1 cells harboring pAST2 revealed that all the plasmid genes were highly expressed under TC conditions, and the expression levels of many host chromosomal genes were modulated by the presence of pAST2. The host energy burden imposed by replication of pAST2 led to (i) lowered ATP concentrations, (ii) downregulated expression of many genes involved in cellular growth, and (iii) reduced growth rate. Interestingly, some phenotypes were restored by deleting the plasmid-encoded efflux pump gene tetH, suggesting that the membrane integrity changes resulting from the incorporation of efflux pump proteins also resulted in altered host response under the tested conditions. Alteration of membrane integrity by tetH deletion was shown by measuring permeability of fluorescent probe and membrane hydrophobicity. The presence of the plasmid conferred peroxide and superoxide resistance to cells, but only peroxide resistance was diminished by tetH gene deletion, suggesting that the plasmid-encoded membrane-bound efflux pump protein provided peroxide resistance. The downregulation of fimbriae-related genes presumably led to reduced swimming motility, but this phenotype was recovered by tetH gene deletion. Our data suggest that not only the plasmid replication burden, but also its encoded efflux pump protein altered host chromosomal gene expression and phenotype, which also alters the ecological fitness of the host in the environment. PMID:25229538

Expression of Arabidopsis FCS-Like Zinc finger genes is differentially regulated by sugars, cellular energy level, and abiotic stress.

PubMed

Jamsheer K, Muhammed; Laxmi, Ashverya

2015-01-01

Cellular energy status is an important regulator of plant growth, development, and stress mitigation. Environmental stresses ultimately lead to energy deficit in the cell which activates the SNF1-RELATED KINASE 1 (SnRK1) signaling cascade which eventually triggering a massive reprogramming of transcription to enable the plant to survive under low-energy conditions. The role of Arabidopsis thaliana FCS-Like Zinc finger (FLZ) gene family in energy and stress signaling is recently come to highlight after their interaction with kinase subunits of SnRK1 were identified. In a detailed expression analysis in different sugars, energy starvation, and replenishment series, we identified that the expression of most of the FLZ genes is differentially modulated by cellular energy level. It was found that FLZ gene family contains genes which are both positively and negatively regulated by energy deficit as well as energy-rich conditions. Genetic and pharmacological studies identified the role of HEXOKINASE 1- dependent and energy signaling pathways in the sugar-induced expression of FLZ genes. Further, these genes were also found to be highly responsive to different stresses as well as abscisic acid. In over-expression of kinase subunit of SnRK1, FLZ genes were found to be differentially regulated in accordance with their response toward energy fluctuation suggesting that these genes may work downstream to the established SnRK1 signaling under low-energy stress. Taken together, the present study provides a conceptual framework for further studies related to SnRK1-FLZ interaction in relation to sugar and energy signaling and stress response.

Heterologous gene expression driven by carbonic anhydrase gene promoter in Dunaliella salina

NASA Astrophysics Data System (ADS)

Chai, Yurong; Lu, Yumin; Wang, Tianyun; Hou, Weihong; Xue, Lexun

2006-12-01

Dunaliella salina, a halotolerant unicellular green alga without a rigid cell wall, can live in salinities ranging from 0.05 to 5 mol/L NaCl. These features of D. salina make it an ideal host for the production of antibodies, oral vaccine, and commercially valuable polypeptides. To produce high level of heterologous proteins from D. salina, highly efficient promoters are required to drive expression of target genes under controlled condition. In the present study, we cloned a 5' franking region of 1.4 kb from the carbonic anhydrase ( CAH) gene of D. salina by genomic walking and PCR. The fragment was ligated to the pMD18-T vector and characterized. Sequence analysis indicated that this region contained conserved motifs, including a TATA- like box and CAAT-box. Tandem (GT)n repeats that had a potential role of transcriptional control, were also found in this region. The transcription start site (TSS) of the CAH gene was determined by 5' RACE and nested PCR method. Transformation assays showed that the 1.4 kb fragment was able to drive expression of the selectable bar (bialaphos resistance) gene when the fusion was transformed into D. salina by biolistics. Northern blotting hybridizations showed that the bar transcript was most abundant in cells grown in 2 mol/L NaCl, and less abundant in 0.5 mol/L NaCl, indicating that expression of the bar gene was induced at high salinity. These results suggest the potential use of the CAH gene promoter to induce the expression of heterologous genes in D. salina under varied salt condition.

Plasmid-encoded tetracycline efflux pump protein alters bacterial stress responses and ecological fitness of Acinetobacter oleivorans.

PubMed

Hong, Hyerim; Jung, Jaejoon; Park, Woojun

2014-01-01

Acquisition of the extracellular tetracycline (TC) resistance plasmid pAST2 affected host gene expression and phenotype in the oil-degrading soil bacterium, Acinetobacter oleivorans DR1. Whole-transcriptome profiling of DR1 cells harboring pAST2 revealed that all the plasmid genes were highly expressed under TC conditions, and the expression levels of many host chromosomal genes were modulated by the presence of pAST2. The host energy burden imposed by replication of pAST2 led to (i) lowered ATP concentrations, (ii) downregulated expression of many genes involved in cellular growth, and (iii) reduced growth rate. Interestingly, some phenotypes were restored by deleting the plasmid-encoded efflux pump gene tetH, suggesting that the membrane integrity changes resulting from the incorporation of efflux pump proteins also resulted in altered host response under the tested conditions. Alteration of membrane integrity by tetH deletion was shown by measuring permeability of fluorescent probe and membrane hydrophobicity. The presence of the plasmid conferred peroxide and superoxide resistance to cells, but only peroxide resistance was diminished by tetH gene deletion, suggesting that the plasmid-encoded membrane-bound efflux pump protein provided peroxide resistance. The downregulation of fimbriae-related genes presumably led to reduced swimming motility, but this phenotype was recovered by tetH gene deletion. Our data suggest that not only the plasmid replication burden, but also its encoded efflux pump protein altered host chromosomal gene expression and phenotype, which also alters the ecological fitness of the host in the environment.

The Effects of Glucosamine Sulfate on Intervertebral Disc Annulus Fibrosus Cells in Vitro

PubMed Central

Sowa, Gwendolyn; Coelho, J. Paulo; Jacobs, Lloydine; Komperda, Kasey; Sherry, Nora; Vo, Nam; Preuss, Harry; Balk, Judith; Kang, Jame

2014-01-01

Background context Glucosamine has gained widespread use among patients, despite inconclusive efficacy data. Inconsistency in the clinical literature may be related to lack of understanding of the effects of glucosamine on the intervertebral disc, and therefore, improper patient selection. Purpose The goal of our study was to investigate the effects of glucosamine on intervertebral disc cells in vitro under the physiological conditions of inflammation and mechanical loading. Study Design Controlled in vitro laboratory setting Methods Intervertebral disc cells isolated from the rabbit annulus fibrosus were exposed to glucosamine sulfate in the presence and absence of interleukin-1beta and tensile strain. Outcome measures included gene expression, measurement of total glycosaminoglycans, new proteoglycan synthesis, prostaglandin E2 production, and matrix metalloproteinase activity. The study was funded by NIH/NCCAM and the authors have no conflicts of interest. Results Under conditions of inflammatory stimulation alone, glucosamine demonstrated a dose dependent effect in decreasing inflammatory and catabolic mediators and increasing anabolic genes. However, under conditions of mechanical stimulation, although inflammatory gene expression was decreased, PGE2 was not. In addition, MMP-3 gene expression was increased and aggrecan expression decreased, both of which would have a detrimental effect on matrix homeostasis. Consistent with this, measurement of total glycosaminoglycans and new proteoglycan synthesis demonstrated detrimental effects of glucosamine under all conditions tested. Conclusions These results may in part help to explain the conflicting reports of efficacy, as there is biological plausibility for a therapeutic effect under conditions of predominate inflammation, but not under conditions where mechanical loading is present or in which matrix synthesis is needed. PMID:24361347

A differential pattern of gene expression in skeletal muscle of tumor-bearing rats reveals dysregulation of excitation–contraction coupling together with additional muscle alterations.

PubMed

Fontes-Oliveira, Cibely Cristine; Busquets, Sílvia; Fuster, Gemma; Ametller, Elisabet; Figueras, Maite; Olivan, Mireia; Toledo, Míriam; López-Soriano, Francisco J; Qu, Xiaoyan; Demuth, Jeffrey; Stevens, Paula; Varbanov, Alex; Wang, Feng; Isfort, Robert J; Argilés, Josep M

2014-02-01

Cachexia is a wasting condition that manifests in several types of cancer. The main characteristic of this condition is a profound loss of muscle mass. By using a microarray system, expression of several hundred genes was screened in skeletal muscle of rats bearing a cachexia-inducing tumor, the AH-130 Yoshida ascites hepatoma. This model induced a strong decrease in muscle mass in the tumor-bearing animals, as compared with their healthy counterparts. The results show important differences in gene expression in EDL skeletal muscle between tumor-bearing animals with cachexia and control animals. The differences observed pertain to genes related to intracellular calcium homeostasis and genes involved in the control of mitochondrial oxidative phosphorylation and protein turnover, both at the level of protein synthesis and proteolysis. Assessment of these differences may be a useful tool for the design of novel therapeutic strategies to fight this devastating syndrome.

Sub-cellular mRNA localization modulates the regulation of gene expression by small RNAs in bacteria

NASA Astrophysics Data System (ADS)

Teimouri, Hamid; Korkmazhan, Elgin; Stavans, Joel; Levine, Erel

2017-10-01

Small non-coding RNAs can exert significant regulatory activity on gene expression in bacteria. In recent years, substantial progress has been made in understanding bacterial gene expression by sRNAs. However, recent findings that demonstrate that families of mRNAs show non-trivial sub-cellular distributions raise the question of how localization may affect the regulatory activity of sRNAs. Here we address this question within a simple mathematical model. We show that the non-uniform spatial distributions of mRNA can alter the threshold-linear response that characterizes sRNAs that act stoichiometrically, and modulate the hierarchy among targets co-regulated by the same sRNA. We also identify conditions where the sub-cellular organization of cofactors in the sRNA pathway can induce spatial heterogeneity on sRNA targets. Our results suggest that under certain conditions, interpretation and modeling of natural and synthetic gene regulatory circuits need to take into account the spatial organization of the transcripts of participating genes.

Microaerobic glycerol formation in Saccharomyces cerevisiae.

PubMed

Costenoble, R; Valadi, H; Gustafsson, L; Niklasson, C; Franzén, C J

2000-12-01

The yeast Saccharomyces cerevisiae produces large amounts of glycerol as an osmoregulator during hyperosmotic stress and as a redox sink at low oxygen availability. NAD(+)-dependent glycerol-3-phosphate dehydrogenase in S. cerevisiae is present in two isoforms, coded for by two different genes, GPD1 and GPD2. Mutants for either one or both of these genes were investigated under carefully controlled static and dynamic conditions in continuous cultures at low oxygen transfer rates. Our results show that S. cerevisiae controls the production of glycerol in response to hypoxic conditions by regulating the expression of several genes. At high demand for NADH reoxidation, a strong induction was seen not only of the GPD2 gene, but also of GPP1, encoding one of the molecular forms of glycerol-3-phosphatase. Induction of the GPP1 gene appears to play a decisive role at elevated growth rates. At low demand for NADH reoxidation via glycerol formation, the GPD1, GPD2, GPP1, and GPP2 genes were all expressed at basal levels. The dynamics of the gene induction and the glycerol formation at low demand for NADH reoxidation point to an important role of the Gpd1p; deletion of the GPD1 gene strongly altered the expression patterns of the GPD2 and GPP1 genes under such conditions. Furthermore, our results indicate that GCY1 and DAK1, tentatively encoding glycerol dehydrogenase and dihydroxyacetone kinase, respectively, may be involved in the redox regulation of S. cerevisiae. Copyright 2000 John Wiley & Sons, Ltd.

Proteomic Characterization of Inbreeding-Related Cold Sensitivity in Drosophila melanogaster

PubMed Central

Beck, Hans C.; Petersen, Jørgen; Gagalova, Kristina Kirilova; Loeschcke, Volker

2013-01-01

Inbreeding depression is a widespread phenomenon of central importance to agriculture, medicine, conservation biology and evolutionary biology. Although the population genetic principles of inbreeding depression are well understood, we know little about its functional genomic causes. To provide insight into the molecular interplay between intrinsic stress responses, inbreeding depression and temperature tolerance, we performed a proteomic characterization of a well-defined conditional inbreeding effect in a single line of Drosophila melanogaster, which suffers from extreme cold sensitivity and lethality. We identified 48 differentially expressed proteins in a conditional lethal line as compared to two control lines. These proteins were enriched for proteins involved in hexose metabolism, in particular pyruvate metabolism, and many were found to be associated with lipid particles. These processes can be linked to known cold tolerance mechanisms, such as the production of cryoprotectants, membrane remodeling and the build-up of energy reserves. We checked mRNA-expression of seven genes with large differential protein expression. Although protein expression poorly correlated with gene expression, we found a single gene (CG18067) that, after cold shock, was upregulated in the conditional lethal line both at the mRNA and protein level. Expression of CG18067 also increased in control flies after cold shock, and has previously been linked to cold exposure and chill coma recovery time. Many differentially expressed proteins in our study appear to be involved in cold tolerance in non-inbred individuals. This suggest the conditional inbreeding effect to be caused by misregulation of physiological cold tolerance mechanisms. PMID:23658762

Emiliania huxleyi increases calcification but not expression of calcification-related genes in long-term exposure to elevated temperature and pCO2

PubMed Central

Benner, Ina; Diner, Rachel E.; Lefebvre, Stephane C.; Li, Dian; Komada, Tomoko; Carpenter, Edward J.; Stillman, Jonathon H.

2013-01-01

Increased atmospheric pCO2 is expected to render future oceans warmer and more acidic than they are at present. Calcifying organisms such as coccolithophores that fix and export carbon into the deep sea provide feedbacks to increasing atmospheric pCO2. Acclimation experiments suggest negative effects of warming and acidification on coccolithophore calcification, but the ability of these organisms to adapt to future environmental conditions is not well understood. Here, we tested the combined effect of pCO2 and temperature on the coccolithophore Emiliania huxleyi over more than 700 generations. Cells increased inorganic carbon content and calcification rate under warm and acidified conditions compared with ambient conditions, whereas organic carbon content and primary production did not show any change. In contrast to findings from short-term experiments, our results suggest that long-term acclimation or adaptation could change, or even reverse, negative calcification responses in E. huxleyi and its feedback to the global carbon cycle. Genome-wide profiles of gene expression using RNA-seq revealed that genes thought to be essential for calcification are not those that are most strongly differentially expressed under long-term exposure to future ocean conditions. Rather, differentially expressed genes observed here represent new targets to study responses to ocean acidification and warming. PMID:23980248

Spermine oxidase maintains basal skeletal muscle gene expression and fiber size and is strongly repressed by conditions that cause skeletal muscle atrophy

PubMed Central

Bongers, Kale S.; Fox, Daniel K.; Kunkel, Steven D.; Stebounova, Larissa V.; Murry, Daryl J.; Pufall, Miles A.; Ebert, Scott M.; Dyle, Michael C.; Bullard, Steven A.; Dierdorff, Jason M.

2014-01-01

Skeletal muscle atrophy is a common and debilitating condition that remains poorly understood at the molecular level. To better understand the mechanisms of muscle atrophy, we used mouse models to search for a skeletal muscle protein that helps to maintain muscle mass and is specifically lost during muscle atrophy. We discovered that diverse causes of muscle atrophy (limb immobilization, fasting, muscle denervation, and aging) strongly reduced expression of the enzyme spermine oxidase. Importantly, a reduction in spermine oxidase was sufficient to induce muscle fiber atrophy. Conversely, forced expression of spermine oxidase increased muscle fiber size in multiple models of muscle atrophy (immobilization, fasting, and denervation). Interestingly, the reduction of spermine oxidase during muscle atrophy was mediated by p21, a protein that is highly induced during muscle atrophy and actively promotes muscle atrophy. In addition, we found that spermine oxidase decreased skeletal muscle mRNAs that promote muscle atrophy (e.g., myogenin) and increased mRNAs that help to maintain muscle mass (e.g., mitofusin-2). Thus, in healthy skeletal muscle, a relatively low level of p21 permits expression of spermine oxidase, which helps to maintain basal muscle gene expression and fiber size; conversely, during conditions that cause muscle atrophy, p21 expression rises, leading to reduced spermine oxidase expression, disruption of basal muscle gene expression, and muscle fiber atrophy. Collectively, these results identify spermine oxidase as an important positive regulator of muscle gene expression and fiber size, and elucidate p21-mediated repression of spermine oxidase as a key step in the pathogenesis of skeletal muscle atrophy. PMID:25406264

Fibroblast extracellular matrix gene expression in response to keratinocyte-releasable stratifin.

PubMed

Ghaffari, Abdi; Li, Yunyaun; Karami, Ali; Ghaffari, Mazyar; Tredget, Edward E; Ghahary, Aziz

2006-05-15

Termination of wound-healing process requires a fine balance between connective tissue deposition and its hydrolysis. Previously, we have demonstrated that keratinocyte-releasable stratifin, also known as 14-3-3 sigma protein, stimulates collagenase (MMP-1) expression in dermal fibroblasts. However, role of extracellular stratifin in regulation of extracellular matrix (ECM) factors and other matrix metalloproteinases (MMPs) in dermal fibroblast remains unexplored. To address this question, large-scale ECM gene expression profile were analyzed in human dermal fibroblasts co-cultured with keratinocytes or treated with recombinant stratifin. Superarray pathway-specific microarrays were utilized to identify upregulation or downregulation of 96 human ECM and adhesion molecule genes. RT-PCR and Western blot were used to validate microarray expression profiles of selected genes. Comparison of gene profiles with the appropriate controls showed a significant (more than twofold) increase in expression of collagenase-1, stromelysin-1 and -2, neutrophil collagenase, and membrane type 5 MMP in dermal fibroblasts treated with stratifin or co-cultured with keratinocytes. Expression of type I collagen and fibronectin genes decreased in the same fibroblasts. The results of a dose-response experiment showed that stratifin stimulates the expression of stromelysin-1 (MMP-3) mRNA by dermal fibroblasts in a concentration-dependent fashion. Furthermore, Western blot analysis of fibroblast-conditioned medium showed a peak in MMP-3 protein levels 48 h following treatment with recombinant stratifin. In a lasting-effect study, MMP-3 protein was detected in fibroblast-condition medium for up to 72 h post removal of stratifin. In conclusion, our results suggest that keratinocyte-releasable stratifin plays a major role in induction of ECM degradation by dermal fibroblasts through stimulation of key MMPs, such as MMP-1 and MMP-3. Therefore, stratifin protein may prove to be a useful target for clinical intervention in controlling excessive wound healing in fibrotic conditions. Copyright 2006 Wiley-Liss, Inc.

Gene expression studies of reference genes for quantitative real-time PCR: an overview in insects.

PubMed

Shakeel, Muhammad; Rodriguez, Alicia; Tahir, Urfa Bin; Jin, Fengliang

2018-02-01

Whenever gene expression is being examined, it is essential that a normalization process is carried out to eliminate non-biological variations. The use of reference genes, such as glyceraldehyde-3-phosphate dehydrogenase, actin, and ribosomal protein genes, is the usual method of choice for normalizing gene expression. Although reference genes are used to normalize target gene expression, a major problem is that the stability of these genes differs among tissues, developmental stages, species, and responses to abiotic factors. Therefore, the use and validation of multiple reference genes are required. This review discusses the reasons that why RT-qPCR has become the preferred method for validating results of gene expression profiles, the use of specific and non-specific dyes and the importance of use of primers and probes for qPCR as well as to discuss several statistical algorithms developed to help the validation of potential reference genes. The conflicts arising in the use of classical reference genes in gene normalization and their replacement with novel references are also discussed by citing the high stability and low stability of classical and novel reference genes under various biotic and abiotic experimental conditions by employing various methods applied for the reference genes amplification.

Decoherence in yeast cell populations and its implications for genome-wide expression noise.

PubMed

Briones, M R S; Bosco, F

2009-01-20

Gene expression "noise" is commonly defined as the stochastic variation of gene expression levels in different cells of the same population under identical growth conditions. Here, we tested whether this "noise" is amplified with time, as a consequence of decoherence in global gene expression profiles (genome-wide microarrays) of synchronized cells. The stochastic component of transcription causes fluctuations that tend to be amplified as time progresses, leading to a decay of correlations of expression profiles, in perfect analogy with elementary relaxation processes. Measuring decoherence, defined here as a decay in the auto-correlation function of yeast genome-wide expression profiles, we found a slowdown in the decay of correlations, opposite to what would be expected if, as in mixing systems, correlations decay exponentially as the equilibrium state is reached. Our results indicate that the populational variation in gene expression (noise) is a consequence of temporal decoherence, in which the slow decay of correlations is a signature of strong interdependence of the transcription dynamics of different genes.

Aberrant Gene Expression in Humans

PubMed Central

Yang, Ence; Ji, Guoli; Brinkmeyer-Langford, Candice L.; Cai, James J.

2015-01-01

Gene expression as an intermediate molecular phenotype has been a focus of research interest. In particular, studies of expression quantitative trait loci (eQTL) have offered promise for understanding gene regulation through the discovery of genetic variants that explain variation in gene expression levels. Existing eQTL methods are designed for assessing the effects of common variants, but not rare variants. Here, we address the problem by establishing a novel analytical framework for evaluating the effects of rare or private variants on gene expression. Our method starts from the identification of outlier individuals that show markedly different gene expression from the majority of a population, and then reveals the contributions of private SNPs to the aberrant gene expression in these outliers. Using population-scale mRNA sequencing data, we identify outlier individuals using a multivariate approach. We find that outlier individuals are more readily detected with respect to gene sets that include genes involved in cellular regulation and signal transduction, and less likely to be detected with respect to the gene sets with genes involved in metabolic pathways and other fundamental molecular functions. Analysis of polymorphic data suggests that private SNPs of outlier individuals are enriched in the enhancer and promoter regions of corresponding aberrantly-expressed genes, suggesting a specific regulatory role of private SNPs, while the commonly-occurring regulatory genetic variants (i.e., eQTL SNPs) show little evidence of involvement. Additional data suggest that non-genetic factors may also underlie aberrant gene expression. Taken together, our findings advance a novel viewpoint relevant to situations wherein common eQTLs fail to predict gene expression when heritable, rare inter-individual variation exists. The analytical framework we describe, taking into consideration the reality of differential phenotypic robustness, may be valuable for investigating complex traits and conditions. PMID:25617623

Suppression of Tla1 gene expression for improved solar conversion efficiency and photosynthetic productivity in plants and algae

DOEpatents

Melis, Anastasios; Mitra, Mautusi

2010-06-29

The invention provides method and compositions to minimize the chlorophyll antenna size of photosynthesis by decreasing TLA1 gene expression, thereby improving solar conversion efficiencies and photosynthetic productivity in plants, e.g., green microalgae, under bright sunlight conditions.

Tumor necrosis a regulation of adipokine gene expression in neonatal adipose tissue

USDA-ARS?s Scientific Manuscript database

The neonatal period is also a time of significant stress and susceptibility to infection, conditions which favor the secretion of tumor necrosis a. The present study was designed to determine if TNFa can alter adipokine gene expression within the adipose tissue of neonatal swine. Primary stromal v...

A novel approach for human whole transcriptome analysis based on absolute gene expression of microarray data.

PubMed

Bikel, Shirley; Jacobo-Albavera, Leonor; Sánchez-Muñoz, Fausto; Cornejo-Granados, Fernanda; Canizales-Quinteros, Samuel; Soberón, Xavier; Sotelo-Mundo, Rogerio R; Del Río-Navarro, Blanca E; Mendoza-Vargas, Alfredo; Sánchez, Filiberto; Ochoa-Leyva, Adrian

2017-01-01

In spite of the emergence of RNA sequencing (RNA-seq), microarrays remain in widespread use for gene expression analysis in the clinic. There are over 767,000 RNA microarrays from human samples in public repositories, which are an invaluable resource for biomedical research and personalized medicine. The absolute gene expression analysis allows the transcriptome profiling of all expressed genes under a specific biological condition without the need of a reference sample. However, the background fluorescence represents a challenge to determine the absolute gene expression in microarrays. Given that the Y chromosome is absent in female subjects, we used it as a new approach for absolute gene expression analysis in which the fluorescence of the Y chromosome genes of female subjects was used as the background fluorescence for all the probes in the microarray. This fluorescence was used to establish an absolute gene expression threshold, allowing the differentiation between expressed and non-expressed genes in microarrays. We extracted the RNA from 16 children leukocyte samples (nine males and seven females, ages 6-10 years). An Affymetrix Gene Chip Human Gene 1.0 ST Array was carried out for each sample and the fluorescence of 124 genes of the Y chromosome was used to calculate the absolute gene expression threshold. After that, several expressed and non-expressed genes according to our absolute gene expression threshold were compared against the expression obtained using real-time quantitative polymerase chain reaction (RT-qPCR). From the 124 genes of the Y chromosome, three genes (DDX3Y, TXLNG2P and EIF1AY) that displayed significant differences between sexes were used to calculate the absolute gene expression threshold. Using this threshold, we selected 13 expressed and non-expressed genes and confirmed their expression level by RT-qPCR. Then, we selected the top 5% most expressed genes and found that several KEGG pathways were significantly enriched. Interestingly, these pathways were related to the typical functions of leukocytes cells, such as antigen processing and presentation and natural killer cell mediated cytotoxicity. We also applied this method to obtain the absolute gene expression threshold in already published microarray data of liver cells, where the top 5% expressed genes showed an enrichment of typical KEGG pathways for liver cells. Our results suggest that the three selected genes of the Y chromosome can be used to calculate an absolute gene expression threshold, allowing a transcriptome profiling of microarray data without the need of an additional reference experiment. Our approach based on the establishment of a threshold for absolute gene expression analysis will allow a new way to analyze thousands of microarrays from public databases. This allows the study of different human diseases without the need of having additional samples for relative expression experiments.

Transcriptomic analysis of inbreeding depression in cold-sensitive Drosophila melanogaster shows upregulation of the immune response.

PubMed

Vermeulen, C J; Sørensen, P; Kirilova Gagalova, K; Loeschcke, V

2013-09-01

In sexually reproducing species, increased homozygosity often causes a decline in fitness, called inbreeding depression. Recently, researchers started describing the functional genomic changes that occur during inbreeding, both in benign conditions and under environmental stress. To further this aim, we have performed a genome-wide gene expression study of inbreeding depression, manifesting as cold sensitivity and conditional lethality. Our focus was to describe general patterns of gene expression during inbreeding depression and to identify specific processes affected in our line. There was a clear difference in gene expression between the stressful restrictive environment and the benign permissive environment in both the affected inbred line and the inbred control line. We noted a strong inbreeding-by-environment interaction, whereby virtually all transcriptional differences between lines were found in the restrictive environment. Functional annotation showed enrichment of transcripts coding for serine proteases and their inhibitors (serpins and BPTI/Kunitz family), which indicates activation of the innate immune response. These genes have previously been shown to respond transcriptionally to cold stress, suggesting the conditional lethal effect is associated with an exaggerated cold stress response. The set of differentially expressed genes significantly overlapped with those found in three other studies of inbreeding depression, demonstrating that it is possible to detect a common signature across different genetic backgrounds. © 2013 The Authors. Journal of Evolutionary Biology © 2013 European Society For Evolutionary Biology.

GAPDH-mediated posttranscriptional regulations of sodium channel Scn1a and Scn3a genes under seizure and ketogenic diet conditions.

PubMed

Lin, Guo-Wang; Lu, Ping; Zeng, Tao; Tang, Hui-Ling; Chen, Yong-Hong; Liu, Shu-Jing; Gao, Mei-Mei; Zhao, Qi-Hua; Yi, Yong-Hong; Long, Yue-Sheng

2017-02-01

Abnormal expressions of sodium channel SCN1A and SCN3A genes alter neural excitability that are believed to contribute to the pathogenesis of epilepsy, a long-term risk of recurrent seizures. Ketogenic diet (KD), a high-fat and low-carbohydrate treatment for difficult-to-control (refractory) epilepsy in children, has been suggested to reverse gene expression patterns. Here, we reveal a novel role of GAPDH on the posttranscriptional regulation of mouse Scn1a and Scn3a expressions under seizure and KD conditions. We show that GAPDH binds to a conserved region in the 3' UTRs of human and mouse SCN1A and SCN3A genes, which decreases and increases genes' expressions by affecting mRNA stability through SCN1A 3' UTR and SCN3A 3' UTR, respectively. In seizure mice, the upregulation and phosphorylation of GAPDH enhance its binding to the 3' UTR, which lead to downregulation of Scn1a and upregulation of Scn3a. Furthermore, administration of KD generates β-hydroxybutyric acid which rescues the abnormal expressions of Scn1a and Scn3a by weakening the GAPDH's binding to the element. Taken together, these data suggest that GAPDH-mediated expression regulation of sodium channel genes may be associated with epilepsy and the anticonvulsant action of KD. Copyright © 2016 Elsevier Ltd. All rights reserved.

Efficient production of antibody Fab fragment by transient gene expression in insect cells.

PubMed

Mori, Keita; Hamada, Hirotsugu; Ogawa, Takafumi; Ohmuro-Matsuyama, Yuki; Katsuda, Tomohisa; Yamaji, Hideki

2017-08-01

Transient gene expression allows a rapid production of diverse recombinant proteins in early-stage preclinical and clinical developments of biologics. Insect cells have proven to be an excellent platform for the production of functional recombinant proteins. In the present study, the production of an antibody Fab fragment by transient gene expression in lepidopteran insect cells was investigated. The DNA fragments encoding heavy-chain (Hc; Fd fragment) and light-chain (Lc) genes of an Fab fragment were individually cloned into the plasmid vector pIHAneo, which contained the Bombyx mori actin promoter downstream of the B. mori nucleopolyhedrovirus (BmNPV) IE-1 transactivator and the BmNPV HR3 enhancer for high-level expression. Trichoplusia ni BTI-TN-5B1-4 (High Five) cells were co-transfected with the resultant plasmid vectors using linear polyethyleneimine. When the transfection efficiency was evaluated, a plasmid vector encoding an enhanced green fluorescent protein (EGFP) gene was also co-transfected. Transfection and culture conditions were optimized based on both the flow cytometry of the EGFP expression in transfected cells and the yield of the secreted Fab fragments determined by enzyme-linked immunosorbent assay (ELISA). Under optimal conditions, a yield of approximately 120 mg/L of Fab fragments was achieved in 5 days in a shake-flask culture. Transient gene expression in insect cells may offer a promising approach to the high-throughput production of recombinant proteins. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Mutants of Neurospora crassa that alter gene expression and conidia development.

PubMed Central

Madi, L; Ebbole, D J; White, B T; Yanofsky, C

1994-01-01

Several genes have been identified that are highly expressed during conidiation. Inactivation of these genes has no observable phenotypic effect. Transcripts of two such genes, con-6 and con-10, are normally absent from vegetative mycelia. To identify regulatory genes that affect con-6 and/or con-10 expression, strains were prepared in which the regulatory regions for these genes were fused to a gene conferring hygromycin resistance. Mutants were then selected that were resistant to the drug during mycelial growth. Mutations in several of the isolates had trans effects; they activated transcription of the corresponding intact gene and, in most isolates, one or more of the other con genes. Most interestingly, resistant mutants were obtained that were defective at different stages of conidiation. One mutant conidiated under conditions that do not permit conidiation in wild type. Images PMID:8016143

Expression Profiling in Bemisia tabaci under Insecticide Treatment: Indicating the Necessity for Custom Reference Gene Selection

PubMed Central

Zhou, Xuguo; Gao, Xiwu

2014-01-01

Finding a suitable reference gene is the key for qRT-PCR analysis. However, none of the reference gene discovered thus far can be utilized universally under various biotic and abiotic experimental conditions. In this study, we further examine the stability of candidate reference genes under a single abiotic factor, insecticide treatment. After being exposed to eight commercially available insecticides, which belong to five different classes, the expression profiles of eight housekeeping genes in the sweetpotato whitefly, Bemisia tabaci, one of the most invasive and destructive pests in the world, were investigated using qRT-PCR analysis. In summary, elongation factor 1α (EF1α), α-tubulin (TUB1α) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were identified as the most stable reference genes under the insecticide treatment. The initial assessment of candidate reference genes was further validated with the expression of two target genes, a P450 (Cyp6cm1) and a glutathione S-transferase (GST). However, ranking of reference genes varied substantially among intra- and inter-classes of insecticides. These combined data strongly suggested the necessity of conducting custom reference gene selection designed for each and every experimental condition, even when examining the same abiotic or biotic factor. PMID:24498122

Reference genes for quantitative real-time PCR analysis in symbiont Entomomyces delphacidicola of Nilaparvata lugens (Stål)

PubMed Central

Wan, Pin-Jun; Tang, Yao-Hua; Yuan, San-Yue; He, Jia-Chun; Wang, Wei-Xia; Lai, Feng-Xiang; Fu, Qiang

2017-01-01

Nilaparvata lugens (Stål) (Hemiptera: Delphacidae) is a major rice pest that harbors an endosymbiont ascomycete fungus, Entomomyces delphacidicola str. NLU (also known as yeast-like symbiont, YLS). Driving by demand of novel population management tactics (e.g. RNAi), the importance of YLS has been studied and revealed, which greatly boosts the interest of molecular level studies related to YLS. The current study focuses on reference genes for RT-qPCR studies related to YLS. Eight previously unreported YLS genes were cloned, and their expressions were evaluated for N. lugens samples of different developmental stages and sexes, and under different nutritional conditions and temperatures. Expression stabilities were analyzed by BestKeeper, geNorm, NormFinder, ΔCt method and RefFinder. Furthermore, the selected reference genes for RT-qPCR of YLS genes were validated using targeted YLS genes that respond to different nutritional conditions (amino acid deprivation) and RNAi. The results suggest that ylsRPS15p/ylsACT are the most suitable reference genes for temporal gene expression profiling, while ylsTUB/ylsACT and ylsRPS15e/ylsGADPH are the most suitable reference gene choices for evaluating nutrition and temperature effects. Validation studies demonstrated the advantage of using endogenous YLS reference genes for YLS studies. PMID:28198810

Core clock, SUB1, and ABAR genes mediate flooding and drought responses via alternative splicing in soybean.

PubMed

Syed, Naeem H; Prince, Silvas J; Mutava, Raymond N; Patil, Gunvant; Li, Song; Chen, Wei; Babu, Valliyodan; Joshi, Trupti; Khan, Saad; Nguyen, Henry T

2015-12-01

Circadian clocks are a great evolutionary innovation and provide competitive advantage during the day/night cycle and under changing environmental conditions. The circadian clock mediates expression of a large proportion of genes in plants, achieving a harmonious relationship between energy metabolism, photosynthesis, and biotic and abiotic stress responses. Here it is shown that multiple paralogues of clock genes are present in soybean (Glycine max) and mediate flooding and drought responses. Differential expression of many clock and SUB1 genes was found under flooding and drought conditions. Furthermore, natural variation in the amplitude and phase shifts in PRR7 and TOC1 genes was also discovered under drought and flooding conditions, respectively. PRR3 exhibited flooding- and drought-specific splicing patterns and may work in concert with PRR7 and TOC1 to achieve energy homeostasis under flooding and drought conditions. Higher expression of TOC1 also coincides with elevated levels of abscisic acid (ABA) and variation in glucose levels in the morning and afternoon, indicating that this response to abiotic stress is mediated by ABA, endogenous sugar levels, and the circadian clock to fine-tune photosynthesis and energy utilization under stress conditions. It is proposed that the presence of multiple clock gene paralogues with variation in DNA sequence, phase, and period could be used to screen exotic germplasm to find sources for drought and flooding tolerance. Furthermore, fine tuning of multiple clock gene paralogues (via a genetic engineering approach) should also facilitate the development of flooding- and drought-tolerant soybean varieties. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Suitable Reference Genes for Accurate Gene Expression Analysis in Parsley (Petroselinum crispum) for Abiotic Stresses and Hormone Stimuli

PubMed Central

Li, Meng-Yao; Song, Xiong; Wang, Feng; Xiong, Ai-Sheng

2016-01-01

Parsley, one of the most important vegetables in the Apiaceae family, is widely used in the food, medicinal, and cosmetic industries. Recent studies on parsley mainly focus on its chemical composition, and further research involving the analysis of the plant's gene functions and expressions is required. qPCR is a powerful method for detecting very low quantities of target transcript levels and is widely used to study gene expression. To ensure the accuracy of results, a suitable reference gene is necessary for expression normalization. In this study, four software, namely geNorm, NormFinder, BestKeeper, and RefFinder were used to evaluate the expression stabilities of eight candidate reference genes of parsley (GAPDH, ACTIN, eIF-4α, SAND, UBC, TIP41, EF-1α, and TUB) under various conditions, including abiotic stresses (heat, cold, salt, and drought) and hormone stimuli treatments (GA, SA, MeJA, and ABA). Results showed that EF-1α and TUB were the most stable genes for abiotic stresses, whereas EF-1α, GAPDH, and TUB were the top three choices for hormone stimuli treatments. Moreover, EF-1α and TUB were the most stable reference genes among all tested samples, and UBC was the least stable one. Expression analysis of PcDREB1 and PcDREB2 further verified that the selected stable reference genes were suitable for gene expression normalization. This study can guide the selection of suitable reference genes in gene expression in parsley. PMID:27746803

Suitable Reference Genes for Accurate Gene Expression Analysis in Parsley (Petroselinum crispum) for Abiotic Stresses and Hormone Stimuli.

PubMed

Li, Meng-Yao; Song, Xiong; Wang, Feng; Xiong, Ai-Sheng

2016-01-01

Parsley, one of the most important vegetables in the Apiaceae family, is widely used in the food, medicinal, and cosmetic industries. Recent studies on parsley mainly focus on its chemical composition, and further research involving the analysis of the plant's gene functions and expressions is required. qPCR is a powerful method for detecting very low quantities of target transcript levels and is widely used to study gene expression. To ensure the accuracy of results, a suitable reference gene is necessary for expression normalization. In this study, four software, namely geNorm, NormFinder, BestKeeper, and RefFinder were used to evaluate the expression stabilities of eight candidate reference genes of parsley ( GAPDH, ACTIN, eIF-4 α, SAND, UBC, TIP41, EF-1 α, and TUB ) under various conditions, including abiotic stresses (heat, cold, salt, and drought) and hormone stimuli treatments (GA, SA, MeJA, and ABA). Results showed that EF-1 α and TUB were the most stable genes for abiotic stresses, whereas EF-1 α, GAPDH , and TUB were the top three choices for hormone stimuli treatments. Moreover, EF-1 α and TUB were the most stable reference genes among all tested samples, and UBC was the least stable one. Expression analysis of PcDREB1 and PcDREB2 further verified that the selected stable reference genes were suitable for gene expression normalization. This study can guide the selection of suitable reference genes in gene expression in parsley.

The heterogeneity of human mesenchymal stem cell preparations--evidence from simultaneous analysis of proteomes and transcriptomes.

PubMed

Wagner, Wolfgang; Feldmann, Robert E; Seckinger, Anja; Maurer, Martin H; Wein, Frederik; Blake, Jonathon; Krause, Ulf; Kalenka, Armin; Bürgers, Heinrich F; Saffrich, Rainer; Wuchter, Patrick; Kuschinsky, Wolfgang; Ho, Anthony D

2006-04-01

Mesenchymal stem cells (MSC) raise high hopes in clinical applications. However, the lack of common standards and a precise definition of MSC preparations remains a major obstacle in research and application of MSC. Whereas surface antigen markers have failed to precisely define this population, a combination of proteomic data and microarray data provides a new dimension for the definition of MSC preparations. In our continuing effort to characterize MSC, we have analyzed the differential transcriptome and proteome expression profiles of MSC preparations isolated from human bone marrow under two different expansion media (BM-MSC-M1 and BM-MSC-M2). In proteomics, 136 protein spots were unambiguously identified by MALDI-TOF-MS and corresponding cDNA spots were selected on our "Human Transcriptome cDNA Microarray." Combination of datasets revealed a correlation in differential gene expression and protein expression of BM-MSC-M1 vs BM-MSC-M2. Genes involved in metabolism were more highly expressed in BM-MSC-M1, whereas genes involved in development, morphogenesis, extracellular matrix, and differentiation were more highly expressed in BM-MSC-M2. Interchanging culture conditions for 8 days revealed that differential expression was retained in several genes whereas it was altered in others. Our results have provided evidence that homogeneous BM-MSC preparations can reproducibly be isolated under standardized conditions, whereas culture conditions exert a prominent impact on transcriptome, proteome, and cellular organization of BM-MSC.

Adapted Resistance to the Knockdown Effect of shRNA-Derived Srsf3 siRNAs in Mouse Littermates | Center for Cancer Research

Cancer.gov

Gene silencing techniques are widely used to control gene expression and have potential for RNAi-based therapeutics. In this report, transgenic mouse lines were created for conditional knockdown of Srsf3 (SRp20) expression in liver and mammary gland tissues by expressing Srsf3-specific shRNAs driven by a U6 promoter.

Expression of the Immediate-Early Gene-Encoded Protein Egr-1 ("zif268") during in Vitro Classical Conditioning

ERIC Educational Resources Information Center

Mokin, Maxim; Keifer, Joyce

2005-01-01

Expression of the immediate-early genes (IEGs) has been shown to be induced by activity-dependent synaptic plasticity or behavioral training and is thought to play an important role in long-term memory. In the present study, we examined the induction and expression of the IEG-encoded protein Egr-1 during an in vitro neural correlate of eyeblink…

The expression patterns of the clock genes period and timeless are affected by photoperiod in the Mediterranean corn stalk borer, Sesamia nonagrioides.

PubMed

Kontogiannatos, Dimitrios; Gkouvitsas, Theodoros; Kourti, Anna

2017-01-01

To obtain clues to the link between the molecular mechanism of circadian and photoperiod clocks, we cloned two circadian clock genes, period (per) and timeless (tim) from the moth Sesamia nonagrioides, which undergoes facultative diapause controlled by photoperiod. Sequence analysis revealed a high degree of conservation among the compared insects fοr both genes. We also investigated the expression patterns of per and tim in brains of larvae growing under 16L:8D (long days), constant darkness (DD) and 10L:14D (short days) conditions by qPCR assays. The results showed that mRNA accumulations encoding both genes exhibited diel oscillations under different photoperiods. The oscillation of per and tim mRNA, under short-day photoperiod differed from long-day. The difference between long-day and short-day conditions in the pattern of mRNA levels of per and tim appears to distinguish photoperiodic conditions clearly and both genes were influenced by photoperiod in different ways. We infer that not all photoperiodic clocks of insects interact with circadian clocks in the same fashion. Our results suggest that transcriptional regulations of the both clock genes act in the diapause programing in S. nonagrioides. The expression patterns of these genes are affected by photoperiod but runs with 24 h by entrainment to daily environmental cues. © 2016 Wiley Periodicals, Inc.

edgeR: a Bioconductor package for differential expression analysis of digital gene expression data.

PubMed

Robinson, Mark D; McCarthy, Davis J; Smyth, Gordon K

2010-01-01

It is expected that emerging digital gene expression (DGE) technologies will overtake microarray technologies in the near future for many functional genomics applications. One of the fundamental data analysis tasks, especially for gene expression studies, involves determining whether there is evidence that counts for a transcript or exon are significantly different across experimental conditions. edgeR is a Bioconductor software package for examining differential expression of replicated count data. An overdispersed Poisson model is used to account for both biological and technical variability. Empirical Bayes methods are used to moderate the degree of overdispersion across transcripts, improving the reliability of inference. The methodology can be used even with the most minimal levels of replication, provided at least one phenotype or experimental condition is replicated. The software may have other applications beyond sequencing data, such as proteome peptide count data. The package is freely available under the LGPL licence from the Bioconductor web site (http://bioconductor.org).

Analysis of global gene expression in Brachypodium distachyon reveals extensive network plasticity in response to abiotic stress.

PubMed

Priest, Henry D; Fox, Samuel E; Rowley, Erik R; Murray, Jessica R; Michael, Todd P; Mockler, Todd C

2014-01-01

Brachypodium distachyon is a close relative of many important cereal crops. Abiotic stress tolerance has a significant impact on productivity of agriculturally important food and feedstock crops. Analysis of the transcriptome of Brachypodium after chilling, high-salinity, drought, and heat stresses revealed diverse differential expression of many transcripts. Weighted Gene Co-Expression Network Analysis revealed 22 distinct gene modules with specific profiles of expression under each stress. Promoter analysis implicated short DNA sequences directly upstream of module members in the regulation of 21 of 22 modules. Functional analysis of module members revealed enrichment in functional terms for 10 of 22 network modules. Analysis of condition-specific correlations between differentially expressed gene pairs revealed extensive plasticity in the expression relationships of gene pairs. Photosynthesis, cell cycle, and cell wall expression modules were down-regulated by all abiotic stresses. Modules which were up-regulated by each abiotic stress fell into diverse and unique gene ontology GO categories. This study provides genomics resources and improves our understanding of abiotic stress responses of Brachypodium.

Expression of glnB and a glnB-Like Gene (glnK) in a Ribulose Bisphosphate Carboxylase/Oxygenase-Deficient Mutant of Rhodobacter sphaeroides

PubMed Central

Qian, Yilei; Tabita, F. Robert

1998-01-01

In a ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO)-deficient mutant of Rhodobacter sphaeroides, strain 16PHC, nitrogenase activity was derepressed in the presence of ammonia under photoheterotrophic growth conditions. Previous studies also showed that reintroduction of a functional RubisCO and Calvin-Benson-Bassham (CBB) pathway suppressed the deregulation of nitrogenase synthesis in this strain. In this study, the derepression of nitrogenase synthesis in the presence of ammonia in strain 16PHC was further explored by using a glnB::lacZ fusion, since the product of the glnB gene is known to have a negative effect on ammonia-regulated nif control. It was found that glnB expression was repressed in strain 16PHC under photoheterotrophic growth conditions with either ammonia or glutamate as the nitrogen source; glutamine synthetase (GS) levels were also affected in this strain. However, when cells regained a functional CBB pathway by trans complementation of the deleted genes, wild-type levels of GS and glnB expression were restored. Furthermore, a glnB-like gene, glnK, was isolated from this organism, and its expression was found to be under tight nitrogen control in the wild type. Surprisingly, glnK expression was found to be derepressed in strain 16PHC under photoheterotrophic conditions in the presence of ammonia. PMID:9721307

Lignin-degrading peroxidases in white-rot fungus Trametes hirsuta 072. Absolute expression quantification of full multigene family

PubMed Central

Vasina, Daria V.; Moiseenko, Konstantin V.; Fedorova, Tatiana V.; Tyazhelova, Tatiana V.

2017-01-01

Ligninolytic heme peroxidases comprise an extensive family of enzymes, which production is characteristic for white-rot Basidiomycota. The majority of fungal heme peroxidases are encoded by multigene families that differentially express closely related proteins. Currently, there were very few attempts to characterize the complete multigene family of heme peroxidases in a single fungus. Here we are focusing on identification and characterization of peroxidase genes, which are transcribed and secreted by basidiomycete Trametes hirsuta 072, an efficient lignin degrader. The T. hirsuta genome contains 18 ligninolytic peroxidase genes encoding 9 putative lignin peroxidases (LiP), 7 putative short manganese peroxidases (MnP) and 2 putative versatile peroxidases (VP). Using ddPCR method we have quantified the absolute expression of the 18 peroxidase genes under different culture conditions and on different growth stages of basidiomycete. It was shown that only two genes (one MnP and one VP) were prevalently expressed as well as secreted into cultural broth under all conditions investigated. However their transcriptome and protein profiles differed in time depending on the effector used. The expression of other peroxidase genes revealed a significant variability, so one can propose the specific roles of these enzymes in fungal development and lifestyle. PMID:28301519

Bacterial evolution through the selective loss of beneficial Genes. Trade-offs in expression involving two loci.

PubMed Central

Zinser, Erik R; Schneider, Dominique; Blot, Michel; Kolter, Roberto

2003-01-01

The loss of preexisting genes or gene activities during evolution is a major mechanism of ecological specialization. Evolutionary processes that can account for gene loss or inactivation have so far been restricted to one of two mechanisms: direct selection for the loss of gene activities that are disadvantageous under the conditions of selection (i.e., antagonistic pleiotropy) and selection-independent genetic drift of neutral (or nearly neutral) mutations (i.e., mutation accumulation). In this study we demonstrate with an evolved strain of Escherichia coli that a third, distinct mechanism exists by which gene activities can be lost. This selection-dependent mechanism involves the expropriation of one gene's upstream regulatory element by a second gene via a homologous recombination event. Resulting from this genetic exchange is the activation of the second gene and a concomitant inactivation of the first gene. This gene-for-gene expression tradeoff provides a net fitness gain, even if the forfeited activity of the first gene can play a positive role in fitness under the conditions of selection. PMID:12930738

[Stability analysis of reference gene based on real-time PCR in Artemisia annua under cadmium treatment].

PubMed

Zhou, Liang-Yun; Mo, Ge; Wang, Sheng; Tang, Jin-Fu; Yue, Hong; Huang, Lu-Qi; Shao, Ai-Juan; Guo, Lan-Ping

2014-03-01

In this study, Actin, 18S rRNA, PAL, GAPDH and CPR of Artemisia annua were selected as candidate reference genes, and their gene-specific primers for real-time PCR were designed, then geNorm, NormFinder, BestKeeper, Delta CT and RefFinder were used to evaluate their expression stability in the leaves of A. annua under treatment of different concentrations of Cd, with the purpose of finding a reliable reference gene to ensure the reliability of gene-expression analysis. The results showed that there were some significant differences among the candidate reference genes under different treatments and the order of expression stability of candidate reference gene was Actin > 18S rRNA > PAL > GAPDH > CPR. These results suggested that Actin, 18S rRNA and PAL could be used as ideal reference genes of gene expression analysis in A. annua and multiple internal control genes were adopted for results calibration. In addition, differences in expression stability of candidate reference genes in the leaves of A. annua under the same concentrations of Cd were observed, which suggested that the screening of candidate reference genes was needed even under the same treatment. To our best knowledge, this study for the first time provided the ideal reference genes under Cd treatment in the leaves of A. annua and offered reference for the gene expression analysis of A. annua under other conditions.

Chilling-Mediated DNA Methylation Changes during Dormancy and Its Release Reveal the Importance of Epigenetic Regulation during Winter Dormancy in Apple (Malus x domestica Borkh.).

PubMed

Kumar, Gulshan; Rattan, Usha Kumari; Singh, Anil Kumar

2016-01-01

Winter dormancy is a well known mechanism adopted by temperate plants, to mitigate the chilling temperature of winters. However, acquisition of sufficient chilling during winter dormancy ensures the normal phenological traits in subsequent growing period. Thus, low temperature appears to play crucial roles in growth and development of temperate plants. Apple, being an important temperate fruit crop, also requires sufficient chilling to release winter dormancy and normal phenological traits, which are often associated with yield and quality of fruits. DNA cytosine methylation is one of the important epigenetic modifications which remarkably affect the gene expression during various developmental and adaptive processes. In present study, methylation sensitive amplified polymorphism was employed to assess the changes in cytosine methylation during dormancy, active growth and fruit set in apple, under differential chilling conditions. Under high chill conditions, total methylation was decreased from 27.2% in dormant bud to 21.0% in fruit set stage, while no significant reduction was found under low chill conditions. Moreover, the demethylation was found to be decreased, while methylation increased from dormant bud to fruit set stage under low chill as compared to high chill conditions. In addition, RNA-Seq analysis showed high expression of DNA methyltransferases and histone methyltransferases during dormancy and fruit set, and low expression of DNA glcosylases during active growth under low chill conditions, which was in accordance with changes in methylation patterns. The RNA-Seq data of 47 genes associated with MSAP fragments involved in cellular metabolism, stress response, antioxidant system and transcriptional regulation showed correlation between methylation and their expression. Similarly, bisulfite sequencing and qRT-PCR analysis of selected genes also showed correlation between gene body methylation and gene expression. Moreover, significant association between chilling and methylation changes was observed, which suggested that chilling acquisition during dormancy in apple is likely to affect the epigenetic regulation through DNA methylation.

Chilling-Mediated DNA Methylation Changes during Dormancy and Its Release Reveal the Importance of Epigenetic Regulation during Winter Dormancy in Apple (Malus x domestica Borkh.)

PubMed Central

Kumar, Gulshan; Rattan, Usha Kumari; Singh, Anil Kumar

2016-01-01

Winter dormancy is a well known mechanism adopted by temperate plants, to mitigate the chilling temperature of winters. However, acquisition of sufficient chilling during winter dormancy ensures the normal phenological traits in subsequent growing period. Thus, low temperature appears to play crucial roles in growth and development of temperate plants. Apple, being an important temperate fruit crop, also requires sufficient chilling to release winter dormancy and normal phenological traits, which are often associated with yield and quality of fruits. DNA cytosine methylation is one of the important epigenetic modifications which remarkably affect the gene expression during various developmental and adaptive processes. In present study, methylation sensitive amplified polymorphism was employed to assess the changes in cytosine methylation during dormancy, active growth and fruit set in apple, under differential chilling conditions. Under high chill conditions, total methylation was decreased from 27.2% in dormant bud to 21.0% in fruit set stage, while no significant reduction was found under low chill conditions. Moreover, the demethylation was found to be decreased, while methylation increased from dormant bud to fruit set stage under low chill as compared to high chill conditions. In addition, RNA-Seq analysis showed high expression of DNA methyltransferases and histone methyltransferases during dormancy and fruit set, and low expression of DNA glcosylases during active growth under low chill conditions, which was in accordance with changes in methylation patterns. The RNA-Seq data of 47 genes associated with MSAP fragments involved in cellular metabolism, stress response, antioxidant system and transcriptional regulation showed correlation between methylation and their expression. Similarly, bisulfite sequencing and qRT-PCR analysis of selected genes also showed correlation between gene body methylation and gene expression. Moreover, significant association between chilling and methylation changes was observed, which suggested that chilling acquisition during dormancy in apple is likely to affect the epigenetic regulation through DNA methylation. PMID:26901339

Unstable Expression of Commonly Used Reference Genes in Rat Pancreatic Islets Early after Isolation Affects Results of Gene Expression Studies.

PubMed

Kosinová, Lucie; Cahová, Monika; Fábryová, Eva; Týcová, Irena; Koblas, Tomáš; Leontovyč, Ivan; Saudek, František; Kříž, Jan

2016-01-01

The use of RT-qPCR provides a powerful tool for gene expression studies; however, the proper interpretation of the obtained data is crucially dependent on accurate normalization based on stable reference genes. Recently, strong evidence has been shown indicating that the expression of many commonly used reference genes may vary significantly due to diverse experimental conditions. The isolation of pancreatic islets is a complicated procedure which creates severe mechanical and metabolic stress leading possibly to cellular damage and alteration of gene expression. Despite of this, freshly isolated islets frequently serve as a control in various gene expression and intervention studies. The aim of our study was to determine expression of 16 candidate reference genes and one gene of interest (F3) in isolated rat pancreatic islets during short-term cultivation in order to find a suitable endogenous control for gene expression studies. We compared the expression stability of the most commonly used reference genes and evaluated the reliability of relative and absolute quantification using RT-qPCR during 0-120 hrs after isolation. In freshly isolated islets, the expression of all tested genes was markedly depressed and it increased several times throughout the first 48 hrs of cultivation. We observed significant variability among samples at 0 and 24 hrs but substantial stabilization from 48 hrs onwards. During the first 48 hrs, relative quantification failed to reflect the real changes in respective mRNA concentrations while in the interval 48-120 hrs, the relative expression generally paralleled the results determined by absolute quantification. Thus, our data call into question the suitability of relative quantification for gene expression analysis in pancreatic islets during the first 48 hrs of cultivation, as the results may be significantly affected by unstable expression of reference genes. However, this method could provide reliable information from 48 hrs onwards.

Serum response factor: positive and negative regulation of an epithelial gene expression network in the destrin mutant cornea

PubMed Central

Kawakami-Schulz, Sharolyn V.; Verdoni, Angela M.; Sattler, Shannon G.; Jessen, Erik; Kao, Winston W.-Y.; Ikeda, Akihiro

2014-01-01

Increased angiogenesis, inflammation, and proliferation are hallmarks of diseased tissues, and in vivo models of these disease phenotypes can provide insight into disease pathology. Dstncorn1 mice, deficient for the actin depolymerizing factor destrin (DSTN), display an increase of serum response factor (SRF) that results in epithelial hyperproliferation, inflammation, and neovascularization in the cornea. Previous work demonstrated that conditional ablation of Srf from the corneal epithelium of Dstncorn1 mice returns the cornea to a wild-type (WT) like state. This result implicated SRF as a major regulator of genes that contributes to abnormal phenotypes in Dstncorn1 cornea. The purpose of this study is to identify gene networks that are affected by increased expression of Srf in the Dstncorn1 cornea. Microarray analysis led to characterization of gene expression changes that occur when conditional knockout of Srf rescues mutant phenotypes in the cornea of Dstncorn1 mice. Comparison of gene expression values from WT, Dstncorn1 mutant, and Dstncorn1 rescued cornea identified >400 differentially expressed genes that are downstream from SRF. Srf ablation had a significant effect on genes associated with epithelial cell-cell junctions and regulation of actin dynamics. The majority of genes affected by SRF are downregulated in the Dstncorn1 mutant cornea, suggesting that increased SRF negatively affects transcription of SRF gene targets. ChIP-seq analysis on Dstncorn1 mutant and WT tissue revealed that, despite being present in higher abundance, SRF binding is significantly decreased in the Dstncorn1 mutant cornea. This study uses a unique model combining genetic and genomic approaches to identify genes that are regulated by SRF. These findings expand current understanding of the role of SRF in both normal and abnormal tissue homeostasis. PMID:24550211

Transcriptional Differences between Diapausing and Non-Diapausing D. montana Females Reared under the Same Photoperiod and Temperature

PubMed Central

Kankare, Maaria; Parker, Darren J.; Merisalo, Mikko; Salminen, Tiina S.; Hoikkala, Anneli

2016-01-01

Background A wide range of insects living at higher latitudes enter diapause at the end of the warm season, which increases their chances of survival through harsh winter conditions. In this study we used RNA sequencing to identify genes involved in adult reproductive diapause in a northern fly species, Drosophila montana. Both diapausing and non-diapausing flies were reared under a critical day length and temperature, where about half of the emerging females enter diapause enabling us to eliminate the effects of varying environmental conditions on gene expression patterns of the two types of female flies. Results RNA sequencing revealed large differences between gene expression patterns of diapausing and non-diapausing females, especially in genes involved with metabolism, fatty acid biosynthesis, and metal and nucleotide binding. Differently expressed genes included several gene groups, including myosin, actin and cytochromeP450 genes, which have been previously associated with diapause. This study also identified new candidate genes, including some involved in cuticular hydrocarbon synthesis or regulation (desat1 and desat2), and acyl-CoA Δ11-desaturase activity (CG9747), and few odorant-binding protein genes (e.g. Obp44A). Also, several transposable elements (TEs) showed differential expression between the two female groups motivating future research on their roles in diapause. Conclusions Our results demonstrate that the adult reproductive diapause in D. montana involves changes in the expression level of a variety of genes involved in key processes (e.g. metabolism and fatty acid biosynthesis) which help diapausing females to cope with overwintering. This is consistent with the view that diapause is a complex adaptive phenotype where not only sexual maturation is arrested, but also changes in adult physiology are required in order to survive over the winter. PMID:27571415

Transcriptome analysis of the Populus trichocarpa-Rhizophagus irregularis Mycorrhizal Symbiosis: Regulation of Plant and Fungal Transportomes under Nitrogen Starvation.

PubMed

Calabrese, Silvia; Kohler, Annegret; Niehl, Annette; Veneault-Fourrey, Claire; Boller, Thomas; Courty, Pierre-Emmanuel

2017-06-01

Nutrient transfer is a key feature of the arbuscular mycorrhizal (AM) symbiosis. Valuable mineral nutrients are transferred from the AM fungus to the plant, increasing its fitness and productivity, and, in exchange, the AM fungus receives carbohydrates as an energy source from the plant. Here, we analyzed the transcriptome of the Populus trichocarpa-Rhizophagus irregularis symbiosis using RNA-sequencing of non-mycorrhizal or mycorrhizal fine roots, with a focus on the effect of nitrogen (N) starvation. In R. irregularis, we identified 1,015 differentially expressed genes, whereby N starvation led to a general induction of gene expression. Genes of the functional classes of cell growth, membrane biogenesis and cell structural components were highly abundant. Interestingly, N starvation also led to a general induction of fungal transporters, indicating increased nutrient demand upon N starvation. In non-mycorrhizal P. trichocarpa roots, 1,341 genes were differentially expressed under N starvation. Among the 953 down-regulated genes in N starvation, most were involved in metabolic processes including amino acids, carbohydrate and inorganic ion transport, while the 342 up-regulated genes included many defense-related genes. Mycorrhization led to the up-regulation of 549 genes mainly involved in secondary metabolite biosynthesis and transport; only 24 genes were down-regulated. Mycorrhization specifically induced expression of three ammonium transporters and one phosphate transporter, independently of the N conditions, corroborating the hypothesis that these transporters are important for symbiotic nutrient exchange. In conclusion, our data establish a framework of gene expression in the two symbiotic partners under high-N and low-N conditions. © The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Divergent gene expression in the conserved dauer stage of the nematodes Pristionchus pacificus and Caenorhabditis elegans.

PubMed

Sinha, Amit; Sommer, Ralf J; Dieterich, Christoph

2012-06-19

An organism can respond to changing environmental conditions by adjusting gene regulation and by forming alternative phenotypes. In nematodes, these mechanisms are coupled because many species will form dauer larvae, a stress-resistant and non-aging developmental stage, when exposed to unfavorable environmental conditions, and execute gene expression programs that have been selected for the survival of the animal in the wild. These dauer larvae represent an environmentally induced, homologous developmental stage across many nematode species, sharing conserved morphological and physiological properties. Hence it can be expected that some core components of the associated transcriptional program would be conserved across species, while others might diverge over the course of evolution. However, transcriptional and metabolic analysis of dauer development has been largely restricted to Caenorhabditis elegans. Here, we use a transcriptomic approach to compare the dauer stage in the evolutionary model system Pristionchus pacificus with the dauer stage in C. elegans. We have employed Agilent microarrays, which represent 20,446 P. pacificus and 20,143 C. elegans genes to show an unexpected divergence in the expression profiles of these two nematodes in dauer and dauer exit samples. P. pacificus and C. elegans differ in the dynamics and function of genes that are differentially expressed. We find that only a small number of orthologous gene pairs show similar expression pattern in the dauers of the two species, while the non-orthologous fraction of genes is a major contributor to the active transcriptome in dauers. Interestingly, many of the genes acquired by horizontal gene transfer and orphan genes in P. pacificus, are differentially expressed suggesting that these genes are of evolutionary and functional importance. Our data set provides a catalog for future functional investigations and indicates novel insight into evolutionary mechanisms. We discuss the limited conservation of core developmental and transcriptional programs as a common aspect of animal evolution.

Divergent gene expression in the conserved dauer stage of the nematodes Pristionchus pacificus and Caenorhabditis elegans

PubMed Central

2012-01-01

Background An organism can respond to changing environmental conditions by adjusting gene regulation and by forming alternative phenotypes. In nematodes, these mechanisms are coupled because many species will form dauer larvae, a stress-resistant and non-aging developmental stage, when exposed to unfavorable environmental conditions, and execute gene expression programs that have been selected for the survival of the animal in the wild. These dauer larvae represent an environmentally induced, homologous developmental stage across many nematode species, sharing conserved morphological and physiological properties. Hence it can be expected that some core components of the associated transcriptional program would be conserved across species, while others might diverge over the course of evolution. However, transcriptional and metabolic analysis of dauer development has been largely restricted to Caenorhabditis elegans. Here, we use a transcriptomic approach to compare the dauer stage in the evolutionary model system Pristionchus pacificus with the dauer stage in C. elegans. Results We have employed Agilent microarrays, which represent 20,446 P. pacificus and 20,143 C. elegans genes to show an unexpected divergence in the expression profiles of these two nematodes in dauer and dauer exit samples. P. pacificus and C. elegans differ in the dynamics and function of genes that are differentially expressed. We find that only a small number of orthologous gene pairs show similar expression pattern in the dauers of the two species, while the non-orthologous fraction of genes is a major contributor to the active transcriptome in dauers. Interestingly, many of the genes acquired by horizontal gene transfer and orphan genes in P. pacificus, are differentially expressed suggesting that these genes are of evolutionary and functional importance. Conclusion Our data set provides a catalog for future functional investigations and indicates novel insight into evolutionary mechanisms. We discuss the limited conservation of core developmental and transcriptional programs as a common aspect of animal evolution. PMID:22712530

Hypoxia/hepatoma dual specific suicide gene expression plasmid delivery using bio-reducible polymer for hepatocellular carcinoma therapy.

PubMed

Kim, Hyun Ah; Nam, Kihoon; Lee, Minhyung; Kim, Sung Wan

2013-10-10

Gene therapy is suggested as a promising alternative strategy of hepatocellular carcinoma (HCC, also called hepatoma) therapy. To achieve a successful and safe gene therapy, tight regulation of gene expression is required to minimize side-effects in normal tissues. In this study, we developed a novel hypoxia and hepatoma dual specific gene expression vector. The constructed vectors were transfected into various cell lines using bio-reducible polymer, PAM-ABP. First, pAFPS-Luc or pAFPL-Luc vector was constructed with the alpha-fectoprotein (AFP) promoter and enhancer for hepatoma tissue specific gene expression. Then, pEpo-AFPL-Luc was constructed by insertion of the erythropoietin (Epo) enhancer for hypoxic cancer specific gene expression. In vitro transfection assay showed that pEpo-AFPL-Luc transfected hepatoma cell increased gene expression under hypoxic condition. To confirm the therapeutic effect of dual specific vector, herpes simplex virus thymidine kinase (HSV-TK) gene was introduced for cancer cell killing. The pEpo-AFPL-TK was transfected into hepatoma cell lines in the presence of ganciclovir (GCV) pro-drug. Caspase-3/7, MTT and TUNEL assays elucidated that pEpo-AFPL-TK transfected cells showed significant increasing of death rate in hypoxic hepatoma cells compared to controls. Therefore, the hypoxia/hepatoma dual specific gene expression vector with the Epo enhancer and AFP promoter may be useful for hepatoma specific gene therapy. © 2013.

Acid environments affect biofilm formation and gene expression in isolates of Salmonella enterica Typhimurium DT104.

PubMed

O'Leary, Denis; McCabe, Evonne M; McCusker, Matthew P; Martins, Marta; Fanning, Séamus; Duffy, Geraldine

2015-08-03

The aim of this study was to examine the survival and potential virulence of biofilm-forming Salmonella Typhimurium DT104 under mild acid conditions. Salmonella Typhimurium DT104 employs an acid tolerance response (ATR) allowing it to adapt to acidic environments. The threat that these acid adapted cells pose to food safety could be enhanced if they also produce biofilms in acidic conditions. The cells were acid-adapted by culturing them in 1% glucose and their ability to form biofilms on stainless steel and on the surface of Luria Bertani (LB) broth at pH7 and pH5 was examined. Plate counts were performed to examine cell survival. RNA was isolated from cells to examine changes in the expression of genes associated with virulence, invasion, biofilm formation and global gene regulation in response to acid stress. Of the 4 isolates that were examined only one (1481) that produced a rigid biofilm in LB broth at pH7 also formed this same structure at pH5. This indicated that the lactic acid severely impeded the biofilm producing capabilities of the other isolates examined under these conditions. Isolate 1481 also had higher expression of genes associated with virulence (hilA) and invasion (invA) with a 24.34-fold and 13.68-fold increase in relative gene expression respectively at pH5 compared to pH7. Although genes associated with biofilm formation had increased expression in response to acid stress for all the isolates this only resulted in the formation of a biofilm by isolate 1481. This suggests that in addition to the range of genes associated with biofilm production at neutral pH, there are genes whose protein products specifically aid in biofilm production in acidic environments. Furthermore, it highlights the potential for the use of lactic acid for the inhibition of Salmonella biofilms. Copyright © 2015 Elsevier B.V. All rights reserved.

The Rice B-Box Zinc Finger Gene Family: Genomic Identification, Characterization, Expression Profiling and Diurnal Analysis

PubMed Central

Huang, Jianyan; Zhao, Xiaobo; Weng, Xiaoyu; Wang, Lei; Xie, Weibo

2012-01-01

Background The B-box (BBX) -containing proteins are a class of zinc finger proteins that contain one or two B-box domains and play important roles in plant growth and development. The Arabidopsis BBX gene family has recently been re-identified and renamed. However, there has not been a genome-wide survey of the rice BBX (OsBBX) gene family until now. Methodology/Principal Findings In this study, we identified 30 rice BBX genes through a comprehensive bioinformatics analysis. Each gene was assigned a uniform nomenclature. We described the chromosome localizations, gene structures, protein domains, phylogenetic relationship, whole life-cycle expression profile and diurnal expression patterns of the OsBBX family members. Based on the phylogeny and domain constitution, the OsBBX gene family was classified into five subfamilies. The gene duplication analysis revealed that only chromosomal segmental duplication contributed to the expansion of the OsBBX gene family. The expression profile of the OsBBX genes was analyzed by Affymetrix GeneChip microarrays throughout the entire life-cycle of rice cultivar Zhenshan 97 (ZS97). In addition, microarray analysis was performed to obtain the expression patterns of these genes under light/dark conditions and after three phytohormone treatments. This analysis revealed that the expression patterns of the OsBBX genes could be classified into eight groups. Eight genes were regulated under the light/dark treatments, and eleven genes showed differential expression under at least one phytohormone treatment. Moreover, we verified the diurnal expression of the OsBBX genes using the data obtained from the Diurnal Project and qPCR analysis, and the results indicated that many of these genes had a diurnal expression pattern. Conclusions/Significance The combination of the genome-wide identification and the expression and diurnal analysis of the OsBBX gene family should facilitate additional functional studies of the OsBBX genes. PMID:23118960

Differential Expression Analysis of a Subset of Drought-Responsive GmNAC Genes in Two Soybean Cultivars Differing in Drought Tolerance

PubMed Central

Thao, Nguyen Phuong; Thu, Nguyen Binh Anh; Hoang, Xuan Lan Thi; Van Ha, Chien; Tran, Lam-Son Phan

2013-01-01

The plant-specific NAC transcription factors play important roles in plant response to drought stress. Here, we have compared the expression levels of a subset of GmNAC genes in drought-tolerant DT51 and drought-sensitive MTD720 under both normal and drought stress conditions aimed at identifying correlation between GmNAC expression levels and drought tolerance degree, as well as potential GmNAC candidates for genetic engineering. The expression of 23 selected dehydration-responsive GmNACs was assessed in both stressed and unstressed root tissues of DT51 and MTD720 using real-time quantitative PCR. The results indicated that expression of GmNACs was genotype-dependent. Seven and 13 of 23 tested GmNACs showed higher expression levels in roots of DT51 in comparison with MTD720 under normal and drought stress conditions, respectively, whereas none of them displayed lower transcript levels under any conditions. This finding suggests that the higher drought tolerance of DT51 might be positively correlated with the higher induction of the GmNAC genes during water deficit. The drought-inducible GmNAC011 needs to be mentioned as its transcript accumulation was more than 76-fold higher in drought-stressed DT51 roots relative to MTD720 roots. Additionally, among the GmNAC genes examined, GmNAC085, 092, 095, 101 and 109 were not only drought-inducible but also more highly up-regulated in DT51 roots than in that of MTD720 under both treatment conditions. These data together suggest that GmNAC011, 085, 092, 095, 101 and 109 might be promising candidates for improvement of drought tolerance in soybean by biotechnological approaches. PMID:24322442

Changes in Gene Expression in the Hippocampus Following Exposure to 56Fe Particles and Protection by Berry Diets

NASA Astrophysics Data System (ADS)

Shukitt-Hale, Barbara; Lau, Francis; Carey, Amanda; Carrihill-Knoll, Kirsty; Rabin, Bernard; Joseph, James

Exposing young rats to particles of high energy and charge (HZE particles), such as 56 Fe, enhances indices of oxidative stress and inflammation and disrupts the functioning of the dopaminergic system and behaviors mediated by this system in a manner similar to that seen in aged animals. Behaviors affected by radiation include deficits in motor performance, spatial learning and memory behavior, amphetamine-induced conditioned taste aversion learning, conditioned place preference, and operant conditioning. Berry fruit diets are high in antioxidant and antiinflammatory activity, and prevent the occurrence of the neurochemical and behavioral changes that occur in aging and by exposure to 56 Fe particles. In the present study, we examined whether gene expression in the hippocampus, an area of the brain important in memory, is affected by exposure to 56 Fe particles 36 hours post-irradiation. We also evaluated whether the blueberry (BB) and strawberry (SB) diets could ameliorate irradiation-induced deficits in gene expression by maintaining rats on these diets or a control diet for 8 weeks prior to being exposed to radiation. Therefore, to measure gene expression, 4 rats/group were euthanized 36 hours post whole-body irradiation with 1.5 Gy or 2.5 Gy of 1 GeV/n high-energy 56 Fe particles. Alterations in gene expression profile induced by radiation were analyzed by pathway-focused microarrays on the inflammatory cytokines and genes involved in NF-κB signal transduction pathways. For the diet studies, 3 rats/group were irradiated with 2.5 Gy of 56 Fe following 8 weeks supplementation with either the 2% BB or the 2% SB diet. We found that genes that directly or indirectly interact in the regulation of growth and differentiation of neurons were changed following irradiation. Genes that regulate apoptosis were up-regulated whereas genes that modulate cellular proliferation were down-regulated, possibly to eliminate damaged cells and to stop cell proliferation to prevent DNA damage caused by radiation to new cells. Supplementation with the berry diets enhanced neuronal communication and cell signaling by altering gene regulation of some of the protective stress signals. Therefore, these data suggest that 56 Fe particle irradiation causes deficits in gene expression in rats which are ameliorated by berry fruit diets.

Effect of UV radiation and its implications on carotenoid pathway in Bixa orellana L.

PubMed

Sankari, M; Hridya, H; Sneha, P; George Priya Doss, C; Ramamoorthy, Siva

2017-11-01

The current study was undertaken to analyse the effect of short-term UV-B and UV-C radiations in provoking carotenoid biosynthesis in Bixa orellana. Seeds of B. orellana were germinated and exposed to the short term UV pre-treatment under controlled environmental condition for 5days. The UV treated young seedlings response in pigment contents; antioxidant enzyme activity and mRNA gene expression level were analysed. The pigment content such as chlorophyll was increased in both UV-B and UV-C treated seedlings, but the total carotenoid level was decreased when compared to the control seedlings this can be attributed to the plant adaptability to survive in a stressed condition. The β-carotene level was increased in UV-B, and UV-C treated young seedlings. No significant changes have occurred in the secondary pigment such as bixin and ABA. The activity of the antioxidant enzymes such as catalase, peroxidase, and superoxide dismutase was significantly increased in UV-B treated seedlings when compared to the UV-C treated seedlings and control. The mRNA expression of the genes involved in bixin biosynthesis pathways such as DXS, PSY, PDS, LCY-β, LCY-ε, CMT, LCD, ADH and CCD genes showed different expression pattern in UV-B and UV-C treated young seedlings. Further we analysed the gene co-expression network to identify the genes which are mainly involved in carotenoid/bixin biosynthesis pathway. Form our findings the CCD, LCY, PDS, ZDS and PSY showed a close interaction. The result of our study shows that the short term UV-B and UV-C radiations induce pigment content, antioxidant enzyme activity and different gene expression pattern allowing the plant to survive in the oxidative stress condition. Copyright © 2017 Elsevier B.V. All rights reserved.

Hybrid coexpression link similarity graph clustering for mining biological modules from multiple gene expression datasets.

PubMed

Salem, Saeed; Ozcaglar, Cagri

2014-01-01

Advances in genomic technologies have enabled the accumulation of vast amount of genomic data, including gene expression data for multiple species under various biological and environmental conditions. Integration of these gene expression datasets is a promising strategy to alleviate the challenges of protein functional annotation and biological module discovery based on a single gene expression data, which suffers from spurious coexpression. We propose a joint mining algorithm that constructs a weighted hybrid similarity graph whose nodes are the coexpression links. The weight of an edge between two coexpression links in this hybrid graph is a linear combination of the topological similarities and co-appearance similarities of the corresponding two coexpression links. Clustering the weighted hybrid similarity graph yields recurrent coexpression link clusters (modules). Experimental results on Human gene expression datasets show that the reported modules are functionally homogeneous as evident by their enrichment with biological process GO terms and KEGG pathways.

Predictable transcriptome evolution in the convergent and complex bioluminescent organs of squid

PubMed Central

Pankey, M. Sabrina; Minin, Vladimir N.; Imholte, Greg C.; Suchard, Marc A.; Oakley, Todd H.

2014-01-01

Despite contingency in life’s history, the similarity of evolutionarily convergent traits may represent predictable solutions to common conditions. However, the extent to which overall gene expression levels (transcriptomes) underlying convergent traits are themselves convergent remains largely unexplored. Here, we show strong statistical support for convergent evolutionary origins and massively parallel evolution of the entire transcriptomes in symbiotic bioluminescent organs (bacterial photophores) from two divergent squid species. The gene expression similarities are so strong that regression models of one species’ photophore can predict organ identity of a distantly related photophore from gene expression levels alone. Our results point to widespread parallel changes in gene expression evolution associated with convergent origins of complex organs. Therefore, predictable solutions may drive not only the evolution of novel, complex organs but also the evolution of overall gene expression levels that underlie them. PMID:25336755

A Genetic Approach to Promoter Recognition during Trans Induction of Viral Gene Expression

NASA Astrophysics Data System (ADS)

Coen, Donald M.; Weinheimer, Steven P.; McKnight, Steven L.

1986-10-01

Viral infection of mammalian cells entails the regulated induction of viral gene expression. The induction of many viral genes, including the herpes simplex virus gene encoding thymidine kinase (tk), depends on viral regulatory proteins that act in trans. Because recognition of the tk promoter by cellular transcription factors is well understood, its trans induction by viral regulatory proteins may serve as a useful model for the regulation of eukaryotic gene expression. A comprehensive set of mutations was therefore introduced into the chromosome of herpes simplex virus at the tk promoter to directly analyze the effects of promoter mutations on tk transcription. The promoter domains required for efficient tk expression under conditions of trans induction corresponded to those important for recognition by cellular transcription factors. Thus, trans induction of tk expression may be catalyzed initially by the interaction of viral regulatory proteins with cellular transcription factors.

Multi-tissue analysis of co-expression networks by higher-order generalized singular value decomposition identifies functionally coherent transcriptional modules.

PubMed

Xiao, Xiaolin; Moreno-Moral, Aida; Rotival, Maxime; Bottolo, Leonardo; Petretto, Enrico

2014-01-01

Recent high-throughput efforts such as ENCODE have generated a large body of genome-scale transcriptional data in multiple conditions (e.g., cell-types and disease states). Leveraging these data is especially important for network-based approaches to human disease, for instance to identify coherent transcriptional modules (subnetworks) that can inform functional disease mechanisms and pathological pathways. Yet, genome-scale network analysis across conditions is significantly hampered by the paucity of robust and computationally-efficient methods. Building on the Higher-Order Generalized Singular Value Decomposition, we introduce a new algorithmic approach for efficient, parameter-free and reproducible identification of network-modules simultaneously across multiple conditions. Our method can accommodate weighted (and unweighted) networks of any size and can similarly use co-expression or raw gene expression input data, without hinging upon the definition and stability of the correlation used to assess gene co-expression. In simulation studies, we demonstrated distinctive advantages of our method over existing methods, which was able to recover accurately both common and condition-specific network-modules without entailing ad-hoc input parameters as required by other approaches. We applied our method to genome-scale and multi-tissue transcriptomic datasets from rats (microarray-based) and humans (mRNA-sequencing-based) and identified several common and tissue-specific subnetworks with functional significance, which were not detected by other methods. In humans we recapitulated the crosstalk between cell-cycle progression and cell-extracellular matrix interactions processes in ventricular zones during neocortex expansion and further, we uncovered pathways related to development of later cognitive functions in the cortical plate of the developing brain which were previously unappreciated. Analyses of seven rat tissues identified a multi-tissue subnetwork of co-expressed heat shock protein (Hsp) and cardiomyopathy genes (Bag3, Cryab, Kras, Emd, Plec), which was significantly replicated using separate failing heart and liver gene expression datasets in humans, thus revealing a conserved functional role for Hsp genes in cardiovascular disease.

Evaluation of a nanotechnology-based approach to induce gene-expression in human THP-1 macrophages under inflammatory conditions.

PubMed

Bernal, Laura; Alvarado-Vázquez, Abigail; Ferreira, David Wilson; Paige, Candler A; Ulecia-Morón, Cristina; Hill, Bailey; Caesar, Marina; Romero-Sandoval, E Alfonso

2017-02-01

Macrophages orchestrate the initiation and resolution of inflammation by producing pro- and anti-inflammatory products. An imbalance in these mediators may originate from a deficient or excessive immune response. Therefore, macrophages are valid therapeutic targets to restore homeostasis under inflammatory conditions. We hypothesize that a specific mannosylated nanoparticle effectively induces gene expression in human macrophages under inflammatory conditions without undesirable immunogenic responses. THP-1 macrophages were challenged with lipopolysaccharide (LPS, 5μg/mL). Polyethylenimine (PEI) nanoparticles grafted with a mannose receptor ligand (Man-PEI) were used as a gene delivery method. Nanoparticle toxicity, Man-PEI cellular uptake rate and gene induction efficiency (GFP, CD14 or CD68) were studied. Potential immunogenic responses were evaluated by measuring the production of tumor necrosis factor-alpha (TNF-α), Interleukin (IL)-6 and IL-10. Man-PEI did not produce cytotoxicity, and it was effectively up-taken by THP-1 macrophages (69%). This approach produced a significant expression of GFP (mRNA and protein), CD14 and CD68 (mRNA), and transiently and mildly reduced IL-6 and IL-10 levels in LPS-challenged macrophages. Our results indicate that Man-PEI is suitable for inducing an efficient gene overexpression in human macrophages under inflammatory conditions with limited immunogenic responses. Our promising results set the foundation to test this technology to induce functional anti-inflammatory genes. Copyright © 2016 Elsevier GmbH. All rights reserved.

Spindle pole body component 25 homolog expressed by ECM stiffening is required for lung cancer cell proliferation.

PubMed

Jeong, Jangho; Keum, Seula; Kim, Daehwan; You, Eunae; Ko, Panseon; Lee, Jieun; Kim, Jaegu; Kim, Jung-Woong; Rhee, Sangmyung

2018-06-12

Accumulating evidence has shown that matrix stiffening in cancer tissue by the deposition of extracellular matrix (ECM) is closely related with severe tumor progression. However, much less is known about the genes affected by matrix stiffness and its signaling for cancer progression. In the current research, we investigated the differential gene expression of a non-small lung adenocarcinoma cell line, H1299, cultured under the conditions of soft (∼0.5 kPa) and stiff (∼40 kPa) matrices, mimicking the mechanical environments of normal and cancerous tissues, respectively. For integrated transcriptome analysis, the genes identified by ECM stiffening were compared with 8248 genes retrieved from The Cancer Genome Atlas Lung Adenocarcinoma (TCGA). In stiff matrix, 29 genes were significantly upregulated, while 75 genes were downregulated. The screening of hazard ratios for these genes using the Kaplan-Meier Plotter identified 8 genes most closely associated with cancer progression under the condition of matrix stiffening. Among these genes, spindle pole body component 25 homolog (SPC25) was one of the most up-regulated genes in stiff matrix and tumor tissue. Knockdown of SPC25 in H1299 cells using shRNA significantly inhibited cell proliferation with downregulation of the expression of checkpoint protein, Cyclin B1, under the condition of stiff matrix whereas the proliferation rate in soft matrix was not affected by SPC25 silencing. Thus, our findings provide novel key molecules for studying the relationship of extracellular matrix stiffening and cancer progression. Copyright © 2018 Elsevier Inc. All rights reserved.

Early activation of quorum sensing in Pseudomonas aeruginosa reveals the architecture of a complex regulon.

PubMed

Schuster, Martin; Greenberg, E Peter

2007-08-22

Quorum-sensing regulation of gene expression in Pseudomonas aeruginosa is complex. Two interconnected acyl-homoserine lactone (acyl-HSL) signal-receptor pairs, 3-oxo-dodecanoyl-HSL-LasR and butanoyl-HSL-RhlR, regulate more than 300 genes. The induction of most of the genes is delayed during growth of P. aeruginosa in complex medium, cannot be advanced by addition of exogenous signal, and requires additional regulatory components. Many of these late genes can be induced by addition of signals early by using specific media conditions. While several factors super-regulate the quorum receptors, others may co-regulate target promoters or may affect expression posttranscriptionally. To better understand the contributions of super-regulation and co-regulation to quorum-sensing gene expression, and to better understand the general structure of the quorum sensing network, we ectopically expressed the two receptors (in the presence of their cognate signals) and another component that affects quorum sensing, the stationary phase sigma factor RpoS, early in growth. We determined the effect on target gene expression by microarray and real-time PCR analysis. Our results show that many target genes (e.g. lasB and hcnABC) are directly responsive to receptor protein levels. Most genes (e.g. lasA, lecA, and phnAB), however, are not significantly affected, although at least some of these genes are directly regulated by quorum sensing. The majority of promoters advanced by RhlR appeared to be regulated directly, which allowed us to build a RhlR consensus sequence. The direct responsiveness of many quorum sensing target genes to receptor protein levels early in growth confirms the role of super-regulation in quorum sensing gene expression. The observation that the induction of most target genes is not affected by signal or receptor protein levels indicates that either target promoters are co-regulated by other transcription factors, or that expression is controlled posttranscriptionally. This architecture permits the integration of multiple signaling pathways resulting in quorum responses that require a "quorum" but are otherwise highly adaptable and receptive to environmental conditions.

Intake of Red Wine in Different Meals Modulates Oxidized LDL Level, Oxidative and Inflammatory Gene Expression in Healthy People: A Randomized Crossover Trial

PubMed Central

Di Renzo, Laura; Valente, Roberto; Colica, Carmen

2014-01-01

Several studies have found that adherence to the Mediterranean Diet, including consumption of red wine, is associated with beneficial effects on oxidative and inflammatory conditions. We evaluate the outcome of consumption of a McDonald's Meal (McD) and a Mediterranean Meal (MM), with and without the additive effect of red wine, in order to ascertain whether the addition of the latter has a positive impact on oxidized (ox-) LDL and on expression of oxidative and inflammatory genes. A total of 24 subjects were analyzed for ox-LDL, CAT, GPX1, SOD2, SIRT2, and CCL5 gene expression levels, before and after consumption of the 4 different meal combinations with washout intervals between each meal. When red wine is associated with McD or MM, values of ox-LDL are lowered (P < 0.05) and expression of antioxidant genes is increased, while CCL5 expression is decreased (P < 0.05). SIRT2 expression after MM and fasting with red wine is significantly correlated with downregulation of CCL5 and upregulation of CAT (P < 0.001). GPX1 increased significantly in the comparison between baseline and all conditions with red wine. We highlighted for the first time the positive effect of red wine intake combined with different but widely consumed meal types on ox-LDL and gene expression. Trial Registration. This trial is registered with ClinicalTrials.gov NCT01890070. PMID:24876915

Sensitivity of housekeeping genes in the suprachiasmatic nucleus of the mouse brain to diet and the daily light-dark cycle.

PubMed

Cleal, Jane K; Shepherd, James N; Shearer, Jasmine L; Bruce, Kimberley D; Cagampang, Felino R

2014-08-05

The endogenous timing system within the suprachiasmatic nuclei (SCN) of the hypothalamus drives the cyclic expression of the clock molecules across the 24h day-night cycle controlling downstream molecular pathways and physiological processes. The developing fetal clock system is sensitive to the environment and physiology of the pregnant mother and as such disruption of this system could lead to altered physiology in the offspring. Characterizing the gene profiles of the endogenous molecular clock system by quantitative reverse transcription polymerase chain reaction is dependent on normalization by appropriate housekeeping genes (HKGs). However, many HKGs commonly used as internal controls, although stably expressed under control conditions, can vary significantly in their expression under certain experimental conditions. Here we analyzed the expression of 10 classic HKG across the 24h light-dark cycle in the SCN of mouse offspring exposed to normal chow or a high fat diet during early development and in postnatal life. We found that the HKGs glyceraldehyde-3-phosphate dehydrogenase, beta actin and adenosine triphosphate synthase subunit to be the most stably expressed genes in the SCN regardless of diet or time within the 24h light-dark cycle, and are therefore suitable to be used as internal controls. However SCN samples collected during the light and dark periods did show differences in expression and as such the timing of collection should be considered when carrying out gene expression studies. Copyright © 2014 Elsevier B.V. All rights reserved.

Response of marine bacterioplankton pH homeostasis gene expression to elevated CO2

NASA Astrophysics Data System (ADS)

Bunse, Carina; Lundin, Daniel; Karlsson, Christofer M. G.; Akram, Neelam; Vila-Costa, Maria; Palovaara, Joakim; Svensson, Lovisa; Holmfeldt, Karin; González, José M.; Calvo, Eva; Pelejero, Carles; Marrasé, Cèlia; Dopson, Mark; Gasol, Josep M.; Pinhassi, Jarone

2016-05-01

Human-induced ocean acidification impacts marine life. Marine bacteria are major drivers of biogeochemical nutrient cycles and energy fluxes; hence, understanding their performance under projected climate change scenarios is crucial for assessing ecosystem functioning. Whereas genetic and physiological responses of phytoplankton to ocean acidification are being disentangled, corresponding functional responses of bacterioplankton to pH reduction from elevated CO2 are essentially unknown. Here we show, from metatranscriptome analyses of a phytoplankton bloom mesocosm experiment, that marine bacteria responded to lowered pH by enhancing the expression of genes encoding proton pumps, such as respiration complexes, proteorhodopsin and membrane transporters. Moreover, taxonomic transcript analysis showed that distinct bacterial groups expressed different pH homeostasis genes in response to elevated CO2. These responses were substantial for numerous pH homeostasis genes under low-chlorophyll conditions (chlorophyll a <2.5 μg l-1) however, the changes in gene expression under high-chlorophyll conditions (chlorophyll a >20 μg l-1) were low. Given that proton expulsion through pH homeostasis mechanisms is energetically costly, these findings suggest that bacterioplankton adaptation to ocean acidification could have long-term effects on the economy of ocean ecosystems.

The mazEF toxin-antitoxin system as an attractive target in clinical isolates of Enterococcus faecium and Enterococcus faecalis.

PubMed

Soheili, Sara; Ghafourian, Sobhan; Sekawi, Zamberi; Neela, Vasantha Kumari; Sadeghifard, Nourkhoda; Taherikalani, Morovat; Khosravi, Afra; Ramli, Ramliza; Hamat, Rukman Awang

2015-01-01

The toxin-antitoxin (TA) system is a regulatory system where two sets of genes encode the toxin and its corresponding antitoxin. In this study, the prevalence of TA systems in independently isolated clinical isolates of Enterococcus faecium and Enterococcus faecalis was determined, the dominant TA system was identified, different virulence genes in E. faecium and E. faecalis were surveyed, the level of expression of the virulence and TA genes in normal and stress conditions was determined, and finally their associations with the TA genes were defined. Remarkably, the analysis demonstrated higBA and mazEF in all clinical isolates, and their locations were on chromosomes and plasmids, respectively. On the other hand, a quantitative analysis of TA and virulence genes revealed that the expression level in both genes is different under normal and stress conditions. The results obtained by anti-mazF peptide nucleic acids demonstrated that the expression level of virulence genes had decreased. These findings demonstrate an association between TA systems and virulence factors. The mazEF on the plasmids and the higBA TA genes on the chromosomes of all E. faecium and E. faecalis strains were dominant. Additionally, there was a decrease in the expression of virulence genes in the presence of anti-mazF peptide nucleic acids. Therefore, it is suggested that mazEF TA systems are potent and sensitive targets in all E. faecium and E. faecalis strains.

The mazEF toxin–antitoxin system as an attractive target in clinical isolates of Enterococcus faecium and Enterococcus faecalis

PubMed Central

Soheili, Sara; Ghafourian, Sobhan; Sekawi, Zamberi; Neela, Vasantha Kumari; Sadeghifard, Nourkhoda; Taherikalani, Morovat; Khosravi, Afra; Ramli, Ramliza; Hamat, Rukman Awang

2015-01-01

The toxin–antitoxin (TA) system is a regulatory system where two sets of genes encode the toxin and its corresponding antitoxin. In this study, the prevalence of TA systems in independently isolated clinical isolates of Enterococcus faecium and Enterococcus faecalis was determined, the dominant TA system was identified, different virulence genes in E. faecium and E. faecalis were surveyed, the level of expression of the virulence and TA genes in normal and stress conditions was determined, and finally their associations with the TA genes were defined. Remarkably, the analysis demonstrated higBA and mazEF in all clinical isolates, and their locations were on chromosomes and plasmids, respectively. On the other hand, a quantitative analysis of TA and virulence genes revealed that the expression level in both genes is different under normal and stress conditions. The results obtained by anti-mazF peptide nucleic acids demonstrated that the expression level of virulence genes had decreased. These findings demonstrate an association between TA systems and virulence factors. The mazEF on the plasmids and the higBA TA genes on the chromosomes of all E. faecium and E. faecalis strains were dominant. Additionally, there was a decrease in the expression of virulence genes in the presence of anti-mazF peptide nucleic acids. Therefore, it is suggested that mazEF TA systems are potent and sensitive targets in all E. faecium and E. faecalis strains. PMID:26005332

Glycosylation-related genes in NS0 cells are insensitive to moderately elevated ammonium concentrations

PubMed Central

Brodsky, Arthur Nathan; Caldwell, Mary; Bae, Sooneon; Harcum, Sarah W.

2014-01-01

NS0 and Chinese hamster ovary (CHO) cell lines are used to produce recombinant proteins for human therapeutics; however, ammonium accumulation can negatively impact cell growth, recombinant protein production, and protein glycosylation. To improve product quality and decrease costs, the relationship between ammonium and protein glycosylation needs to be elucidated. While ammonium has been shown to adversely affect glycosylation-related gene expression in CHO cells, NS0 studies have not been performed. Therefore, this study sought to determine if glycosylation in NS0 cells were ammonium-sensitive at the gene expression level. Using a DNA microarray that contained mouse glycosylation-related and housekeeping genes, the of these genes was analysed in response to various culture conditions – elevated ammonium, elevated salt, and elevated ammonium with proline. Surprisingly, no significant differences in gene expression levels were observed between the control and these conditions. Further, the elevated ammonium cultures were analysed using real-time quantitative reverse transcriptase PCR (qRT-PCR) for key glycosylation genes, and the qRT-PCR results corroborated the DNA microarray results, demonstrating that NS0 cells are ammonium-insensitive at the gene expression level. Since NS0 are known to have elevated nucleotide sugar pools under ammonium stress, and none of the genes directly responsible for these metabolic pools were changed, consequently cellular control at the translational or substrate-level must be responsible for the universally observed decreased glycosylation quality under elevated ammonium. PMID:25062658

Epipodial Tentacle Gene Expression and Predetermined Resilience to Summer Mortality in the Commercially Important Greenlip Abalone, Haliotis laevigata.

PubMed

Shiel, Brett P; Hall, Nathan E; Cooke, Ira R; Robinson, Nicholas A; Strugnell, Jan M

2017-04-01

"Summer mortality" is a phenomenon that occurs during warm water temperature spikes that results in the mass mortality of many ecologically and economically important mollusks such as abalone. This study aimed to determine whether the baseline gene expression of abalone before a laboratory-induced summer mortality event was associated with resilience to summer mortality. Tentacle transcriptomes of 35 greenlip abalone (Haliotis laevigata) were sequenced prior to the animals being exposed to an increase in water temperature-simulating conditions which have previously resulted in summer mortality. Abalone derived from three source locations with different environmental conditions were categorized as susceptible or resistant to summer mortality depending on whether they died or survived after the water temperature was increased. We detected two genes showing significantly higher expression in resilient abalone relative to susceptible abalone prior to the laboratory-induced summer mortality event. One of these genes was annotated through the NCBI non-redundant protein database using BLASTX to an anemone (Exaiptasia pallida) Transposon Ty3-G Gag Pol polyprotein. Distinct gene expression signatures were also found between resilient and susceptible abalone depending on the population origin, which may suggest divergence in local adaptation mechanisms for resilience. Many of these genes have been suggested to be involved in antioxidant and immune-related functions. The identification of these genes and their functional roles have enhanced our understanding of processes that may contribute to summer mortality in abalone. Our study supports the hypothesis that prestress gene expression signatures are indicative of the likelihood of summer mortality.

Heterologous Expression of the Carrot Hsp17.7 gene Increased Growth, Cell Viability, and Protein Solubility in Transformed Yeast (Saccharomyces cerevisiae) under Heat, Cold, Acid, and Osmotic Stress Conditions.

PubMed

Ko, Eunhye; Kim, Minhye; Park, Yunho; Ahn, Yeh-Jin

2017-08-01

In industrial fermentation of yeast (Saccharomyces cerevisiae), culture conditions are often modified from the optimal growth conditions of the cells to maintain large-scale cultures and/or to increase recombinant protein production. However, altered growth conditions can be stressful to yeast cells resulting in reduced cell growth and viability. In this study, a small heat shock protein gene from carrot (Daucus carota L.), Hsp17.7, was inserted into the yeast genome via homologous recombination to increase tolerance to stress conditions that can occur during industrial culture. A DNA construct, Translational elongation factor gene promoter-carrot Hsp17.7 gene-Phosphoribosyl-anthranilate isomerase gene (an auxotrophic marker), was generated by a series of PCRs and introduced into the chromosome IV of the yeast genome. Immunoblot analysis showed that carrot Hsp17.7 accumulated in the transformed yeast cell lines. Growth rates and cell viability of these cell lines were higher than control cell lines under heat, cold, acid, and hyperosmotic stress conditions. Soluble protein levels were higher in the transgenic cell lines than control cell lines under heat and cold conditions, suggesting the molecular chaperone function of the recombinant Hsp17.7. This study showed that a recombinant DNA construct containing a HSP gene from carrot was successfully expressed in yeast by homologous recombination and increased tolerances to abiotic stress conditions.

Transcriptome and gene expression analysis of DHA producer Aurantiochytrium under low temperature conditions

PubMed Central

Ma, Zengxin; Tan, Yanzhen; Cui, Guzhen; Feng, Yingang; Cui, Qiu; Song, Xiaojin

2015-01-01

Aurantiochytrium is a promising docosahexaenoic acid (DHA) production candidate due to its fast growth rate and high proportions of lipid and DHA content. In this study, high-throughput RNA sequencing technology was employed to explore the acclimatization of this DHA producer under cold stress at the transcriptional level. The overall de novo assembly of the cDNA sequence data generated 29,783 unigenes, with an average length of 1,200 bp. In total, 13,245 unigenes were annotated in at least one database. A comparative genomic analysis between normal conditions and cold stress revealed that 2,013 genes were differentially expressed during the growth stage, while 2,071 genes were differentially expressed during the lipid accumulation stage. Further functional categorization and analyses showed some differentially expressed genes were involved in processes crucial to cold acclimation, such as signal transduction, cellular component biogenesis, and carbohydrate and lipid metabolism. A brief survey of the transcripts obtained in response to cold stress underlines the survival strategy of Aurantiochytrium; of these transcripts, many directly or indirectly influence the lipid composition. This is the first study to perform a transcriptomic analysis of the Aurantiochytrium under low temperature conditions. Our results will help to enhance DHA production by Aurantiochytrium in the future. PMID:26403200

Transcriptome and gene expression analysis of DHA producer Aurantiochytrium under low temperature conditions.

PubMed

Ma, Zengxin; Tan, Yanzhen; Cui, Guzhen; Feng, Yingang; Cui, Qiu; Song, Xiaojin

2015-09-25

Aurantiochytrium is a promising docosahexaenoic acid (DHA) production candidate due to its fast growth rate and high proportions of lipid and DHA content. In this study, high-throughput RNA sequencing technology was employed to explore the acclimatization of this DHA producer under cold stress at the transcriptional level. The overall de novo assembly of the cDNA sequence data generated 29,783 unigenes, with an average length of 1,200 bp. In total, 13,245 unigenes were annotated in at least one database. A comparative genomic analysis between normal conditions and cold stress revealed that 2,013 genes were differentially expressed during the growth stage, while 2,071 genes were differentially expressed during the lipid accumulation stage. Further functional categorization and analyses showed some differentially expressed genes were involved in processes crucial to cold acclimation, such as signal transduction, cellular component biogenesis, and carbohydrate and lipid metabolism. A brief survey of the transcripts obtained in response to cold stress underlines the survival strategy of Aurantiochytrium; of these transcripts, many directly or indirectly influence the lipid composition. This is the first study to perform a transcriptomic analysis of the Aurantiochytrium under low temperature conditions. Our results will help to enhance DHA production by Aurantiochytrium in the future.

Low-Dose, Long-Wave UV Light Does Not Affect Gene Expression of Human Mesenchymal Stem Cells

PubMed Central

Wong, Darice Y.; Ranganath, Thanmayi; Kasko, Andrea M.

2015-01-01

Light is a non-invasive tool that is widely used in a range of biomedical applications. Techniques such as photopolymerization, photodegradation, and photouncaging can be used to alter the chemical and physical properties of biomaterials in the presence of live cells. Long-wave UV light (315 nm–400 nm) is an easily accessible and commonly used energy source for triggering biomaterial changes. Although exposure to low doses of long-wave UV light is generally accepted as biocompatible, most studies employing this wavelength only establish cell viability, ignoring other possible (non-toxic) effects. Since light exposure of wavelengths longer than 315 nm may potentially induce changes in cell behavior, we examined changes in gene expression of human mesenchymal stem cells exposed to light under both 2D and 3D culture conditions, including two different hydrogel fabrication techniques, decoupling UV exposure and radical generation. While exposure to long-wave UV light did not induce significant changes in gene expression regardless of culture conditions, significant changes were observed due to scaffold fabrication chemistry and between cells plated in 2D versus encapsulated in 3D scaffolds. In order to facilitate others in searching for more specific changes between the many conditions, the full data set is available on Gene Expression Omnibus for querying. PMID:26418040

Opposite nuclear level and binding activity of STAT5B and STAT3 proteins with rat haptoglobin gene under normal and turpentine induced acute phase conditions.

PubMed

Grigorov, I; Lazić, T; Cvetković, I; Milosavljević, T; Petrović, M

2001-01-01

Transcription of the rat gene encoding haptoglobin (Hp) is highly induced during acute phase (AP) response which has been previously shown to be mediated by inducible STAT3 member of the Signal Transducer and Activators of Transcription (STATs) family proteins. In this study, we observed that under normal but not in the turpentine induced AP conditions, another member of the STAT family proteins, STAT5b is expressed and binds to the hormone regulatory element (HRE) of the rat Hp gene. We found that the nuclear amounts of constitutively active STAT5b in rat liver decreased significantly with time of turpentine treatment as opposed to that of cytosol STAT5b, suggesting possible export of constitutive STAT5b from the nucleus. Nuclear accumulation and binding of inducible STAT3 proteins to the rat Hp gene HRE following turpentine treatment implicated that STAT5b negatively regulates Hp gene expression during normal conditions.

Heat shock protein Hsp90-2 expression in the Arabidopsis thaliana seedlings under clinorotation

NASA Astrophysics Data System (ADS)

Kozeko, Liudmyla

Heat shock proteins 90 kDa (Hsp90) are abundant under normal conditions and induced by stress. This family is distinguished from other chaperones in that most of its substrates are signal transduction proteins. Previously, we determined some time-dependent increase in the Hsp90 level in pea seedlings in response to simulated microgravity that indicated a stress-reaction. However, expression of the individual members of the Hsp90 family have specific pattern. The purpose of this study was to investigate possible alterations in the gene expression pattern of cytosolic Hsp90-2 in Arabidopsis thaliana seedlings under 2D-clinorotation. To obtain detailed expression pattern of the HSP90-2 genes we used seeds that provides a resource of loss-of-function mutations gene expression patterns via translational fusions with the reporter gene, GUS (a line N 166718, NASC). There were two variants of the experiment: 1) seedlings grew under clinorotation for 10, 12, 14 d; 2) seedlings grew in the stationary conditions for 10 d followed by clinorotation for 3 h -at 22o C and 16h light cycle. The seedlings grown in the stationary conditions were used as a control. GUS staining showed that HSP90-2 expression was regulated during seedling development and affected by clinorotation in the heterozygous mutant plants. In the homozygous for the mutation plants, HSP90-2 expression was stable during seedling development and not affected by clinorotation. GUS staining was observed in cotyledons, leaves and hypocotyls of the seedlings (especially intense in vascular bundles), indicating intensive cellular processes with participation of this chaperone. Possible pathways of influence of clinorotation on HSP90-2 expression are discussed.

Genome-wide identification of gene expression in contrasting maize inbred lines under field drought conditions reveals the significance of transcription factors in drought tolerance

PubMed Central

Zhang, Xiaojing; Liu, Xuyang; Zhang, Dengfeng; Tang, Huaijun; Sun, Baocheng; Li, Chunhui; Hao, Luyang; Liu, Cheng; Li, Yongxiang; Shi, Yunsu; Xie, Xiaoqing; Song, Yanchun; Wang, Tianyu; Li, Yu

2017-01-01

Drought is a major threat to maize growth and production. Understanding the molecular regulation network of drought tolerance in maize is of great importance. In this study, two maize inbred lines with contrasting drought tolerance were tested in the field under natural soil drought and well-watered conditions. In addition, the transcriptomes of their leaves was analyzed by RNA-Seq. In total, 555 and 2,558 genes were detected to specifically respond to drought in the tolerant and the sensitive line, respectively, with a more positive regulation tendency in the tolerant genotype. Furthermore, 4,700, 4,748, 4,403 and 4,288 genes showed differential expression between the two lines under moderate drought, severe drought and their well-watered controls, respectively. Transcription factors were enriched in both genotypic differentially expressed genes and specifically responsive genes of the tolerant line. It was speculated that the genotype-specific response of 20 transcription factors in the tolerance line and the sustained genotypically differential expression of 22 transcription factors might enhance tolerance to drought in maize. Our results provide new insight into maize drought tolerance-related regulation systems and provide gene resources for subsequent studies and drought tolerance improvement. PMID:28700592

Transgenic mouse models in the study of reproduction: insights into GATA protein function.

PubMed

Tevosian, Sergei G

2014-07-01

For the past 2 decades, transgenic technology in mice has allowed for an unprecedented insight into the transcriptional control of reproductive development and function. The key factor among the mouse genetic tools that made this rapid advance possible is a conditional transgenic approach, a particularly versatile method of creating gene deletions and substitutions in the mouse genome. A centerpiece of this strategy is an enzyme, Cre recombinase, which is expressed from defined DNA regulatory elements that are active in the tissue of choice. The regulatory DNA element (either genetically engineered or natural) assures Cre expression only in predetermined cell types, leading to the guided deletion of genetically modified (flanked by loxP or 'floxed' by loxP) gene loci. This review summarizes and compares the studies in which genes encoding GATA family transcription factors were targeted either globally or by Cre recombinases active in the somatic cells of ovaries and testes. The conditional gene loss experiments require detailed knowledge of the spatial and temporal expression of Cre activity, and the challenges in interpreting the outcomes are highlighted. These studies also expose the complexity of GATA-dependent regulation of gonadal gene expression and suggest that gene function is highly context dependent. © 2014 Society for Reproduction and Fertility.

Validation of Endogenous Internal Real-Time PCR Controls in Renal Tissues

PubMed Central

Cui, Xiangqin; Zhou, Juling; Qiu, Jing; Johnson, Martin R.; Mrug, Michal

2009-01-01

Background Endogenous internal controls (‘reference’ or ‘housekeeping’ genes) are widely used in real-time PCR (RT-PCR) analyses. Their use relies on the premise of consistently stable expression across studied experimental conditions. Unfortunately, none of these controls fulfills this premise across a wide range of experimental conditions; consequently, none of them can be recommended for universal use. Methods To determine which endogenous RT-PCR controls are suitable for analyses of renal tissues altered by kidney disease, we studied the expression of 16 commonly used ‘reference genes’ in 7 mildly and 7 severely affected whole kidney tissues from a well-characterized cystic kidney disease model. Expression levels of these 16 genes, determined by TaqMan® RT-PCR analyses and Affymetrix GeneChip® arrays, were normalized and tested for overall variance and equivalence of the means. Results Both statistical approaches and both TaqMan- and GeneChip-based methods converged on 3 out of the 4 top-ranked genes (Ppia, Gapdh and Pgk1) that had the most constant expression levels across the studied phenotypes. Conclusion A combination of the top-ranked genes will provide a suitable endogenous internal control for similar studies of kidney tissues across a wide range of disease severity. PMID:19729889

Gene silencing in Escherichia coli using antisense RNAs expressed from doxycycline-inducible vectors.

PubMed

Nakashima, N; Tamura, T

2013-06-01

Here, we report on the construction of doxycycline (tetracycline analogue)-inducible vectors that express antisense RNAs in Escherichia coli. Using these vectors, the expression of genes of interest can be silenced conditionally. The expression of antisense RNAs from the vectors was more tightly regulated than the previously constructed isopropyl-β-D-galactopyranoside-inducible vectors. Furthermore, expression levels of antisense RNAs were enhanced by combining the doxycycline-inducible promoter with the T7 promoter-T7 RNA polymerase system; the T7 RNA polymerase gene, under control of the doxycycline-inducible promoter, was integrated into the lacZ locus of the genome without leaving any antibiotic marker. These vectors are useful for investigating gene functions or altering cell phenotypes for biotechnological and industrial applications. A gene silencing method using antisense RNAs in Escherichia coli is described, which facilitates the investigation of bacterial gene function. In particular, the method is suitable for comprehensive analyses or phenotypic analyses of genes essential for growth. Here, we describe expansion of vector variations for expressing antisense RNAs, allowing choice of a vector appropriate for the target genes or experimental purpose. © 2013 The Society for Applied Microbiology.

Identification of Genes Differentially Expressed During Heat Shock Treatment in Aedes aegypti

DTIC Science & Technology

2009-01-01

Chan, C. W. Cheng, and R. S. Wu. 2003. Cloning of theHSP70 gene in barnacle larvae and its expression under hypoxic conditions. Mar. Pollut. Bull. 46...665Ð671. Chuang, K. H., S. H. Ho, and Y. L. Song. 2007. Cloning and expression analysis of heat shock cognate 70 gene pro- moter in tiger shrimp ...in larvae , but not adults, of a polar insect. Proc. Natl. Acad. Sci. U.S.A. 103: 14223Ð14227. Robich, R. M., J. P. Rinehart, L. J. Kitchen, and D. L

Interactions between genetic variation and cellular environment in skeletal muscle gene expression.

PubMed

Taylor, D Leland; Knowles, David A; Scott, Laura J; Ramirez, Andrea H; Casale, Francesco Paolo; Wolford, Brooke N; Guan, Li; Varshney, Arushi; Albanus, Ricardo D'Oliveira; Parker, Stephen C J; Narisu, Narisu; Chines, Peter S; Erdos, Michael R; Welch, Ryan P; Kinnunen, Leena; Saramies, Jouko; Sundvall, Jouko; Lakka, Timo A; Laakso, Markku; Tuomilehto, Jaakko; Koistinen, Heikki A; Stegle, Oliver; Boehnke, Michael; Birney, Ewan; Collins, Francis S

2018-01-01

From whole organisms to individual cells, responses to environmental conditions are influenced by genetic makeup, where the effect of genetic variation on a trait depends on the environmental context. RNA-sequencing quantifies gene expression as a molecular trait, and is capable of capturing both genetic and environmental effects. In this study, we explore opportunities of using allele-specific expression (ASE) to discover cis-acting genotype-environment interactions (GxE)-genetic effects on gene expression that depend on an environmental condition. Treating 17 common, clinical traits as approximations of the cellular environment of 267 skeletal muscle biopsies, we identify 10 candidate environmental response expression quantitative trait loci (reQTLs) across 6 traits (12 unique gene-environment trait pairs; 10% FDR per trait) including sex, systolic blood pressure, and low-density lipoprotein cholesterol. Although using ASE is in principle a promising approach to detect GxE effects, replication of such signals can be challenging as validation requires harmonization of environmental traits across cohorts and a sufficient sampling of heterozygotes for a transcribed SNP. Comprehensive discovery and replication will require large human transcriptome datasets, or the integration of multiple transcribed SNPs, coupled with standardized clinical phenotyping.

Identification of reference genes and validation for gene expression studies in diverse axolotl (Ambystoma mexicanum) tissues.

PubMed

Guelke, Eileen; Bucan, Vesna; Liebsch, Christina; Lazaridis, Andrea; Radtke, Christine; Vogt, Peter M; Reimers, Kerstin

2015-04-10

For the precise quantitative RT-PCR normalization a set of valid reference genes is obligatory. Moreover have to be taken into concern the experimental conditions as they bias the regulation of reference genes. Up till now, no reference targets have been described for the axolotl (Ambystoma mexicanum). In a search in the public database SalSite for genetic information of the axolotl we identified fourteen presumptive reference genes, eleven of which were further tested for their gene expression stability. This study characterizes the expressional patterns of 11 putative endogenous control genes during axolotl limb regeneration and in an axolotl tissue panel. All 11 reference genes showed variable expression. Strikingly, ACTB was to be found most stable expressed in all comparative tissue groups, so we reason it to be suitable for all different kinds of axolotl tissue-type investigations. Moreover do we suggest GAPDH and RPLP0 as suitable for certain axolotl tissue analysis. When it comes to axolotl limb regeneration, a validated pair of reference genes is ODC and RPLP0. With these findings, new insights into axolotl gene expression profiling might be gained. Copyright © 2015 Elsevier B.V. All rights reserved.

Time-series RNA-seq analysis package (TRAP) and its application to the analysis of rice, Oryza sativa L. ssp. Japonica, upon drought stress.

PubMed

Jo, Kyuri; Kwon, Hawk-Bin; Kim, Sun

2014-06-01

Measuring expression levels of genes at the whole genome level can be useful for many purposes, especially for revealing biological pathways underlying specific phenotype conditions. When gene expression is measured over a time period, we have opportunities to understand how organisms react to stress conditions over time. Thus many biologists routinely measure whole genome level gene expressions at multiple time points. However, there are several technical difficulties for analyzing such whole genome expression data. In addition, these days gene expression data is often measured by using RNA-sequencing rather than microarray technologies and then analysis of expression data is much more complicated since the analysis process should start with mapping short reads and produce differentially activated pathways and also possibly interactions among pathways. In addition, many useful tools for analyzing microarray gene expression data are not applicable for the RNA-seq data. Thus a comprehensive package for analyzing time series transcriptome data is much needed. In this article, we present a comprehensive package, Time-series RNA-seq Analysis Package (TRAP), integrating all necessary tasks such as mapping short reads, measuring gene expression levels, finding differentially expressed genes (DEGs), clustering and pathway analysis for time-series data in a single environment. In addition to implementing useful algorithms that are not available for RNA-seq data, we extended existing pathway analysis methods, ORA and SPIA, for time series analysis and estimates statistical values for combined dataset by an advanced metric. TRAP also produces visual summary of pathway interactions. Gene expression change labeling, a practical clustering method used in TRAP, enables more accurate interpretation of the data when combined with pathway analysis. We applied our methods on a real dataset for the analysis of rice (Oryza sativa L. Japonica nipponbare) upon drought stress. The result showed that TRAP was able to detect pathways more accurately than several existing methods. TRAP is available at http://biohealth.snu.ac.kr/software/TRAP/. Copyright © 2014 Elsevier Inc. All rights reserved.

[Analysis of gene expression pattern in peripheral blood leukocytes during experimental heat wave].

PubMed

Feoktistova, E S; Skamrov, A V; Goryunova, L E; Khaspekov, G L; Osyaeva, M K; Rodnenkov, O V; Beabealashvilli, R Sh

2017-03-01

The conditions of Moscow 2010 summer heat wave were simulated in an accommodation module. Six healthy men aged from 22 to 46 years stayed in the module for 30 days. Measurements of gene expression in peripheral blood leukocytes before, during and 3 day after simulated heat wave were performed using qRT-PCR. We observed a shift in the expression level of certain genes after heat exposure for a long time, and rapid return to the initial level, when volunteers leaved the accommodation module. Eight genes were chosen to form the "heat expression signature". EGR2, EGR3 were upregulated in all six volunteers, EGR1, SIRT1, CYP51A1, MAPK9, BAG5, MNDA were upregulated in 5 volunteers.

Kindness in the blood: A randomized controlled trial of the gene regulatory impact of prosocial behavior.

PubMed

Nelson-Coffey, S Katherine; Fritz, Megan M; Lyubomirsky, Sonja; Cole, Steve W

2017-07-01

Prosocial behavior is linked to longevity, but few studies have experimentally manipulated prosocial behavior to identify the causal mechanisms underlying this association. One possible mediating pathway involves changes in gene expression that may subsequently influence disease development or resistance. In the current study, we examined changes in a leukocyte gene expression profile known as the Conserved Transcriptional Response to Adversity (CTRA) in 159 adults who were randomly assigned for 4 weeks to engage in prosocial behavior directed towards specific others, prosocial behavior directed towards the world in general, self-focused kindness, or a neutral control task. Those randomized to prosocial behavior towards specific others demonstrated improvements (i.e., reductions) in leukocyte expression of CTRA indicator genes. No significant changes in CTRA gene expression were observed in the other 3 conditions. These findings suggest that prosocial behavior can causally impact leukocyte gene expression profiles in ways that might potentially help explain the previously observed health advantages associated with social ties. Copyright © 2017 Elsevier Ltd. All rights reserved.

Blockage of hemichannels alters gene expression in osteocytes in a high magneto-gravitational environment

PubMed Central

Xu, Huiyun; Ning, Dandan; Zhao, Dezhi; Chen, Yunhe; Zhao, Dongdong; Gu, Sumin; Jiang, Jean X.; Shang, Peng

2017-01-01

Osteocytes, the most abundant cells in bone, are highly responsive to external environmental changes. We tested how Cx43 hemichannels which mediate the exchange of small molecules between cells and extracellular environment impact genome wide gene expression under conditions of abnormal gravity and magnetic field. To this end, we subjected osteocytic MLO-Y4 cells to a high magneto-gravitational environment and used microarray to examine global gene expression and a specific blocking antibody was used to assess the role of Cx43 hemichannels. While 3 hr exposure to abnormal gravity and magnetic field had relatively minor effects on global gene expression, blocking hemichannels significantly impacted the expression of a number of genes which are involved in cell viability, apoptosis, mineral absorption, protein absorption and digestion, and focal adhesion. Also, blocking of hemichannels enriched genes in multiple signaling pathways which are enaged by TGF-beta, Jak-STAT and VEGF. These results show the role of connexin hemichannels in bone cells in high magneto-gravitational environments. PMID:27814646

The ESC/E(Z) complex, an effector of response to ovarian steroids, manifests an intrinsic difference in cells from women with Premenstrual Dysphoric Disorder

PubMed Central

Dubey, Neelima; Hoffman, Jessica F.; Schuebel, Kornel; Yuan, Qiaoping; Martinez, Pedro E.; Nieman, Lynnette K.; Rubinow, David R.; Schmidt, Peter J.; Goldman, David

2016-01-01

Clinical evidence suggests that mood and behavioral symptoms in Premenstrual Dysphoric Disorder (PMDD), a common, recently recognized, psychiatric condition among women, reflect abnormal responsivity to ovarian steroids. This differential sensitivity could be due to an unrecognized aspect of hormonal signaling or a difference in cellular response. In this study, lymphoblastoid cell line cultures (LCLs) from women with PMDD and asymptomatic Controls were compared via whole transcriptome sequencing (RNA-seq) during untreated (ovarian steroid-free) conditions and following hormone treatment. The women with PMDD manifested ovarian steroid-triggered behavioral sensitivity during a hormone suppression and add-back clinical trial, and Controls did not, leading us to hypothesize that women with PMDD might differ in their cellular response to ovarian steroids. In untreated LCLs, our results overall suggest a divergence between mRNA (e.g., gene transcription) and protein (e.g., RNA translation in proteins) for the same genes. Pathway analysis of the LCL transcriptome revealed, among others, over-expression of ESC/E(Z) complex genes (an ovarian steroid-regulated gene silencing complex) in untreated LCLs from women with PMDD, with more than half of these genes over-expressed as compared to Controls, and with significant effects for MTF2, PHF19, and SIRT1 (p<0.05). RNA and protein expression of the 13 ESC/E(Z) complex genes were individually quantitated. This pattern of increased ESC/E(Z) mRNA expression was confirmed in a larger cohort by qRT-PCR. In contrast, protein expression of ESC/E(Z) genes was decreased in untreated PMDD LCLs with MTF2, PHF19, and SIRT1 all significantly decreased (p<0.05). Finally, mRNA expression of several ESC/E(Z) complex genes were increased by progesterone in Controls only, and decreased by estradiol in PMDD LCLs. These findings demonstrate that LCLs from women with PMDD manifest a cellular difference in ESC/E(Z) complex function both in the untreated condition and in response to ovarian hormones. Dysregulation of ESC/E(Z) complex function could contribute to PMDD. PMID:28044059

Cell-cell contact regulates gene expression in CDK4-transformed mouse podocytes.

PubMed

Sakairi, Toru; Abe, Yoshifusa; Jat, Parmijit S; Kopp, Jeffrey B

2010-10-01

We transformed mouse podocytes by ectopic expression of cyclin-dependent kinase 4 (CDK4). Compared with podocytes transformed with a thermo-sensitive SV40 large T antigen mutant tsA58U19 (tsT podocytes), podocytes transformed with CDK4 (CDK4 podocytes) exhibited significantly higher expression of nephrin mRNA. Synaptopodin mRNA expression was significantly lower in CDK4 podocytes and in tsT podocytes under growth-permissive conditions (33°C) compared with tsT podocytes under growth-restricted conditions (37°C), which suggests a role for cell cycle arrest in synaptopodin mRNA expression. Confluent CDK4 podocytes showed significantly higher mRNA expression levels for nephrin, synaptopodin, Wilms tumor 1, podocalyxin, and P-cadherin compared with subconfluent cultures. We carried out experiments to clarify roles of various factors in the confluent podocyte cultures; our findings indicate that cell-cell contact promotes expression of five podocyte marker genes studied, that cellular quiescence increases synaptopodin and podocalyxin mRNA expression, and that soluble factors play a role in nephrin mRNA expression. Our findings suggest that CDK4 podocytes are useful tools to study podocyte biology. Furthermore, the role of cell-cell contact in podocyte gene expression may have relevance for podocyte function in vivo.

Environmental effects on resistance gene expression in milk stage popcorn kernels and associations with mycotoxin production

USDA-ARS?s Scientific Manuscript database

Like other forms of maize, popcorn is subject to increased levels of contamination by a variety of different mycotoxins under stress conditions, although levels generally are less than dent maize under comparable stress. Gene array analysis was used to determine expression differences of disease res...

Transcriptome and secretome analyses of Phanerochaete chrysosporium reveal complex patterns of gene expression

Treesearch

Amber J. Vanden Wymelenberg; Jill A. Gaskell; Michael D. Mozuch; Philip J. Kersten; Grzegorz Sabat; Diego Martinez; Daniel Cullen

2009-01-01

The wood decay basidiomycete Phanerochaete chrysosporium was grown under standard ligninolytic or cellulolytic conditions and subjected to whole-genome expression microarray analysis and liquid chromatography-tandem mass spectrometry of extracellular proteins. A total of 545 genes were flagged on the basis of significant changes in transcript accumulation and/or...

Expression Profile of Drug and Nutrient Absorption Related Genes in Madin-Darby Canine Kidney (MDCK) Cells Grown under Differentiation Conditions.

PubMed

Quan, Yong; Jin, Yisheng; Faria, Teresa N; Tilford, Charles A; He, Aiqing; Wall, Doris A; Smith, Ronald L; Vig, Balvinder S

2012-06-18

The expression levels of genes involved in drug and nutrient absorption were evaluated in the Madin-Darby Canine Kidney (MDCK) in vitro drug absorption model. MDCK cells were grown on plastic surfaces (for 3 days) or on Transwell® membranes (for 3, 5, 7, and 9 days). The expression profile of genes including ABC transporters, SLC transporters, and cytochrome P450 (CYP) enzymes was determined using the Affymetrix® Canine GeneChip®. Expression of genes whose probe sets passed a stringent confirmation process was examined. Expression of a few transporter (MDR1, PEPT1 and PEPT2) genes in MDCK cells was confirmed by RT-PCR. The overall gene expression profile was strongly influenced by the type of support the cells were grown on. After 3 days of growth, expression of 28% of the genes was statistically different (1.5-fold cutoff, p < 0.05) between the cells grown on plastic and Transwell® membranes. When cells were differentiated on Transwell® membranes, large changes in gene expression profile were observed during the early stages, which then stabilized after 5-7 days. Only a small number of genes encoding drug absorption related SLC, ABC, and CYP were detected in MDCK cells, and most of them exhibited low hybridization signals. Results from this study provide valuable reference information on endogenous gene expression in MDCK cells that could assist in design of drug-transporter and/or drug-enzyme interaction studies, and help interpret the contributions of various transporters and metabolic enzymes in studies with MDCK cells.

Expression Profile of Drug and Nutrient Absorption Related Genes in Madin-Darby Canine Kidney (MDCK) Cells Grown under Differentiation Conditions

PubMed Central

Quan, Yong; Jin, Yisheng; Faria, Teresa N.; Tilford, Charles A.; He, Aiqing; Wall, Doris A.; Smith, Ronald L.; Vig, Balvinder S.

2012-01-01

The expression levels of genes involved in drug and nutrient absorption were evaluated in the Madin-Darby Canine Kidney (MDCK) in vitro drug absorption model. MDCK cells were grown on plastic surfaces (for 3 days) or on Transwell® membranes (for 3, 5, 7, and 9 days). The expression profile of genes including ABC transporters, SLC transporters, and cytochrome P450 (CYP) enzymes was determined using the Affymetrix® Canine GeneChip®. Expression of genes whose probe sets passed a stringent confirmation process was examined. Expression of a few transporter (MDR1, PEPT1 and PEPT2) genes in MDCK cells was confirmed by RT-PCR. The overall gene expression profile was strongly influenced by the type of support the cells were grown on. After 3 days of growth, expression of 28% of the genes was statistically different (1.5-fold cutoff, p < 0.05) between the cells grown on plastic and Transwell® membranes. When cells were differentiated on Transwell® membranes, large changes in gene expression profile were observed during the early stages, which then stabilized after 5–7 days. Only a small number of genes encoding drug absorption related SLC, ABC, and CYP were detected in MDCK cells, and most of them exhibited low hybridization signals. Results from this study provide valuable reference information on endogenous gene expression in MDCK cells that could assist in design of drug-transporter and/or drug-enzyme interaction studies, and help interpret the contributions of various transporters and metabolic enzymes in studies with MDCK cells. PMID:24300234

Gene expression of H+-pumps in plasma and vacuolar membranes of corn root cells under the effect of sodium ions and bioactive preparations.

PubMed

Kovalenko, N O; Palladina, T A

2016-01-01

Four isoforms of H+-ATPase of plasma membrane: MHA1, MHA2, MHA3, MHA4 are expressed in the corn seedling roots with prevalence of genes MHA3 і MHA4. The exposure of seedlings in the presence of 0.1 M NaCl activated the expression of MHA4 gene isoform, that demonstrates its important role in the processes of adaptation to salinization conditions. In vacuolar membrane, where potential is created by two Н+-pumps, sodium ions activated gene expression of only Н+-АТРase of V-type, taking no effect on the expression of Н+-pyrophosphatase. The seeds pretreatment by synthetic preparations Methyure and Ivine did not affect gene expression of Н+-pumps. Thus we can suppose that the ability of the above preparations to activate functioning of Н+-pumps in the presence of sodium ions is realized at the post-tranlation level.

CMV induces HERV-K and HERV-W expression in kidney transplant recipients.

PubMed

Bergallo, Massimiliano; Galliano, Ilaria; Montanari, Paola; Gambarino, Stefano; Mareschi, Katia; Ferro, Francesca; Fagioli, Franca; Tovo, Pier-Angelo; Ravanini, Paolo

2015-07-01

Human endogenous retrovirus (HERVs) constitute approximately 8% of the human genome. Induction of HERV transcription is possible under certain circumstances, and may have a possible role in some pathological conditions. The aim of this study was to evaluate HERV-K and -W pol gene expression in kidney transplant recipients and to investigate the possible relationship between HERVs gene expression and CMV infection in these patients. Thirty-three samples of kidney transplant patients and twenty healthy blood donors were used to analyze, HERV-K and -W pol gene RNA expression by relative quantitative relative Real-Time PCR. We demonstrated that HERVs pol gene expression levels were higher in kidney transplant recipients than in healthy subjects. Moreover, HERV-K and -W pol gene expression was significantly higher in the group of kidney transplant recipients with high CMV viral load than in the groups with no or moderate CMV viral load. Our data suggest that CMV may facilitate in vivo HERV activation. Published by Elsevier B.V.

Uterine progesterone signaling is a target for metformin therapy in PCOS-like rats.

PubMed

Hu, Min; Zhang, Yuehui; Feng, Jiaxing; Xu, Xue; Zhang, Jiao; Zhao, Wei; Guo, Xiaozhu; Li, Juan; Vestin, Edvin; Cui, Peng; Li, Xin; Wu, Xiao-Ke; Brännström, Mats; Shao, Linus R; Billig, Håkan

2018-05-01

Impaired progesterone (P4) signaling is linked to endometrial dysfunction and infertility in women with polycystic ovary syndrome (PCOS). Here, we report for the first time that elevated expression of progesterone receptor (PGR) isoforms A and B parallels increased estrogen receptor (ER) expression in PCOS-like rat uteri. The aberrant PGR-targeted gene expression in PCOS-like rats before and after implantation overlaps with dysregulated expression of Fkbp52 and Ncoa2 , two genes that contribute to the development of uterine P4 resistance. In vivo and in vitro studies of the effects of metformin on the regulation of the uterine P4 signaling pathway under PCOS conditions showed that metformin directly inhibits the expression of PGR and ER along with the regulation of several genes that are targeted dependently or independently of PGR-mediated uterine implantation. Functionally, metformin treatment corrected the abnormal expression of cell-specific PGR and ER and some PGR-target genes in PCOS-like rats with implantation. Additionally, we documented how metformin contributes to the regulation of the PGR-associated MAPK/ERK/p38 signaling pathway in the PCOS-like rat uterus. Our data provide novel insights into how metformin therapy regulates uterine P4 signaling molecules under PCOS conditions. © 2018 Society for Endocrinology.

tCRISPRi: tunable and reversible, one-step control of gene expression

NASA Astrophysics Data System (ADS)

Li, Xin-Tian; Jun, Yonggun; Erickstad, Michael J.; Brown, Steven D.; Parks, Adam; Court, Donald L.; Jun, Suckjoon

2016-12-01

The ability to control the level of gene expression is a major quest in biology. A widely used approach employs deletion of a nonessential gene of interest (knockout), or multi-step recombineering to move a gene of interest under a repressible promoter (knockdown). However, these genetic methods are laborious, and limited for quantitative study. Here, we report a tunable CRISPR-cas system, “tCRISPRi”, for precise and continuous titration of gene expression by more than 30-fold. Our tCRISPRi system employs various previous advancements into a single strain: (1) We constructed a new strain containing a tunable arabinose operon promoter PBAD to quantitatively control the expression of CRISPR-(d)Cas protein over two orders of magnitude in a plasmid-free system. (2) tCRISPRi is reversible, and gene expression is repressed under knockdown conditions. (3) tCRISPRi shows significantly less than 10% leaky expression. (4) Most important from a practical perspective, construction of tCRISPRi to target a new gene requires only one-step of oligo recombineering. Our results show that tCRISPRi, in combination with recombineering, provides a simple and easy-to-implement tool for gene expression control, and is ideally suited for construction of both individual strains and high-throughput tunable knockdown libraries.

Global Expression Profiling in Atopic Eczema Reveals Reciprocal Expression of Inflammatory and Lipid Genes

PubMed Central

Sääf, Annika M.; Tengvall-Linder, Maria; Chang, Howard Y.; Adler, Adam S.; Wahlgren, Carl-Fredrik; Scheynius, Annika; Nordenskjöld, Magnus; Bradley, Maria

2008-01-01

Background Atopic eczema (AE) is a common chronic inflammatory skin disorder. In order to dissect the genetic background several linkage and genetic association studies have been performed. Yet very little is known about specific genes involved in this complex skin disease, and the underlying molecular mechanisms are not fully understood. Methodology/Findings We used human DNA microarrays to identify a molecular picture of the programmed responses of the human genome to AE. The transcriptional program was analyzed in skin biopsy samples from lesional and patch-tested skin from AE patients sensitized to Malassezia sympodialis (M. sympodialis), and corresponding biopsies from healthy individuals. The most notable feature of the global gene-expression pattern observed in AE skin was a reciprocal expression of induced inflammatory genes and repressed lipid metabolism genes. The overall transcriptional response in M. sympodialis patch-tested AE skin was similar to the gene-expression signature identified in lesional AE skin. In the constellation of genes differentially expressed in AE skin compared to healthy control skin, we have identified several potential susceptibility genes that may play a critical role in the pathological condition of AE. Many of these genes, including genes with a role in immune responses, lipid homeostasis, and epidermal differentiation, are localized on chromosomal regions previously linked to AE. Conclusions/Significance Through genome-wide expression profiling, we were able to discover a distinct reciprocal expression pattern of induced inflammatory genes and repressed lipid metabolism genes in skin from AE patients. We found a significant enrichment of differentially expressed genes in AE with cytobands associated to the disease, and furthermore new chromosomal regions were found that could potentially guide future region-specific linkage mapping in AE. The full data set is available at http://microarray-pubs.stanford.edu/eczema. PMID:19107207

Expression of Arabidopsis FCS-Like Zinc finger genes is differentially regulated by sugars, cellular energy level, and abiotic stress

PubMed Central

Jamsheer K, Muhammed; Laxmi, Ashverya

2015-01-01

Cellular energy status is an important regulator of plant growth, development, and stress mitigation. Environmental stresses ultimately lead to energy deficit in the cell which activates the SNF1-RELATED KINASE 1 (SnRK1) signaling cascade which eventually triggering a massive reprogramming of transcription to enable the plant to survive under low-energy conditions. The role of Arabidopsis thaliana FCS-Like Zinc finger (FLZ) gene family in energy and stress signaling is recently come to highlight after their interaction with kinase subunits of SnRK1 were identified. In a detailed expression analysis in different sugars, energy starvation, and replenishment series, we identified that the expression of most of the FLZ genes is differentially modulated by cellular energy level. It was found that FLZ gene family contains genes which are both positively and negatively regulated by energy deficit as well as energy-rich conditions. Genetic and pharmacological studies identified the role of HEXOKINASE 1- dependent and energy signaling pathways in the sugar-induced expression of FLZ genes. Further, these genes were also found to be highly responsive to different stresses as well as abscisic acid. In over-expression of kinase subunit of SnRK1, FLZ genes were found to be differentially regulated in accordance with their response toward energy fluctuation suggesting that these genes may work downstream to the established SnRK1 signaling under low-energy stress. Taken together, the present study provides a conceptual framework for further studies related to SnRK1-FLZ interaction in relation to sugar and energy signaling and stress response. PMID:26442059

The Regulation of Cytokine Networks in Hippocampal CA1 Differentiates Extinction from Those Required for the Maintenance of Contextual Fear Memory after Recall

PubMed Central

Scholz, Birger; Doidge, Amie N.; Barnes, Philip; Hall, Jeremy; Wilkinson, Lawrence S.; Thomas, Kerrie L.

2016-01-01

We investigated the distinctiveness of gene regulatory networks in CA1 associated with the extinction of contextual fear memory (CFM) after recall using Affymetrix GeneChip Rat Genome 230 2.0 Arrays. These data were compared to previously published retrieval and reconsolidation-attributed, and consolidation datasets. A stringent dual normalization and pareto-scaled orthogonal partial least-square discriminant multivariate analysis together with a jack-knifing-based cross-validation approach was used on all datasets to reduce false positives. Consolidation, retrieval and extinction were correlated with distinct patterns of gene expression 2 hours later. Extinction-related gene expression was most distinct from the profile accompanying consolidation. A highly specific feature was the discrete regulation of neuroimmunological gene expression associated with retrieval and extinction. Immunity–associated genes of the tyrosine kinase receptor TGFβ and PDGF, and TNF families’ characterized extinction. Cytokines and proinflammatory interleukins of the IL-1 and IL-6 families were enriched with the no-extinction retrieval condition. We used comparative genomics to predict transcription factor binding sites in proximal promoter regions of the retrieval-regulated genes. Retrieval that does not lead to extinction was associated with NF-κB-mediated gene expression. We confirmed differential NF-κBp65 expression, and activity in all of a representative sample of our candidate genes in the no-extinction condition. The differential regulation of cytokine networks after the acquisition and retrieval of CFM identifies the important contribution that neuroimmune signalling plays in normal hippocampal function. Further, targeting cytokine signalling upon retrieval offers a therapeutic strategy to promote extinction mechanisms in human disorders characterised by dysregulation of associative memory. PMID:27224427

Integrated pathway-based transcription regulation network mining and visualization based on gene expression profiles.

PubMed

Kibinge, Nelson; Ono, Naoaki; Horie, Masafumi; Sato, Tetsuo; Sugiura, Tadao; Altaf-Ul-Amin, Md; Saito, Akira; Kanaya, Shigehiko

2016-06-01

Conventionally, workflows examining transcription regulation networks from gene expression data involve distinct analytical steps. There is a need for pipelines that unify data mining and inference deduction into a singular framework to enhance interpretation and hypotheses generation. We propose a workflow that merges network construction with gene expression data mining focusing on regulation processes in the context of transcription factor driven gene regulation. The pipeline implements pathway-based modularization of expression profiles into functional units to improve biological interpretation. The integrated workflow was implemented as a web application software (TransReguloNet) with functions that enable pathway visualization and comparison of transcription factor activity between sample conditions defined in the experimental design. The pipeline merges differential expression, network construction, pathway-based abstraction, clustering and visualization. The framework was applied in analysis of actual expression datasets related to lung, breast and prostrate cancer. Copyright © 2016 Elsevier Inc. All rights reserved.

Isolation and selection of suitable reference genes for real-time PCR analyses in the skeletal muscle of the fine flounder in response to nutritional status: assessment and normalization of gene expression of growth-related genes.

PubMed

Fuentes, Eduardo N; Safian, Diego; Valdés, Juan Antonio; Molina, Alfredo

2013-08-01

In the present study, different reference genes were isolated, and their stability in the skeletal muscle of fine flounder subjected to different nutritional states was assessed using geNorm and NormFinder. The combinations between 18S and ActB; Fau and 18S; and Fau and Tubb were chosen as the most stable gene combinations in feeding, long-term fasting and refeeding, and short-term refeeding conditions, respectively. In all periods, ActB was identified as the single least stable gene. Subsequently, the expression of the myosin heavy chain (MYH) and the insulin-like growth factor-I receptor (IGF-IR) was assessed. A large variation in MYH and IGF-IR expression was found depending on the reference gene that was chosen for normalizing the expression of both genes. Using the most stable reference genes, mRNA levels of MYH decreased and IGF-IR increased during fasting, with both returning to basal levels during refeeding. However, the drop in mRNA levels for IGF-IR occurred during short-term refeeding, in contrast with the observed events in the expression of MYH, which occurred during long-term refeeding. The present study highlights the vast differences incurred when using unsuitable versus suitable reference genes for normalizing gene expression, pointing out that normalization without proper validation could result in a bias of gene expression.

A novel approach for human whole transcriptome analysis based on absolute gene expression of microarray data

PubMed Central

Bikel, Shirley; Jacobo-Albavera, Leonor; Sánchez-Muñoz, Fausto; Cornejo-Granados, Fernanda; Canizales-Quinteros, Samuel; Soberón, Xavier; Sotelo-Mundo, Rogerio R.; del Río-Navarro, Blanca E.; Mendoza-Vargas, Alfredo; Sánchez, Filiberto

2017-01-01

Background In spite of the emergence of RNA sequencing (RNA-seq), microarrays remain in widespread use for gene expression analysis in the clinic. There are over 767,000 RNA microarrays from human samples in public repositories, which are an invaluable resource for biomedical research and personalized medicine. The absolute gene expression analysis allows the transcriptome profiling of all expressed genes under a specific biological condition without the need of a reference sample. However, the background fluorescence represents a challenge to determine the absolute gene expression in microarrays. Given that the Y chromosome is absent in female subjects, we used it as a new approach for absolute gene expression analysis in which the fluorescence of the Y chromosome genes of female subjects was used as the background fluorescence for all the probes in the microarray. This fluorescence was used to establish an absolute gene expression threshold, allowing the differentiation between expressed and non-expressed genes in microarrays. Methods We extracted the RNA from 16 children leukocyte samples (nine males and seven females, ages 6–10 years). An Affymetrix Gene Chip Human Gene 1.0 ST Array was carried out for each sample and the fluorescence of 124 genes of the Y chromosome was used to calculate the absolute gene expression threshold. After that, several expressed and non-expressed genes according to our absolute gene expression threshold were compared against the expression obtained using real-time quantitative polymerase chain reaction (RT-qPCR). Results From the 124 genes of the Y chromosome, three genes (DDX3Y, TXLNG2P and EIF1AY) that displayed significant differences between sexes were used to calculate the absolute gene expression threshold. Using this threshold, we selected 13 expressed and non-expressed genes and confirmed their expression level by RT-qPCR. Then, we selected the top 5% most expressed genes and found that several KEGG pathways were significantly enriched. Interestingly, these pathways were related to the typical functions of leukocytes cells, such as antigen processing and presentation and natural killer cell mediated cytotoxicity. We also applied this method to obtain the absolute gene expression threshold in already published microarray data of liver cells, where the top 5% expressed genes showed an enrichment of typical KEGG pathways for liver cells. Our results suggest that the three selected genes of the Y chromosome can be used to calculate an absolute gene expression threshold, allowing a transcriptome profiling of microarray data without the need of an additional reference experiment. Discussion Our approach based on the establishment of a threshold for absolute gene expression analysis will allow a new way to analyze thousands of microarrays from public databases. This allows the study of different human diseases without the need of having additional samples for relative expression experiments. PMID:29230367

Different HER2 protein expression profiles aid in the histologic differential diagnosis between urothelial carcinoma in situ (CIS) and non-CIS conditions (dysplasia and reactive atypia) of the urinary bladder mucosa.

PubMed

Gunia, Sven; Koch, Stefan; Hakenberg, Oliver W; May, Matthias; Kakies, Christoph; Erbersdobler, Andreas

2011-12-01

We evaluated HER2 expression profiles in 32 carcinoma in situ (CIS) and 31 non-CIS conditions (5 dysplasia and 26 reactive atypia) of the urinary bladder mucosa by applying breast cancer scoring rules. In situ hybridization was performed on tissue microarrays to assess HER2 gene amplification status. Our immunoprofiling data disclosed moderate to strong HER2 expression in CIS, including the basal layer of the urothelium, and absent to weak HER2 expression in non-CIS conditions. From the histologic differential diagnostic standpoint, immunostaining for HER2 protein represents a useful adjunct to aid in the delineation between CIS and non-CIS conditions of the bladder mucosa. Pathogenically, aberrant HER2 protein expression in CIS seems to be more commonly associated with polysomy than with gene amplification. From a therapeutic viewpoint, prospective clinical studies should investigate the potential benefit of HER2-targeted therapies in CIS, particularly in cases unresponsive to conventional therapeutic regimens.