Use of CYP52A2A promoter to increase gene expression in yeast

DOEpatents

Craft, David L.; Wilson, C. Ron; Eirich, Dudley; Zhang, Yeyan

2004-01-06

A nucleic acid sequence including a CYP promoter operably linked to nucleic acid encoding a heterologous protein is provided to increase transcription of the nucleic acid. Expression vectors and host cells containing the nucleic acid sequence are also provided. The methods and compositions described herein are especially useful in the production of polycarboxylic acids by yeast cells.

Alu sequence involvement in transcriptional insulation of the keratin 18 gene in transgenic mice.

PubMed Central

Thorey, I S; Ceceña, G; Reynolds, W; Oshima, R G

1993-01-01

The human keratin 18 (K18) gene is expressed in a variety of adult simple epithelial tissues, including liver, intestine, lung, and kidney, but is not normally found in skin, muscle, heart, spleen, or most of the brain. Transgenic animals derived from the cloned K18 gene express the transgene in appropriate tissues at levels directly proportional to the copy number and independently of the sites of integration. We have investigated in transgenic mice the dependence of K18 gene expression on the distal 5' and 3' flanking sequences and upon the RNA polymerase III promoter of an Alu repetitive DNA transcription unit immediately upstream of the K18 promoter. Integration site-independent expression of tandemly duplicated K18 transgenes requires the presence of either an 825-bp fragment of the 5' flanking sequence or the 3.5-kb 3' flanking sequence. Mutation of the RNA polymerase III promoter of the Alu element within the 825-bp fragment abolishes copy number-dependent expression in kidney but does not abolish integration site-independent expression when assayed in the absence of the 3' flanking sequence of the K18 gene. The characteristics of integration site-independent expression and copy number-dependent expression are separable. In addition, the formation of the chromatin state of the K18 gene, which likely restricts the tissue-specific expression of this gene, is not dependent upon the distal flanking sequences of the 10-kb K18 gene but rather may depend on internal regulatory regions of the gene. Images PMID:7692231

dictyExpress: a web-based platform for sequence data management and analytics in Dictyostelium and beyond.

PubMed

Stajdohar, Miha; Rosengarten, Rafael D; Kokosar, Janez; Jeran, Luka; Blenkus, Domen; Shaulsky, Gad; Zupan, Blaz

2017-06-02

Dictyostelium discoideum, a soil-dwelling social amoeba, is a model for the study of numerous biological processes. Research in the field has benefited mightily from the adoption of next-generation sequencing for genomics and transcriptomics. Dictyostelium biologists now face the widespread challenges of analyzing and exploring high dimensional data sets to generate hypotheses and discovering novel insights. We present dictyExpress (2.0), a web application designed for exploratory analysis of gene expression data, as well as data from related experiments such as Chromatin Immunoprecipitation sequencing (ChIP-Seq). The application features visualization modules that include time course expression profiles, clustering, gene ontology enrichment analysis, differential expression analysis and comparison of experiments. All visualizations are interactive and interconnected, such that the selection of genes in one module propagates instantly to visualizations in other modules. dictyExpress currently stores the data from over 800 Dictyostelium experiments and is embedded within a general-purpose software framework for management of next-generation sequencing data. dictyExpress allows users to explore their data in a broader context by reciprocal linking with dictyBase-a repository of Dictyostelium genomic data. In addition, we introduce a companion application called GenBoard, an intuitive graphic user interface for data management and bioinformatics analysis. dictyExpress and GenBoard enable broad adoption of next generation sequencing based inquiries by the Dictyostelium research community. Labs without the means to undertake deep sequencing projects can mine the data available to the public. The entire information flow, from raw sequence data to hypothesis testing, can be accomplished in an efficient workspace. The software framework is generalizable and represents a useful approach for any research community. To encourage more wide usage, the backend is open-source, available for extension and further development by bioinformaticians and data scientists.

Efficient experimental design and analysis strategies for the detection of differential expression using RNA-Sequencing

PubMed Central

2012-01-01

Background RNA sequencing (RNA-Seq) has emerged as a powerful approach for the detection of differential gene expression with both high-throughput and high resolution capabilities possible depending upon the experimental design chosen. Multiplex experimental designs are now readily available, these can be utilised to increase the numbers of samples or replicates profiled at the cost of decreased sequencing depth generated per sample. These strategies impact on the power of the approach to accurately identify differential expression. This study presents a detailed analysis of the power to detect differential expression in a range of scenarios including simulated null and differential expression distributions with varying numbers of biological or technical replicates, sequencing depths and analysis methods. Results Differential and non-differential expression datasets were simulated using a combination of negative binomial and exponential distributions derived from real RNA-Seq data. These datasets were used to evaluate the performance of three commonly used differential expression analysis algorithms and to quantify the changes in power with respect to true and false positive rates when simulating variations in sequencing depth, biological replication and multiplex experimental design choices. Conclusions This work quantitatively explores comparisons between contemporary analysis tools and experimental design choices for the detection of differential expression using RNA-Seq. We found that the DESeq algorithm performs more conservatively than edgeR and NBPSeq. With regard to testing of various experimental designs, this work strongly suggests that greater power is gained through the use of biological replicates relative to library (technical) replicates and sequencing depth. Strikingly, sequencing depth could be reduced as low as 15% without substantial impacts on false positive or true positive rates. PMID:22985019

Efficient experimental design and analysis strategies for the detection of differential expression using RNA-Sequencing.

PubMed

Robles, José A; Qureshi, Sumaira E; Stephen, Stuart J; Wilson, Susan R; Burden, Conrad J; Taylor, Jennifer M

2012-09-17

RNA sequencing (RNA-Seq) has emerged as a powerful approach for the detection of differential gene expression with both high-throughput and high resolution capabilities possible depending upon the experimental design chosen. Multiplex experimental designs are now readily available, these can be utilised to increase the numbers of samples or replicates profiled at the cost of decreased sequencing depth generated per sample. These strategies impact on the power of the approach to accurately identify differential expression. This study presents a detailed analysis of the power to detect differential expression in a range of scenarios including simulated null and differential expression distributions with varying numbers of biological or technical replicates, sequencing depths and analysis methods. Differential and non-differential expression datasets were simulated using a combination of negative binomial and exponential distributions derived from real RNA-Seq data. These datasets were used to evaluate the performance of three commonly used differential expression analysis algorithms and to quantify the changes in power with respect to true and false positive rates when simulating variations in sequencing depth, biological replication and multiplex experimental design choices. This work quantitatively explores comparisons between contemporary analysis tools and experimental design choices for the detection of differential expression using RNA-Seq. We found that the DESeq algorithm performs more conservatively than edgeR and NBPSeq. With regard to testing of various experimental designs, this work strongly suggests that greater power is gained through the use of biological replicates relative to library (technical) replicates and sequencing depth. Strikingly, sequencing depth could be reduced as low as 15% without substantial impacts on false positive or true positive rates.

Bacterial inosine 5'-monophosphate dehydrogenase ("IMPDH") DNA as a dominant selectable marker in mammals and other eukaryotes

DOEpatents

Huberman, Eliezer [Chicago, IL; Baccam, Mekhine J [Woodridge, IL

2007-02-27

The present invention relates to a nucleic acid sequence and its corresponding protein sequence useful as a dominant selectable marker in eukaryotes. More specifically the invention relates to a nucleic acid encoding a bacterial IMPDH gene that has been engineered into a eukaryotic expression vectors, thereby permitting bacterial IMPDH expression in mammalian cells. Bacterial IMPDH expression confers resistance to MPA which can be used as dominant selectable marker in eukaryotes including mammals. The invention also relates to expression vectors and cells that express the bacterial IMPDH gene as well as gene therapies and protein synthesis.

Protein Sequencing with Tandem Mass Spectrometry

NASA Astrophysics Data System (ADS)

Ziady, Assem G.; Kinter, Michael

The recent introduction of electrospray ionization techniques that are suitable for peptides and whole proteins has allowed for the design of mass spectrometric protocols that provide accurate sequence information for proteins. The advantages gained by these approaches over traditional Edman Degradation sequencing include faster analysis and femtomole, sometimes attomole, sensitivity. The ability to efficiently identify proteins has allowed investigators to conduct studies on their differential expression or modification in response to various treatments or disease states. In this chapter, we discuss the use of electrospray tandem mass spectrometry, a technique whereby protein-derived peptides are subjected to fragmentation in the gas phase, revealing sequence information for the protein. This powerful technique has been instrumental for the study of proteins and markers associated with various disorders, including heart disease, cancer, and cystic fibrosis. We use the study of protein expression in cystic fibrosis as an example.

A diverse family of serine proteinase genes expressed in cotton boll weevil (Anthonomus grandis): implications for the design of pest-resistant transgenic cotton plants.

PubMed

Oliveira-Neto, Osmundo B; Batista, João A N; Rigden, Daniel J; Fragoso, Rodrigo R; Silva, Rodrigo O; Gomes, Eliane A; Franco, Octávio L; Dias, Simoni C; Cordeiro, Célia M T; Monnerat, Rose G; Grossi-De-Sá, Maria F

2004-09-01

Fourteen different cDNA fragments encoding serine proteinases were isolated by reverse transcription-PCR from cotton boll weevil (Anthonomus grandis) larvae. A large diversity between the sequences was observed, with a mean pairwise identity of 22% in the amino acid sequence. The cDNAs encompassed 11 trypsin-like sequences classifiable into three families and three chymotrypsin-like sequences belonging to a single family. Using a combination of 5' and 3' RACE, the full-length sequence was obtained for five of the cDNAs, named Agser2, Agser5, Agser6, Agser10 and Agser21. The encoded proteins included amino acid sequence motifs of serine proteinase active sites, conserved cysteine residues, and both zymogen activation and signal peptides. Southern blotting analysis suggested that one or two copies of these serine proteinase genes exist in the A. grandis genome. Northern blotting analysis of Agser2 and Agser5 showed that for both genes, expression is induced upon feeding and is concentrated in the gut of larvae and adult insects. Reverse northern analysis of the 14 cDNA fragments showed that only two trypsin-like and two chymotrypsin-like were expressed at detectable levels. Under the effect of the serine proteinase inhibitors soybean Kunitz trypsin inhibitor and black-eyed pea trypsin/chymotrypsin inhibitor, expression of one of the trypsin-like sequences was upregulated while expression of the two chymotrypsin-like sequences was downregulated. Copyright 2004 Elsevier Ltd.

Association of coral algal symbionts with a diverse viral community responsive to heat shock.

PubMed

Brüwer, Jan D; Agrawal, Shobhit; Liew, Yi Jin; Aranda, Manuel; Voolstra, Christian R

2017-08-17

Stony corals provide the structural foundation of coral reef ecosystems and are termed holobionts given they engage in symbioses, in particular with photosynthetic dinoflagellates of the genus Symbiodinium. Besides Symbiodinium, corals also engage with bacteria affecting metabolism, immunity, and resilience of the coral holobiont, but the role of associated viruses is largely unknown. In this regard, the increase of studies using RNA sequencing (RNA-Seq) to assess gene expression provides an opportunity to elucidate viral signatures encompassed within the data via careful delineation of sequence reads and their source of origin. Here, we re-analyzed an RNA-Seq dataset from a cultured coral symbiont (Symbiodinium microadriaticum, Clade A1) across four experimental treatments (control, cold shock, heat shock, dark shock) to characterize associated viral diversity, abundance, and gene expression. Our approach comprised the filtering and removal of host sequence reads, subsequent phylogenetic assignment of sequence reads of putative viral origin, and the assembly and analysis of differentially expressed viral genes. About 15.46% (123 million) of all sequence reads were non-host-related, of which <1% could be classified as archaea, bacteria, or virus. Of these, 18.78% were annotated as virus and comprised a diverse community consistent across experimental treatments. Further, non-host related sequence reads assembled into 56,064 contigs, including 4856 contigs of putative viral origin that featured 43 differentially expressed genes during heat shock. The differentially expressed genes included viral kinases, ubiquitin, and ankyrin repeat proteins (amongst others), which are suggested to help the virus proliferate and inhibit the algal host's antiviral response. Our results suggest that a diverse viral community is associated with coral algal endosymbionts of the genus Symbiodinium, which prompts further research on their ecological role in coral health and resilience.

Comprehensive processing of high-throughput small RNA sequencing data including quality checking, normalization, and differential expression analysis using the UEA sRNA Workbench

PubMed Central

Beckers, Matthew; Mohorianu, Irina; Stocks, Matthew; Applegate, Christopher; Dalmay, Tamas; Moulton, Vincent

2017-01-01

Recently, high-throughput sequencing (HTS) has revealed compelling details about the small RNA (sRNA) population in eukaryotes. These 20 to 25 nt noncoding RNAs can influence gene expression by acting as guides for the sequence-specific regulatory mechanism known as RNA silencing. The increase in sequencing depth and number of samples per project enables a better understanding of the role sRNAs play by facilitating the study of expression patterns. However, the intricacy of the biological hypotheses coupled with a lack of appropriate tools often leads to inadequate mining of the available data and thus, an incomplete description of the biological mechanisms involved. To enable a comprehensive study of differential expression in sRNA data sets, we present a new interactive pipeline that guides researchers through the various stages of data preprocessing and analysis. This includes various tools, some of which we specifically developed for sRNA analysis, for quality checking and normalization of sRNA samples as well as tools for the detection of differentially expressed sRNAs and identification of the resulting expression patterns. The pipeline is available within the UEA sRNA Workbench, a user-friendly software package for the processing of sRNA data sets. We demonstrate the use of the pipeline on a H. sapiens data set; additional examples on a B. terrestris data set and on an A. thaliana data set are described in the Supplemental Information. A comparison with existing approaches is also included, which exemplifies some of the issues that need to be addressed for sRNA analysis and how the new pipeline may be used to do this. PMID:28289155

ArrayExpress update--trends in database growth and links to data analysis tools.

PubMed

Rustici, Gabriella; Kolesnikov, Nikolay; Brandizi, Marco; Burdett, Tony; Dylag, Miroslaw; Emam, Ibrahim; Farne, Anna; Hastings, Emma; Ison, Jon; Keays, Maria; Kurbatova, Natalja; Malone, James; Mani, Roby; Mupo, Annalisa; Pedro Pereira, Rui; Pilicheva, Ekaterina; Rung, Johan; Sharma, Anjan; Tang, Y Amy; Ternent, Tobias; Tikhonov, Andrew; Welter, Danielle; Williams, Eleanor; Brazma, Alvis; Parkinson, Helen; Sarkans, Ugis

2013-01-01

The ArrayExpress Archive of Functional Genomics Data (http://www.ebi.ac.uk/arrayexpress) is one of three international functional genomics public data repositories, alongside the Gene Expression Omnibus at NCBI and the DDBJ Omics Archive, supporting peer-reviewed publications. It accepts data generated by sequencing or array-based technologies and currently contains data from almost a million assays, from over 30 000 experiments. The proportion of sequencing-based submissions has grown significantly over the last 2 years and has reached, in 2012, 15% of all new data. All data are available from ArrayExpress in MAGE-TAB format, which allows robust linking to data analysis and visualization tools, including Bioconductor and GenomeSpace. Additionally, R objects, for microarray data, and binary alignment format files, for sequencing data, have been generated for a significant proportion of ArrayExpress data.

Bone morphogenetic protein-binding endothelial regulator of liver sinusoidal endothelial cells induces iron overload in a fatty liver mouse model.

PubMed

Hasebe, Takumu; Tanaka, Hiroki; Sawada, Koji; Nakajima, Shunsuke; Ohtake, Takaaki; Fujiya, Mikihiro; Kohgo, Yutaka

2017-03-01

Non-alcoholic fatty liver disease (NAFLD) is frequently accompanied by iron overload. However, because of the complex hepcidin-regulating molecules, the molecular mechanism underlying iron overload remains unknown. To identify the key molecule involved in NAFLD-associated iron dysregulation, we performed whole-RNA sequencing on the livers of obese mice. Male C57BL/6 mice were fed a regular or high-fat diet for 16 or 48 weeks. Internal iron was evaluated by plasma iron, ferritin or hepatic iron content. Whole-RNA sequencing was performed by transcriptome analysis using semiconductor high-throughput sequencer. Mouse liver tissues or isolated hepatocytes and sinusoidal endothelial cells were used to assess the expression of iron-regulating molecules. Mice fed a high-fat diet for 16 weeks showed excess iron accumulation. Longer exposure to a high-fat diet increased hepatic fibrosis and intrahepatic iron accumulation. A pathway analysis of the sequencing data showed that several inflammatory pathways, including bone morphogenetic protein (BMP)-SMAD signaling, were significantly affected. Sequencing analysis showed 2314 altered genes, including decreased mRNA expression of the hepcidin-coding gene Hamp. Hepcidin protein expression and SMAD phosphorylation, which induces Hamp, were found to be reduced. The expression of BMP-binding endothelial regulator (BMPER), which inhibits BMP-SMAD signaling by binding BMP extracellularly, was up-regulated in fatty livers. In addition, immunohistochemical and cell isolation analyses showed that BMPER was primarily expressed in the liver sinusoidal endothelial cells (LSECs) rather than hepatocytes. BMPER secretion by LSECs inhibits BMP-SMAD signaling in hepatocytes and further reduces hepcidin protein expression. These intrahepatic molecular interactions suggest a novel molecular basis of iron overload in NAFLD.

Diversity, expression and mRNA targeting abilities of Argonaute-targeting miRNAs among selected vascular plants.

PubMed

Jagtap, Soham; Shivaprasad, Padubidri V

2014-12-02

Micro (mi)RNAs are important regulators of plant development. Across plant lineages, Dicer-like 1 (DCL1) proteins process long ds-like structures to produce micro (mi) RNA duplexes in a stepwise manner. These miRNAs are incorporated into Argonaute (AGO) proteins and influence expression of RNAs that have sequence complementarity with miRNAs. Expression levels of AGOs are greatly regulated by plants in order to minimize unwarranted perturbations using miRNAs to target mRNAs coding for AGOs. AGOs may also have high promoter specificity-sometimes expression of AGO can be limited to just a few cells in a plant. Viral pathogens utilize various means to counter antiviral roles of AGOs including hijacking the host encoded miRNAs to target AGOs. Two host encoded miRNAs namely miR168 and miR403 that target AGOs have been described in the model plant Arabidopsis and such a mechanism is thought to be well conserved across plants because AGO sequences are well conserved. We show that the interaction between AGO mRNAs and miRNAs is species-specific due to the diversity in sequences of two miRNAs that target AGOs, sequence diversity among corresponding target regions in AGO mRNAs and variable expression levels of these miRNAs among vascular plants. We used miRNA sequences from 68 plant species representing 31 plant families for this analysis. Sequences of miR168 and miR403 are not conserved among plant lineages, but surprisingly they differ drastically in their sequence diversity and expression levels even among closely related plants. Variation in miR168 expression among plants correlates well with secondary structures/length of loop sequences of their precursors. Our data indicates a complex AGO targeting interaction among plant lineages due to miRNA sequence diversity and sequences of miRNA targeting regions among AGO mRNAs, thus leading to the assumption that the perturbations by viruses that use host miRNAs to target antiviral AGOs can only be species-specific. We also show that rapid evolution and likely loss of expression of miR168 isoforms in tobacco is related to the insertion of MITE-like transposons between miRNA and miRNA* sequences, a possible mechanism showing how miRNAs are lost in few plant lineages even though other close relatives have abundantly expressing miRNAs.

Using Poisson mixed-effects model to quantify transcript-level gene expression in RNA-Seq.

PubMed

Hu, Ming; Zhu, Yu; Taylor, Jeremy M G; Liu, Jun S; Qin, Zhaohui S

2012-01-01

RNA sequencing (RNA-Seq) is a powerful new technology for mapping and quantifying transcriptomes using ultra high-throughput next-generation sequencing technologies. Using deep sequencing, gene expression levels of all transcripts including novel ones can be quantified digitally. Although extremely promising, the massive amounts of data generated by RNA-Seq, substantial biases and uncertainty in short read alignment pose challenges for data analysis. In particular, large base-specific variation and between-base dependence make simple approaches, such as those that use averaging to normalize RNA-Seq data and quantify gene expressions, ineffective. In this study, we propose a Poisson mixed-effects (POME) model to characterize base-level read coverage within each transcript. The underlying expression level is included as a key parameter in this model. Since the proposed model is capable of incorporating base-specific variation as well as between-base dependence that affect read coverage profile throughout the transcript, it can lead to improved quantification of the true underlying expression level. POME can be freely downloaded at http://www.stat.purdue.edu/~yuzhu/pome.html. yuzhu@purdue.edu; zhaohui.qin@emory.edu Supplementary data are available at Bioinformatics online.

New extension software modules to enhance searching and display of transcriptome data in Tripal databases

PubMed Central

Chen, Ming; Henry, Nathan; Almsaeed, Abdullah; Zhou, Xiao; Wegrzyn, Jill; Ficklin, Stephen

2017-01-01

Abstract Tripal is an open source software package for developing biological databases with a focus on genetic and genomic data. It consists of a set of core modules that deliver essential functions for loading and displaying data records and associated attributes including organisms, sequence features and genetic markers. Beyond the core modules, community members are encouraged to contribute extension modules to build on the Tripal core and to customize Tripal for individual community needs. To expand the utility of the Tripal software system, particularly for RNASeq data, we developed two new extension modules. Tripal Elasticsearch enables fast, scalable searching of the entire content of a Tripal site as well as the construction of customized advanced searches of specific data types. We demonstrate the use of this module for searching assembled transcripts by functional annotation. A second module, Tripal Analysis Expression, houses and displays records from gene expression assays such as RNA sequencing. This includes biological source materials (biomaterials), gene expression values and protocols used to generate the data. In the case of an RNASeq experiment, this would reflect the individual organisms and tissues used to produce sequencing libraries, the normalized gene expression values derived from the RNASeq data analysis and a description of the software or code used to generate the expression values. The module will load data from common flat file formats including standard NCBI Biosample XML. Data loading, display options and other configurations can be controlled by authorized users in the Drupal administrative backend. Both modules are open source, include usage documentation, and can be found in the Tripal organization’s GitHub repository. Database URL: Tripal Elasticsearch module: https://github.com/tripal/tripal_elasticsearch Tripal Analysis Expression module: https://github.com/tripal/tripal_analysis_expression PMID:29220446

Transcriptome Assembly, Gene Annotation and Tissue Gene Expression Atlas of the Rainbow Trout

PubMed Central

Salem, Mohamed; Paneru, Bam; Al-Tobasei, Rafet; Abdouni, Fatima; Thorgaard, Gary H.; Rexroad, Caird E.; Yao, Jianbo

2015-01-01

Efforts to obtain a comprehensive genome sequence for rainbow trout are ongoing and will be complemented by transcriptome information that will enhance genome assembly and annotation. Previously, transcriptome reference sequences were reported using data from different sources. Although the previous work added a great wealth of sequences, a complete and well-annotated transcriptome is still needed. In addition, gene expression in different tissues was not completely addressed in the previous studies. In this study, non-normalized cDNA libraries were sequenced from 13 different tissues of a single doubled haploid rainbow trout from the same source used for the rainbow trout genome sequence. A total of ~1.167 billion paired-end reads were de novo assembled using the Trinity RNA-Seq assembler yielding 474,524 contigs > 500 base-pairs. Of them, 287,593 had homologies to the NCBI non-redundant protein database. The longest contig of each cluster was selected as a reference, yielding 44,990 representative contigs. A total of 4,146 contigs (9.2%), including 710 full-length sequences, did not match any mRNA sequences in the current rainbow trout genome reference. Mapping reads to the reference genome identified an additional 11,843 transcripts not annotated in the genome. A digital gene expression atlas revealed 7,678 housekeeping and 4,021 tissue-specific genes. Expression of about 16,000–32,000 genes (35–71% of the identified genes) accounted for basic and specialized functions of each tissue. White muscle and stomach had the least complex transcriptomes, with high percentages of their total mRNA contributed by a small number of genes. Brain, testis and intestine, in contrast, had complex transcriptomes, with a large numbers of genes involved in their expression patterns. This study provides comprehensive de novo transcriptome information that is suitable for functional and comparative genomics studies in rainbow trout, including annotation of the genome. PMID:25793877

Improving membrane protein expression by optimizing integration efficiency

PubMed Central

2017-01-01

The heterologous overexpression of integral membrane proteins in Escherichia coli often yields insufficient quantities of purifiable protein for applications of interest. The current study leverages a recently demonstrated link between co-translational membrane integration efficiency and protein expression levels to predict protein sequence modifications that improve expression. Membrane integration efficiencies, obtained using a coarse-grained simulation approach, robustly predicted effects on expression of the integral membrane protein TatC for a set of 140 sequence modifications, including loop-swap chimeras and single-residue mutations distributed throughout the protein sequence. Mutations that improve simulated integration efficiency were 4-fold enriched with respect to improved experimentally observed expression levels. Furthermore, the effects of double mutations on both simulated integration efficiency and experimentally observed expression levels were cumulative and largely independent, suggesting that multiple mutations can be introduced to yield higher levels of purifiable protein. This work provides a foundation for a general method for the rational overexpression of integral membrane proteins based on computationally simulated membrane integration efficiencies. PMID:28918393

Genome sequence of the necrotrophic plant pathogen Pythium ultimum reveals original pathogenicity mechanisms and effector repertoire.

USDA-ARS?s Scientific Manuscript database

The P. ultimum DAOM BR144 (=CBS 805.95 = ATCC200006) genome (42.8 Mb) encodes 15,290 genes, and has extensive sequence similarity and synteny with related Phytophthora spp., including the potato late blight pathogen Phytophthora infestans. Whole transcriptome sequencing revealed expression of 86 % o...

Repressor-mediated tissue-specific gene expression in plants

DOEpatents

Meagher, Richard B [Athens, GA; Balish, Rebecca S [Oxford, OH; Tehryung, Kim [Athens, GA; McKinney, Elizabeth C [Athens, GA

2009-02-17

Plant tissue specific gene expression by way of repressor-operator complexes, has enabled outcomes including, without limitation, male sterility and engineered plants having root-specific gene expression of relevant proteins to clean environmental pollutants from soil and water. A mercury hyperaccumulation strategy requires that mercuric ion reductase coding sequence is strongly expressed. The actin promoter vector, A2pot, engineered to contain bacterial lac operator sequences, directed strong expression in all plant vegetative organs and tissues. In contrast, the expression from the A2pot construct was restricted primarily to root tissues when a modified bacterial repressor (LacIn) was coexpressed from the light-regulated rubisco small subunit promoter in above-ground tissues. Also provided are analogous repressor operator complexes for selective expression in other plant tissues, for example, to produce male sterile plants.

Structural and functional analysis of mouse Msx1 gene promoter: sequence conservation with human MSX1 promoter points at potential regulatory elements.

PubMed

Gonzalez, S M; Ferland, L H; Robert, B; Abdelhay, E

1998-06-01

Vertebrate Msx genes are related to one of the most divergent homeobox genes of Drosophila, the muscle segment homeobox (msh) gene, and are expressed in a well-defined pattern at sites of tissue interactions. This pattern of expression is conserved in vertebrates as diverse as quail, zebrafish, and mouse in a range of sites including neural crest, appendages, and craniofacial structures. In the present work, we performed structural and functional analyses in order to identify potential cis-acting elements that may be regulating Msx1 gene expression. To this end, a 4.9-kb segment of the 5'-flanking region was sequenced and analyzed for transcription-factor binding sites. Four regions showing a high concentration of these sites were identified. Transfection assays with fragments of regulatory sequences driving the expression of the bacterial lacZ reporter gene showed that a region of 4 kb upstream of the transcription start site contains positive and negative elements responsible for controlling gene expression. Interestingly, a fragment of 130 bp seems to contain the minimal elements necessary for gene expression, as its removal completely abolishes gene expression in cultured cells. These results are reinforced by comparison of this region with the human Msx1 gene promoter, which shows extensive conservation, including many consensus binding sites, suggesting a regulatory role for them.

Transposon Variants and Their Effects on Gene Expression in Arabidopsis

PubMed Central

Wang, Xi; Weigel, Detlef; Smith, Lisa M.

2013-01-01

Transposable elements (TEs) make up the majority of many plant genomes. Their transcription and transposition is controlled through siRNAs and epigenetic marks including DNA methylation. To dissect the interplay of siRNA–mediated regulation and TE evolution, and to examine how TE differences affect nearby gene expression, we investigated genome-wide differences in TEs, siRNAs, and gene expression among three Arabidopsis thaliana accessions. Both TE sequence polymorphisms and presence of linked TEs are positively correlated with intraspecific variation in gene expression. The expression of genes within 2 kb of conserved TEs is more stable than that of genes next to variant TEs harboring sequence polymorphisms. Polymorphism levels of TEs and closely linked adjacent genes are positively correlated as well. We also investigated the distribution of 24-nt-long siRNAs, which mediate TE repression. TEs targeted by uniquely mapping siRNAs are on average farther from coding genes, apparently because they more strongly suppress expression of adjacent genes. Furthermore, siRNAs, and especially uniquely mapping siRNAs, are enriched in TE regions missing in other accessions. Thus, targeting by uniquely mapping siRNAs appears to promote sequence deletions in TEs. Overall, our work indicates that siRNA–targeting of TEs may influence removal of sequences from the genome and hence evolution of gene expression in plants. PMID:23408902

Environmental Control Of A Genetic Process

NASA Technical Reports Server (NTRS)

Khosla, Chaitan; Bailey, James E.

1991-01-01

E. coli bacteria altered to contain DNA sequence encoding production of hemoglobin made to produce hemoglobin at rates decreasing with increases in concentration of oxygen in culture media. Represents amplification of part of method described in "Cloned Hemoglobin Genes Enhance Growth Of Cells" (NPO-17517). Manipulation of promoter/regulator DNA sequences opens promising new subfield of recombinant-DNA technology for environmental control of expression of selected DNA sequences. New recombinant-DNA fusion gene products, expression vectors, and nucleotide-base sequences will emerge. Likely applications include such aerobic processes as manufacture of cloned proteins and synthesis of metabolites, production of chemicals by fermentation, enzymatic degradation, treatment of wastes, brewing, and variety of oxidative chemical reactions.

Isolated Polynucleotides and Methods of Promoting a Morphology in a Fungus

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

Lasure, Linda L; Dai, Ziyu

2008-10-21

The invention includes isolated polynucleotide molecules that are differentially expressed in a native fungus exhibiting a first morphology relative to the native fungus exhibiting a second morphology. The invention includes a method of enhancing a bioprocess utilizing a fungus. A transformed fungus is produced by transforming a fungus with a recombinant polynucleotide molecule. The recombinant polynucleotide molecule contains an isolated polynucleotide sequence linked operably to a promoter. The polynucleotide sequence is expressed to promote a first morphology. The first morphology of the transformed fungus enhances a bioprocess relative to the bioprocess utilizing a second morphology.

A detailed gene expression study of the Miscanthus genus reveals changes in the transcriptome associated with the rejuvenation of spring rhizomes.

PubMed

Barling, Adam; Swaminathan, Kankshita; Mitros, Therese; James, Brandon T; Morris, Juliette; Ngamboma, Ornella; Hall, Megan C; Kirkpatrick, Jessica; Alabady, Magdy; Spence, Ashley K; Hudson, Matthew E; Rokhsar, Daniel S; Moose, Stephen P

2013-12-09

The Miscanthus genus of perennial C4 grasses contains promising biofuel crops for temperate climates. However, few genomic resources exist for Miscanthus, which limits understanding of its interesting biology and future genetic improvement. A comprehensive catalog of expressed sequences were generated from a variety of Miscanthus species and tissue types, with an emphasis on characterizing gene expression changes in spring compared to fall rhizomes. Illumina short read sequencing technology was used to produce transcriptome sequences from different tissues and organs during distinct developmental stages for multiple Miscanthus species, including Miscanthus sinensis, Miscanthus sacchariflorus, and their interspecific hybrid Miscanthus × giganteus. More than fifty billion base-pairs of Miscanthus transcript sequence were produced. Overall, 26,230 Sorghum gene models (i.e., ~ 96% of predicted Sorghum genes) had at least five Miscanthus reads mapped to them, suggesting that a large portion of the Miscanthus transcriptome is represented in this dataset. The Miscanthus × giganteus data was used to identify genes preferentially expressed in a single tissue, such as the spring rhizome, using Sorghum bicolor as a reference. Quantitative real-time PCR was used to verify examples of preferential expression predicted via RNA-Seq. Contiguous consensus transcript sequences were assembled for each species and annotated using InterProScan. Sequences from the assembled transcriptome were used to amplify genomic segments from a doubled haploid Miscanthus sinensis and from Miscanthus × giganteus to further disentangle the allelic and paralogous variations in genes. This large expressed sequence tag collection creates a valuable resource for the study of Miscanthus biology by providing detailed gene sequence information and tissue preferred expression patterns. We have successfully generated a database of transcriptome assemblies and demonstrated its use in the study of genes of interest. Analysis of gene expression profiles revealed biological pathways that exhibit altered regulation in spring compared to fall rhizomes, which are consistent with their different physiological functions. The expression profiles of the subterranean rhizome provides a better understanding of the biological activities of the underground stem structures that are essentials for perenniality and the storage or remobilization of carbon and nutrient resources.

cDNA sequence and expression of a cold-responsive gene in Citrus unshiu.

PubMed

Hara, M; Wakasugi, Y; Ikoma, Y; Yano, M; Ogawa, K; Kuboi, T

1999-02-01

A cDNA clone encoding a protein (CuCOR19), the sequence of which is similar to Poncirus COR19, of the dehydrin family was isolated from the epicarp of Citrus unshiu. The molecular mass of the predicted protein was 18,980 daltons. CuCOR19 was highly hydrophilic and contained three repeating elements including Lys-rich motifs. The gene expression in leaves increased by cold stress.

The opportunities and challenges of large-scale molecular approaches to songbird neurobiology

PubMed Central

Mello, C.V.; Clayton, D.F.

2014-01-01

High-through put methods for analyzing genome structure and function are having a large impact in song-bird neurobiology. Methods include genome sequencing and annotation, comparative genomics, DNA microarrays and transcriptomics, and the development of a brain atlas of gene expression. Key emerging findings include the identification of complex transcriptional programs active during singing, the robust brain expression of non-coding RNAs, evidence of profound variations in gene expression across brain regions, and the identification of molecular specializations within song production and learning circuits. Current challenges include the statistical analysis of large datasets, effective genome curations, the efficient localization of gene expression changes to specific neuronal circuits and cells, and the dissection of behavioral and environmental factors that influence brain gene expression. The field requires efficient methods for comparisons with organisms like chicken, which offer important anatomical, functional and behavioral contrasts. As sequencing costs plummet, opportunities emerge for comparative approaches that may help reveal evolutionary transitions contributing to vocal learning, social behavior and other properties that make songbirds such compelling research subjects. PMID:25280907

Molecular cloning of chitinase 33 (chit33) gene from Trichoderma atroviride

PubMed Central

Matroudi, S.; Zamani, M.R.; Motallebi, M.

2008-01-01

In this study Trichoderma atroviride was selected as over producer of chitinase enzyme among 30 different isolates of Trichoderma sp. on the basis of chitinase specific activity. From this isolate the genomic and cDNA clones encoding chit33 have been isolated and sequenced. Comparison of genomic and cDNA sequences for defining gene structure indicates that this gene contains three short introns and also an open reading frame coding for a protein of 321 amino acids. The deduced amino acid sequence includes a 19 aa putative signal peptide. Homology between this sequence and other reported Trichoderma Chit33 proteins are discussed. The coding sequence of chit33 gene was cloned in pEt26b(+) expression vector and expressed in E. coli. PMID:24031242

Analysis of Epstein-Barr Virus Genomes and Expression Profiles in Gastric Adenocarcinoma.

PubMed

Borozan, Ivan; Zapatka, Marc; Frappier, Lori; Ferretti, Vincent

2018-01-15

Epstein-Barr virus (EBV) is a causative agent of a variety of lymphomas, nasopharyngeal carcinoma (NPC), and ∼9% of gastric carcinomas (GCs). An important question is whether particular EBV variants are more oncogenic than others, but conclusions are currently hampered by the lack of sequenced EBV genomes. Here, we contribute to this question by mining whole-genome sequences of 201 GCs to identify 13 EBV-positive GCs and by assembling 13 new EBV genome sequences, almost doubling the number of available GC-derived EBV genome sequences and providing the first non-Asian EBV genome sequences from GC. Whole-genome sequence comparisons of all EBV isolates sequenced to date (85 from tumors and 57 from healthy individuals) showed that most GC and NPC EBV isolates were closely related although American Caucasian GC samples were more distant, suggesting a geographical component. However, EBV GC isolates were found to contain some consistent changes in protein sequences regardless of geographical origin. In addition, transcriptome data available for eight of the EBV-positive GCs were analyzed to determine which EBV genes are expressed in GC. In addition to the expected latency proteins (EBNA1, LMP1, and LMP2A), specific subsets of lytic genes were consistently expressed that did not reflect a typical lytic or abortive lytic infection, suggesting a novel mechanism of EBV gene regulation in the context of GC. These results are consistent with a model in which a combination of specific latent and lytic EBV proteins promotes tumorigenesis. IMPORTANCE Epstein-Barr virus (EBV) is a widespread virus that causes cancer, including gastric carcinoma (GC), in a small subset of individuals. An important question is whether particular EBV variants are more cancer associated than others, but more EBV sequences are required to address this question. Here, we have generated 13 new EBV genome sequences from GC, almost doubling the number of EBV sequences from GC isolates and providing the first EBV sequences from non-Asian GC. We further identify sequence changes in some EBV proteins common to GC isolates. In addition, gene expression analysis of eight of the EBV-positive GCs showed consistent expression of both the expected latency proteins and a subset of lytic proteins that was not consistent with typical lytic or abortive lytic expression. These results suggest that novel mechanisms activate expression of some EBV lytic proteins and that their expression may contribute to oncogenesis. Copyright © 2018 American Society for Microbiology.

Inhibition of hepatitis B virus replication with linear DNA sequences expressing antiviral micro-RNA shuttles

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

Chattopadhyay, Saket; Ely, Abdullah; Bloom, Kristie

2009-11-20

RNA interference (RNAi) may be harnessed to inhibit viral gene expression and this approach is being developed to counter chronic infection with hepatitis B virus (HBV). Compared to synthetic RNAi activators, DNA expression cassettes that generate silencing sequences have advantages of sustained efficacy and ease of propagation in plasmid DNA (pDNA). However, the large size of pDNAs and inclusion of sequences conferring antibiotic resistance and immunostimulation limit delivery efficiency and safety. To develop use of alternative DNA templates that may be applied for therapeutic gene silencing, we assessed the usefulness of PCR-generated linear expression cassettes that produce anti-HBV micro-RNA (miR)more » shuttles. We found that silencing of HBV markers of replication was efficient (>75%) in cell culture and in vivo. miR shuttles were processed to form anti-HBV guide strands and there was no evidence of induction of the interferon response. Modification of terminal sequences to include flanking human adenoviral type-5 inverted terminal repeats was easily achieved and did not compromise silencing efficacy. These linear DNA sequences should have utility in the development of gene silencing applications where modifications of terminal elements with elimination of potentially harmful and non-essential sequences are required.« less

ESTuber db: an online database for Tuber borchii EST sequences.

PubMed

Lazzari, Barbara; Caprera, Andrea; Cosentino, Cristian; Stella, Alessandra; Milanesi, Luciano; Viotti, Angelo

2007-03-08

The ESTuber database (http://www.itb.cnr.it/estuber) includes 3,271 Tuber borchii expressed sequence tags (EST). The dataset consists of 2,389 sequences from an in-house prepared cDNA library from truffle vegetative hyphae, and 882 sequences downloaded from GenBank and representing four libraries from white truffle mycelia and ascocarps at different developmental stages. An automated pipeline was prepared to process EST sequences using public software integrated by in-house developed Perl scripts. Data were collected in a MySQL database, which can be queried via a php-based web interface. Sequences included in the ESTuber db were clustered and annotated against three databases: the GenBank nr database, the UniProtKB database and a third in-house prepared database of fungi genomic sequences. An algorithm was implemented to infer statistical classification among Gene Ontology categories from the ontology occurrences deduced from the annotation procedure against the UniProtKB database. Ontologies were also deduced from the annotation of more than 130,000 EST sequences from five filamentous fungi, for intra-species comparison purposes. Further analyses were performed on the ESTuber db dataset, including tandem repeats search and comparison of the putative protein dataset inferred from the EST sequences to the PROSITE database for protein patterns identification. All the analyses were performed both on the complete sequence dataset and on the contig consensus sequences generated by the EST assembly procedure. The resulting web site is a resource of data and links related to truffle expressed genes. The Sequence Report and Contig Report pages are the web interface core structures which, together with the Text search utility and the Blast utility, allow easy access to the data stored in the database.

Digital gene expression analysis of the zebra finch genome

PubMed Central

2010-01-01

Background In order to understand patterns of adaptation and molecular evolution it is important to quantify both variation in gene expression and nucleotide sequence divergence. Gene expression profiling in non-model organisms has recently been facilitated by the advent of massively parallel sequencing technology. Here we investigate tissue specific gene expression patterns in the zebra finch (Taeniopygia guttata) with special emphasis on the genes of the major histocompatibility complex (MHC). Results Almost 2 million 454-sequencing reads from cDNA of six different tissues were assembled and analysed. A total of 11,793 zebra finch transcripts were represented in this EST data, indicating a transcriptome coverage of about 65%. There was a positive correlation between the tissue specificity of gene expression and non-synonymous to synonymous nucleotide substitution ratio of genes, suggesting that genes with a specialised function are evolving at a higher rate (or with less constraint) than genes with a more general function. In line with this, there was also a negative correlation between overall expression levels and expression specificity of contigs. We found evidence for expression of 10 different genes related to the MHC. MHC genes showed relatively tissue specific expression levels and were in general primarily expressed in spleen. Several MHC genes, including MHC class I also showed expression in brain. Furthermore, for all genes with highest levels of expression in spleen there was an overrepresentation of several gene ontology terms related to immune function. Conclusions Our study highlights the usefulness of next-generation sequence data for quantifying gene expression in the genome as a whole as well as in specific candidate genes. Overall, the data show predicted patterns of gene expression profiles and molecular evolution in the zebra finch genome. Expression of MHC genes in particular, corresponds well with expression patterns in other vertebrates. PMID:20359325

Expression of young HERV-H loci in the course of colorectal carcinoma and correlation with molecular subtypes

PubMed Central

Naville, Magali; Bressan, Cédric; Hühns, Maja; Gock, Michael; Kühn, Florian; Volff, Jean-Nicolas; Trillet-Lenoir, Véronique

2015-01-01

Background Expression of the human endogenous retrovirus (HERV)-H family has been associated with colorectal carcinomas (CRC), yet no individual HERV-H locus expression has been thoroughly correlated with clinical data. Here, we characterized HERV-H reactivations in clinical CRC samples by integrating expression profiles, molecular patterns and clinical data. Expression of relevant HERV-H sequences was analyzed by qRT-PCR on two well-defined clinical cohorts (n = 139 pairs of tumor and adjacent normal colon tissue) including samples from adenomas (n = 21) and liver metastases (n = 16). Correlations with clinical and molecular data were assessed. Results CRC specific HERV-H sequences were validated and found expressed throughout CRC disease progression. Correlations between HERV-H expression and lymph node invasion of tumor cells (p = 0.0006) as well as microsatellite instable tumors (p < 0.0001) were established. No association with regard to age, tumor localization, grading or common mutations became apparent. Interestingly, CRC expressed elements belonged to specific young HERV-H subfamilies and their 5′ LTR often presented active histone marks. Conclusion These results suggest a functional role of HERV-H sequences in colorectal carcinogenesis. The pronounced connection with microsatellite instability warrants a more detailed investigation. Thus, HERV-H sequences in addition to tumor specific mutations may represent clinically relevant, truly CRC specific markers for diagnostic, prognostic and therapeutic purposes. PMID:26517682

Expression of young HERV-H loci in the course of colorectal carcinoma and correlation with molecular subtypes.

PubMed

Pérot, Philippe; Mullins, Christina Susanne; Naville, Magali; Bressan, Cédric; Hühns, Maja; Gock, Michael; Kühn, Florian; Volff, Jean-Nicolas; Trillet-Lenoir, Véronique; Linnebacher, Michael; Mallet, François

2015-11-24

Expression of the human endogenous retrovirus (HERV)-H family has been associated with colorectal carcinomas (CRC), yet no individual HERV-H locus expression has been thoroughly correlated with clinical data.Here, we characterized HERV-H reactivations in clinical CRC samples by integrating expression profiles, molecular patterns and clinical data. Expression of relevant HERV-H sequences was analyzed by qRT-PCR on two well-defined clinical cohorts (n = 139 pairs of tumor and adjacent normal colon tissue) including samples from adenomas (n = 21) and liver metastases (n = 16). Correlations with clinical and molecular data were assessed. CRC specific HERV-H sequences were validated and found expressed throughout CRC disease progression. Correlations between HERV-H expression and lymph node invasion of tumor cells (p = 0.0006) as well as microsatellite instable tumors (p < 0.0001) were established. No association with regard to age, tumor localization, grading or common mutations became apparent. Interestingly, CRC expressed elements belonged to specific young HERV-H subfamilies and their 5' LTR often presented active histone marks. These results suggest a functional role of HERV-H sequences in colorectal carcinogenesis. The pronounced connection with microsatellite instability warrants a more detailed investigation. Thus, HERV-H sequences in addition to tumor specific mutations may represent clinically relevant, truly CRC specific markers for diagnostic, prognostic and therapeutic purposes.

Molecular cloning, overexpression, purification, and sequence analysis of the giant panda (Ailuropoda melanoleuca) ferritin light polypeptide.

PubMed

Fu, L; Hou, Y L; Ding, X; Du, Y J; Zhu, H Q; Zhang, N; Hou, W R

2016-08-30

The complementary DNA (cDNA) of the giant panda (Ailuropoda melanoleuca) ferritin light polypeptide (FTL) gene was successfully cloned using reverse transcription-polymerase chain reaction technology. We constructed a recombinant expression vector containing FTL cDNA and overexpressed it in Escherichia coli using pET28a plasmids. The expressed protein was then purified by nickel chelate affinity chromatography. The cloned cDNA fragment was 580 bp long and contained an open reading frame of 525 bp. The deduced protein sequence was composed of 175 amino acids and had an estimated molecular weight of 19.90 kDa, with an isoelectric point of 5.53. Topology prediction revealed one N-glycosylation site, two casein kinase II phosphorylation sites, one N-myristoylation site, two protein kinase C phosphorylation sites, and one cell attachment sequence. Alignment indicated that the nucleotide and deduced amino acid sequences are highly conserved across several mammals, including Homo sapiens, Cavia porcellus, Equus caballus, and Felis catus, among others. The FTL gene was readily expressed in E. coli, which gave rise to the accumulation of a polypeptide of the expected size (25.50 kDa, including an N-terminal polyhistidine tag).

Inferring the expression variability of human transposable element-derived exons by linear model analysis of deep RNA sequencing data.

PubMed

Zhang, Wensheng; Edwards, Andrea; Fan, Wei; Fang, Zhide; Deininger, Prescott; Zhang, Kun

2013-08-28

The exonization of transposable elements (TEs) has proven to be a significant mechanism for the creation of novel exons. Existing knowledge of the retention patterns of TE exons in mRNAs were mainly established by the analysis of Expressed Sequence Tag (EST) data and microarray data. This study seeks to validate and extend previous studies on the expression of TE exons by an integrative statistical analysis of high throughput RNA sequencing data. We collected 26 RNA-seq datasets spanning multiple tissues and cancer types. The exon-level digital expressions (indicating retention rates in mRNAs) were quantified by a double normalized measure, called the rescaled RPKM (Reads Per Kilobase of exon model per Million mapped reads). We analyzed the distribution profiles and the variability (across samples and between tissue/disease groups) of TE exon expressions, and compared them with those of other constitutive or cassette exons. We inferred the effects of four genomic factors, including the location, length, cognate TE family and TE nucleotide proportion (RTE, see Methods section) of a TE exon, on the exons' expression level and expression variability. We also investigated the biological implications of an assembly of highly-expressed TE exons. Our analysis confirmed prior studies from the following four aspects. First, with relatively high expression variability, most TE exons in mRNAs, especially those without exact counterparts in the UCSC RefSeq (Reference Sequence) gene tables, demonstrate low but still detectable expression levels in most tissue samples. Second, the TE exons in coding DNA sequences (CDSs) are less highly expressed than those in 3' (5') untranslated regions (UTRs). Third, the exons derived from chronologically ancient repeat elements, such as MIRs, tend to be highly expressed in comparison with those derived from younger TEs. Fourth, the previously observed negative relationship between the lengths of exons and the inclusion levels in transcripts is also true for exonized TEs. Furthermore, our study resulted in several novel findings. They include: (1) for the TE exons with non-zero expression and as shown in most of the studied biological samples, a high TE nucleotide proportion leads to their lower retention rates in mRNAs; (2) the considered genomic features (i.e. a continuous variable such as the exon length or a category indicator such as 3'UTR) influence the expression level and the expression variability (CV) of TE exons in an inverse manner; (3) not only the exons derived from Alu elements but also the exons from the TEs of other families were preferentially established in zinc finger (ZNF) genes.

Expression profiling of the mouse early embryo: Reflections and Perspectives

PubMed Central

Ko, Minoru S. H.

2008-01-01

Laboratory mouse plays important role in our understanding of early mammalian development and provides invaluable model for human early embryos, which are difficult to study for ethical and technical reasons. Comprehensive collection of cDNA clones, their sequences, and complete genome sequence information, which have been accumulated over last two decades, have provided even more advantages to mouse models. Here the progress in global gene expression profiling in early mouse embryos and, to some extent, stem cells are reviewed and the future directions and challenges are discussed. The discussions include the restatement of global gene expression profiles as snapshot of cellular status, and subsequent distinction between the differentiation state and physiological state of the cells. The discussions then extend to the biological problems that can be addressed only through global expression profiling, which include: bird’s-eye view of global gene expression changes, molecular index for developmental potency, cell lineage trajectory, microarray-guided cell manipulation, and the possibility of delineating gene regulatory cascades and networks. PMID:16739220

Identification of cDNAs encoding HSP70 and HSP90 in the abalone Haliotis tuberculata: Transcriptional induction in response to thermal stress in hemocyte primary culture.

PubMed

Farcy, Emilie; Serpentini, Antoine; Fiévet, Bruno; Lebel, Jean-Marc

2007-04-01

Heat-shock proteins are a multigene family of proteins whose expression is induced by a variety of stress factors. This work reports the cloning and sequencing of HSP70 and HSP90 cDNAs in the gastropod Haliotis tuberculata. The deduced amino acid sequences of both HSP70 and HSP90 from H. tuberculata shared a high degree of homology with their homologues in other species, including typical eukaryotic HSP70 and HSP90 signature sequences. We examined their transcription expression pattern in abalone hemocytes exposed to thermal stress. Real-time PCR analysis indicated that both HSP70 and HSP90 mRNA were expressed in control animals but rapidly increased after heat-shock.

Characterization of Adelphocoris suturalis (Hemiptera: Miridae) Transcriptome from Different Developmental Stages

NASA Astrophysics Data System (ADS)

Tian, Caihong; Tek Tay, Wee; Feng, Hongqiang; Wang, Ying; Hu, Yongmin; Li, Guoping

2015-06-01

Adelphocoris suturalis is one of the most serious pest insects of Bt cotton in China, however its molecular genetics, biochemistry and physiology are poorly understood. We used high throughput sequencing platform to perform de novo transcriptome assembly and gene expression analyses across different developmental stages (eggs, 2nd and 5th instar nymphs, female and male adults). We obtained 20 GB of clean data and revealed 88,614 unigenes, including 23,830 clusters and 64,784 singletons. These unigene sequences were annotated and classified by Gene Ontology, Clusters of Orthologous Groups, and Kyoto Encyclopedia of Genes and Genomes databases. A large number of differentially expressed genes were discovered through pairwise comparisons between these developmental stages. Gene expression profiles were dramatically different between life stage transitions, with some of these most differentially expressed genes being associated with sex difference, metabolism and development. Quantitative real-time PCR results confirm deep-sequencing findings based on relative expression levels of nine randomly selected genes. Furthermore, over 791,390 single nucleotide polymorphisms and 2,682 potential simple sequence repeats were identified. Our study provided comprehensive transcriptional gene expression information for A. suturalis that will form the basis to better understanding of development pathways, hormone biosynthesis, sex differences and wing formation in mirid bugs.

Characterization of Adelphocoris suturalis (Hemiptera: Miridae) Transcriptome from Different Developmental Stages

PubMed Central

Tian, Caihong; Tek Tay, Wee; Feng, Hongqiang; Wang, Ying; Hu, Yongmin; Li, Guoping

2015-01-01

Adelphocoris suturalis is one of the most serious pest insects of Bt cotton in China, however its molecular genetics, biochemistry and physiology are poorly understood. We used high throughput sequencing platform to perform de novo transcriptome assembly and gene expression analyses across different developmental stages (eggs, 2nd and 5th instar nymphs, female and male adults). We obtained 20 GB of clean data and revealed 88,614 unigenes, including 23,830 clusters and 64,784 singletons. These unigene sequences were annotated and classified by Gene Ontology, Clusters of Orthologous Groups, and Kyoto Encyclopedia of Genes and Genomes databases. A large number of differentially expressed genes were discovered through pairwise comparisons between these developmental stages. Gene expression profiles were dramatically different between life stage transitions, with some of these most differentially expressed genes being associated with sex difference, metabolism and development. Quantitative real-time PCR results confirm deep-sequencing findings based on relative expression levels of nine randomly selected genes. Furthermore, over 791,390 single nucleotide polymorphisms and 2,682 potential simple sequence repeats were identified. Our study provided comprehensive transcriptional gene expression information for A. suturalis that will form the basis to better understanding of development pathways, hormone biosynthesis, sex differences and wing formation in mirid bugs. PMID:26047353

Characterization of Cer-1 cis-regulatory region during early Xenopus development.

PubMed

Silva, Ana Cristina; Filipe, Mário; Steinbeisser, Herbert; Belo, José António

2011-05-01

Cerberus-related molecules are well-known Wnt, Nodal, and BMP inhibitors that have been implicated in different processes including anterior–posterior patterning and left–right asymmetry. In both mouse and frog, two Cerberus-related genes have been isolated, mCer-1 and mCer-2, and Xcer and Xcoco, respectively. Until now, little is known about the mechanisms involved in their transcriptional regulation. Here, we report a heterologous analysis of the mouse Cerberus-1 gene upstream regulatory regions, responsible for its expression in the visceral endodermal cells. Our analysis showed that the consensus sequences for a TATA, CAAT, or GC boxes were absent but a TGTGG sequence was present at position -172 to -168 bp, relative to the ATG. Using a series of deletion constructs and transient expression in Xenopus embryos, we found that a fragment of 1.4 kb of Cer-1 promoter sequence could reproduce the endogenous expression pattern of Xenopus cerberus. A 0.7-kb mcer-1 upstream region was able to drive reporter expression to the involuting mesendodermal cells, while further deletions abolished reporter gene expression. Our results suggest that although no sequence similarity was found between mouse and Xenopus cerberus cis-regulatory regions, the signaling cascades regulating cerberus expression, during gastrulation, is conserved.

Functionally Convergent B Cell Receptor Sequences in Transgenic Rats Expressing a Human B Cell Repertoire in Response to Tetanus Toxoid and Measles Antigens.

PubMed

Bürckert, Jean-Philippe; Dubois, Axel R S X; Faison, William J; Farinelle, Sophie; Charpentier, Emilie; Sinner, Regina; Wienecke-Baldacchino, Anke; Muller, Claude P

2017-01-01

The identification and tracking of antigen-specific immunoglobulin (Ig) sequences within total Ig repertoires is central to high-throughput sequencing (HTS) studies of infections or vaccinations. In this context, public Ig sequences shared by different individuals exposed to the same antigen could be valuable markers for tracing back infections, measuring vaccine immunogenicity, and perhaps ultimately allow the reconstruction of the immunological history of an individual. Here, we immunized groups of transgenic rats expressing human Ig against tetanus toxoid (TT), Modified Vaccinia virus Ankara (MVA), measles virus hemagglutinin and fusion proteins expressed on MVA, and the environmental carcinogen benzo[a]pyrene, coupled to TT. We showed that these antigens impose a selective pressure causing the Ig heavy chain (IgH) repertoires of the rats to converge toward the expression of antibodies with highly similar IgH CDR3 amino acid sequences. We present a computational approach, similar to differential gene expression analysis, that selects for clusters of CDR3s with 80% similarity, significantly overrepresented within the different groups of immunized rats. These IgH clusters represent antigen-induced IgH signatures exhibiting stereotypic amino acid patterns including previously described TT- and measles-specific IgH sequences. Our data suggest that with the presented methodology, transgenic Ig rats can be utilized as a model to identify antigen-induced, human IgH signatures to a variety of different antigens.

Genomic resources for Myzus persicae: EST sequencing, SNP identification, and microarray design

PubMed Central

Ramsey, John S; Wilson, Alex CC; de Vos, Martin; Sun, Qi; Tamborindeguy, Cecilia; Winfield, Agnese; Malloch, Gaynor; Smith, Dawn M; Fenton, Brian; Gray, Stewart M; Jander, Georg

2007-01-01

Background The green peach aphid, Myzus persicae (Sulzer), is a world-wide insect pest capable of infesting more than 40 plant families, including many crop species. However, despite the significant damage inflicted by M. persicae in agricultural systems through direct feeding damage and by its ability to transmit plant viruses, limited genomic information is available for this species. Results Sequencing of 16 M. persicae cDNA libraries generated 26,669 expressed sequence tags (ESTs). Aphids for library construction were raised on Arabidopsis thaliana, Nicotiana benthamiana, Brassica oleracea, B. napus, and Physalis floridana (with and without Potato leafroll virus infection). The M. persicae cDNA libraries include ones made from sexual and asexual whole aphids, guts, heads, and salivary glands. In silico comparison of cDNA libraries identified aphid genes with tissue-specific expression patterns, and gene expression that is induced by feeding on Nicotiana benthamiana. Furthermore, 2423 genes that are novel to science and potentially aphid-specific were identified. Comparison of cDNA data from three aphid lineages identified single nucleotide polymorphisms that can be used as genetic markers and, in some cases, may represent functional differences in the protein products. In particular, non-conservative amino acid substitutions in a highly expressed gut protease may be of adaptive significance for M. persicae feeding on different host plants. The Agilent eArray platform was used to design an M. persicae oligonucleotide microarray representing over 10,000 unique genes. Conclusion New genomic resources have been developed for M. persicae, an agriculturally important insect pest. These include previously unknown sequence data, a collection of expressed genes, molecular markers, and a DNA microarray that can be used to study aphid gene expression. These resources will help elucidate the adaptations that allow M. persicae to develop compatible interactions with its host plants, complementing ongoing work illuminating plant molecular responses to phloem-feeding insects. PMID:18021414

Potential Direct Regulators of the Drosophila yellow Gene Identified by Yeast One-Hybrid and RNAi Screens

PubMed Central

Kalay, Gizem; Lusk, Richard; Dome, Mackenzie; Hens, Korneel; Deplancke, Bart; Wittkopp, Patricia J.

2016-01-01

The regulation of gene expression controls development, and changes in this regulation often contribute to phenotypic evolution. Drosophila pigmentation is a model system for studying evolutionary changes in gene regulation, with differences in expression of pigmentation genes such as yellow that correlate with divergent pigment patterns among species shown to be caused by changes in cis- and trans-regulation. Currently, much more is known about the cis-regulatory component of divergent yellow expression than the trans-regulatory component, in part because very few trans-acting regulators of yellow expression have been identified. This study aims to improve our understanding of the trans-acting control of yellow expression by combining yeast-one-hybrid and RNAi screens for transcription factors binding to yellow cis-regulatory sequences and affecting abdominal pigmentation in adults, respectively. Of the 670 transcription factors included in the yeast-one-hybrid screen, 45 showed evidence of binding to one or more sequence fragments tested from the 5′ intergenic and intronic yellow sequences from D. melanogaster, D. pseudoobscura, and D. willistoni, suggesting that they might be direct regulators of yellow expression. Of the 670 transcription factors included in the yeast-one-hybrid screen, plus another TF previously shown to be genetically upstream of yellow, 125 were also tested using RNAi, and 32 showed altered abdominal pigmentation. Nine transcription factors were identified in both screens, including four nuclear receptors related to ecdysone signaling (Hr78, Hr38, Hr46, and Eip78C). This finding suggests that yellow expression might be directly controlled by nuclear receptors influenced by ecdysone during early pupal development when adult pigmentation is forming. PMID:27527791

Characteristics of the Lotus japonicus gene repertoire deduced from large-scale expressed sequence tag (EST) analysis.

PubMed

Asamizu, Erika; Nakamura, Yasukazu; Sato, Shusei; Tabata, Satoshi

2004-02-01

To perform a comprehensive analysis of genes expressed in a model legume, Lotus japonicus, a total of 74472 3'-end expressed sequence tags (EST) were generated from cDNA libraries produced from six different organs. Clustering of sequences was performed with an identity criterion of 95% for 50 bases, and a total of 20457 non-redundant sequences, 8503 contigs and 11954 singletons were generated. EST sequence coverage was analyzed by using the annotated L. japonicus genomic sequence and 1093 of the 1889 predicted protein-encoding genes (57.9%) were hit by the EST sequence(s). Gene content was compared to several plant species. Among the 8503 contigs, 471 were identified as sequences conserved only in leguminous species and these included several disease resistance-related genes. This suggested that in legumes, these genes may have evolved specifically to resist pathogen attack. The rate of gene sequence divergence was assessed by comparing similarity level and functional category based on the Gene Ontology (GO) annotation of Arabidopsis genes. This revealed that genes encoding ribosomal proteins, as well as those related to translation, photosynthesis, and cellular structure were more abundantly represented in the highly conserved class, and that genes encoding transcription factors and receptor protein kinases were abundantly represented in the less conserved class. To make the sequence information and the cDNA clones available to the research community, a Web database with useful services was created at http://www.kazusa.or.jp/en/plant/lotus/EST/.

Solexa-Sequencing Based Transcriptome Study of Plaice Skin Phenotype in Rex Rabbits (Oryctolagus cuniculus)

PubMed Central

Pan, Lei; Liu, Yan; Wei, Qiang; Xiao, Chenwen; Ji, Quanan; Bao, Guolian; Wu, Xinsheng

2015-01-01

Background Fur is an important genetically-determined characteristic of domestic rabbits; rabbit furs are of great economic value. We used the Solexa sequencing technology to assess gene expression in skin tissues from full-sib Rex rabbits of different phenotypes in order to explore the molecular mechanisms associated with fur determination. Methodology/Principal Findings Transcriptome analysis included de novo assembly, gene function identification, and gene function classification and enrichment. We obtained 74,032,912 and 71,126,891 short reads of 100 nt, which were assembled into 377,618 unique sequences by Trinity strategy (N50=680 nt). Based on BLAST results with known proteins, 50,228 sequences were identified at a cut-off E-value ≥ 10-5. Using Blast to Gene Ontology (GO), Clusters of Orthologous Groups (KOG) and Kyoto Encyclopedia of Genes and Genomes (KEGG), we obtained several genes with important protein functions. A total of 308 differentially expressed genes were obtained by transcriptome analysis of plaice and un-plaice phenotype animals; 209 additional differentially expressed genes were not found in any database. These genes included 49 that were only expressed in plaice skin rabbits. The novel genes may play important roles during skin growth and development. In addition, 99 known differentially expressed genes were assigned to PI3K-Akt signaling, focal adhesion, and ECM-receptor interactin, among others. Growth factors play a role in skin growth and development by regulating these signaling pathways. We confirmed the altered expression levels of seven target genes by qRT-PCR. And chosen a key gene for SNP to found the differentially between plaice and un-plaice phenotypes rabbit. Conclusions/Significance The rabbit transcriptome profiling data provide new insights in understanding the molecular mechanisms underlying rabbit skin growth and development. PMID:25955442

The Silkworm (Bombyx mori) microRNAs and Their Expressions in Multiple Developmental Stages

PubMed Central

Luo, Qibin; Cai, Yimei; Lin, Wen-chang; Chen, Huan; Yang, Yue; Hu, Songnian; Yu, Jun

2008-01-01

Background MicroRNAs (miRNAs) play crucial roles in various physiological processes through post-transcriptional regulation of gene expressions and are involved in development, metabolism, and many other important molecular mechanisms and cellular processes. The Bombyx mori genome sequence provides opportunities for a thorough survey for miRNAs as well as comparative analyses with other sequenced insect species. Methodology/Principal Findings We identified 114 non-redundant conserved miRNAs and 148 novel putative miRNAs from the B. mori genome with an elaborate computational protocol. We also sequenced 6,720 clones from 14 developmental stage-specific small RNA libraries in which we identified 35 unique miRNAs containing 21 conserved miRNAs (including 17 predicted miRNAs) and 14 novel miRNAs (including 11 predicted novel miRNAs). Among the 114 conserved miRNAs, we found six pairs of clusters evolutionarily conserved cross insect lineages. Our observations on length heterogeneity at 5′ and/or 3′ ends of nine miRNAs between cloned and predicted sequences, and three mature forms deriving from the same arm of putative pre-miRNAs suggest a mechanism by which miRNAs gain new functions. Analyzing development-related miRNAs expression at 14 developmental stages based on clone-sampling and stem-loop RT PCR, we discovered an unusual abundance of 33 sequences representing 12 different miRNAs and sharply fluctuated expression of miRNAs at larva-molting stage. The potential functions of several stage-biased miRNAs were also analyzed in combination with predicted target genes and silkworm's phenotypic traits; our results indicated that miRNAs may play key regulatory roles in specific developmental stages in the silkworm, such as ecdysis. Conclusions/Significance Taking a combined approach, we identified 118 conserved miRNAs and 151 novel miRNA candidates from the B. mori genome sequence. Our expression analyses by sampling miRNAs and real-time PCR over multiple developmental stages allowed us to pinpoint molting stages as hotspots of miRNA expression both in sorts and quantities. Based on the analysis of target genes, we hypothesized that miRNAs regulate development through a particular emphasis on complex stages rather than general regulatory mechanisms. PMID:18714353

Streptomyces griseus streptomycin phosphotransferase: expression of its gene in Escherichia coli and sequence homology with other antibiotic phosphotransferases and with eukaryotic protein kinases.

PubMed

Lim, C K; Smith, M C; Petty, J; Baumberg, S; Wootton, J C

1989-12-01

The aphD gene of Streptomyces griseus, encoding a streptomycin 6-phosphotransferase (SPH), was sub-cloned in the pBR322-based expression vector pRK9 (which contains the Serratia marcescens trp promoter) with selection for expression of streptomycin resistance in Escherichia coli. Two hybrid plasmids, pCKL631 and pCKL711, were isolated which conferred resistance. Both contained a approximately 2 kbp fragment already suspected to include aphD. The properties of in vitro deletion derivatives of these plasmids were consistent with the presumed location of aphD. In vitro deletion of a sequence including most of the trp promoter largely, but not quite completely, abolished the ability of the plasmid to confer streptomycin resistance, confirming that expression was indeed principally from the trp promoter. A polypeptide of approximately 34.5 kDa was present in minicells containing plasmids that conferred streptomycin resistance, but was absent when the plasmids contained in vitro deletions removing streptomycin resistance. Part of the fragment was sequenced and an open reading frame corresponding to aphD identified. A computer-assisted comparison of the deduced SPH sequence with those of other antibiotic phosphotransferases suggested a common structure A-B-C-D-E, where B and D were conserved between all sequences compared while A, C and E divided between the streptomycin and hygromycin B phosphotransferases on one hand and kanamycin/neomycin ones on the other. A composite sequence data base was searched for homologues to consensus matrices constructed from five approximately 12-residue subsequences within blocks B and D. For one subsequence, corresponding to the N-terminal portion of block D, those sequences from the database that yielded the highest homology scores comprised almost entirely either antibiotic phosphotransferases or eukaryotic protein kinases. Possible evolutionary implications of this homology, previously described by other groups, are discussed.

Transcriptome sequencing and de novo analysis of the copepod Calanus sinicus using 454 GS FLX.

PubMed

Ning, Juan; Wang, Minxiao; Li, Chaolun; Sun, Song

2013-01-01

Despite their species abundance and primary economic importance, genomic information about copepods is still limited. In particular, genomic resources are lacking for the copepod Calanus sinicus, which is a dominant species in the coastal waters of East Asia. In this study, we performed de novo transcriptome sequencing to produce a large number of expressed sequence tags for the copepod C. sinicus. Copepodid larvae and adults were used as the basic material for transcriptome sequencing. Using 454 pyrosequencing, a total of 1,470,799 reads were obtained, which were assembled into 56,809 high quality expressed sequence tags. Based on their sequence similarity to known proteins, about 14,000 different genes were identified, including members of all major conserved signaling pathways. Transcripts that were putatively involved with growth, lipid metabolism, molting, and diapause were also identified among these genes. Differentially expressed genes related to several processes were found in C. sinicus copepodid larvae and adults. We detected 284,154 single nucleotide polymorphisms (SNPs) that provide a resource for gene function studies. Our data provide the most comprehensive transcriptome resource available for C. sinicus. This resource allowed us to identify genes associated with primary physiological processes and SNPs in coding regions, which facilitated the quantitative analysis of differential gene expression. These data should provide foundation for future genetic and genomic studies of this and related species.

Cell cycle, differentiation and tissue-independent expression of ribosomal protein L37.

PubMed

Su, S; Bird, R C

1995-09-15

A unique human cDNA (hG1.16) that encodes a mRNA of 450 nucleotides was isolated from a subtractive library derived from HeLa cells. The relative expression level of hG1.16 during different cell-cycle phases was determined by Northern-blot analysis of cells synchronized by double-thymidine block and serum deprivation/refeeding. hG1.16 was constitutively expressed during all phases of the cell cycle, including the quiescent phase when even most constitutively expressed genes experience some suppression of expression. The expression level of hG1.16 did not change during terminal differentiation of myoblasts to myotubes, during which cells become permanently post-mitotic. Examination of other tissues revealed that the relative expression level of hG1.16 was constitutive in all embryonic mouse tissues examined, including brain, eye, heart, kidney, liver, lung and skeletal muscle. This was unusual in that expression was not down-modulated during differentiation and did not vary appreciably between tissue types. Analysis by inter-species Northern-blot analysis revealed that hG1.16 was highly conserved among all vertebrates studied (from fish to humans but not in insects). DNA sequence analysis of hG1.16 revealed a high level of similarity to rat ribosomal protein L37, identifying hG1.16 as a new member of this multigene family. The deduced amino acid sequence of hG1.16 was identical to rat ribosomal protein L37 that contained 97 amino acids, many of which are highly positively charged (15 arginine and 14 lysine residues with a predicted M(r) of 11,065). hG1.16 protein has a single C2-C2 zinc-finger-like motif which is also present in rat ribosomal protein L37. Using primers designed from the sequence of hG1.16, unique bovine and rat cDNAs were also isolated by 5'-rapid-amplification of cDNA ends. DNA sequences of bovine and rat G1.16, clones were 92.8% and 92.2% similar to human G1.16 while the deduced amino acid sequences derived from bovine and rat cDNAs each differed by a single amino acid from the sequence of hG1.16 and the published rat L37 sequence. Southern-blot analysis revealed that hG1.16 exists in multiple copies in human, rat and mouse genomes. These G1.16 clones encode unique human, rat and bovine members of the ribosomal protein L37 gene family, which are constitutively expressed even during transitions from quiescence to active cell proliferation or terminal differentiation, in all tissues and all vertebrates investigated.

OSG-GEM: Gene Expression Matrix Construction Using the Open Science Grid.

PubMed

Poehlman, William L; Rynge, Mats; Branton, Chris; Balamurugan, D; Feltus, Frank A

2016-01-01

High-throughput DNA sequencing technology has revolutionized the study of gene expression while introducing significant computational challenges for biologists. These computational challenges include access to sufficient computer hardware and functional data processing workflows. Both these challenges are addressed with our scalable, open-source Pegasus workflow for processing high-throughput DNA sequence datasets into a gene expression matrix (GEM) using computational resources available to U.S.-based researchers on the Open Science Grid (OSG). We describe the usage of the workflow (OSG-GEM), discuss workflow design, inspect performance data, and assess accuracy in mapping paired-end sequencing reads to a reference genome. A target OSG-GEM user is proficient with the Linux command line and possesses basic bioinformatics experience. The user may run this workflow directly on the OSG or adapt it to novel computing environments.

OSG-GEM: Gene Expression Matrix Construction Using the Open Science Grid

PubMed Central

Poehlman, William L.; Rynge, Mats; Branton, Chris; Balamurugan, D.; Feltus, Frank A.

2016-01-01

High-throughput DNA sequencing technology has revolutionized the study of gene expression while introducing significant computational challenges for biologists. These computational challenges include access to sufficient computer hardware and functional data processing workflows. Both these challenges are addressed with our scalable, open-source Pegasus workflow for processing high-throughput DNA sequence datasets into a gene expression matrix (GEM) using computational resources available to U.S.-based researchers on the Open Science Grid (OSG). We describe the usage of the workflow (OSG-GEM), discuss workflow design, inspect performance data, and assess accuracy in mapping paired-end sequencing reads to a reference genome. A target OSG-GEM user is proficient with the Linux command line and possesses basic bioinformatics experience. The user may run this workflow directly on the OSG or adapt it to novel computing environments. PMID:27499617

Analysis of expressed sequence tags for Frankliniella occidentalis, the western flower thrips.

PubMed

Rotenberg, D; Whitfield, A E

2010-08-01

Thrips are members of the insect order Thysanoptera and Frankliniella occidentalis (the western flower thrips) is the most economically important pest within this order. F. occidentalis is both a direct pest of crops and an efficient vector of plant viruses, including Tomato spotted wilt virus (TSWV). Despite the world-wide importance of thrips in agriculture, there is little knowledge of the F. occidentalis genome or gene functions at this time. A normalized cDNA library was constructed from first instar thrips and 13 839 expressed sequence tags (ESTs) were obtained. Our EST data assembled into 894 contigs and 11 806 singletons (12 700 nonredundant sequences). We found that 31% of these sequences had significant similarity (E< or = 10(-10)) to protein sequences in the National Center for Biotechnology Information nonredundant (nr) protein database, and 25% were functionally annotated using Blast 2GO. We identified 74 sequences with putative homology to proteins associated with insect innate immunity. Sixteen sequences had significant similarity to proteins associated with small RNA-mediated gene silencing pathways (RNA interference; RNAi), including the antiviral pathway (short interfering RNA-mediated pathway). Our EST collection provides new sequence resources for characterizing gene functions in F. occidentalis and other thrips species with regards to vital biological processes, studying the mechanism of interactions with the viruses harboured and transmitted by the vector, and identifying new insect gene-centred targets for plant disease and insect control.

Comparative transcriptome analysis of microsclerotia development in Nomuraea rileyi.

PubMed

Song, Zhangyong; Yin, Youping; Jiang, Shasha; Liu, Juanjuan; Chen, Huan; Wang, Zhongkang

2013-06-19

Nomuraea rileyi is used as an environmental-friendly biopesticide. However, mass production and commercialization of this organism are limited due to its fastidious growth and sporulation requirements. When cultured in amended medium, we found that N. rileyi could produce microsclerotia bodies, replacing conidiophores as the infectious agent. However, little is known about the genes involved in microsclerotia development. In the present study, the transcriptomes were analyzed using next-generation sequencing technology to find the genes involved in microsclerotia development. A total of 4.69 Gb of clean nucleotides comprising 32,061 sequences was obtained, and 20,919 sequences were annotated (about 65%). Among the annotated sequences, only 5928 were annotated with 34 gene ontology (GO) functional categories, and 12,778 sequences were mapped to 165 pathways by searching against the Kyoto Encyclopedia of Genes and Genomes pathway (KEGG) database. Furthermore, we assessed the transcriptomic differences between cultures grown in minimal and amended medium. In total, 4808 sequences were found to be differentially expressed; 719 differentially expressed unigenes were assigned to 25 GO classes and 1888 differentially expressed unigenes were assigned to 161 KEGG pathways, including 25 enrichment pathways. Subsequently, we examined the up-regulation or uniquely expressed genes following amended medium treatment, which were also expressed on the enrichment pathway, and found that most of them participated in mediating oxidative stress homeostasis. To elucidate the role of oxidative stress in microsclerotia development, we analyzed the diversification of unigenes using quantitative reverse transcription-PCR (RT-qPCR). Our findings suggest that oxidative stress occurs during microsclerotia development, along with a broad metabolic activity change. Our data provide the most comprehensive sequence resource available for the study of N. rileyi. We believe that the transcriptome datasets will serve as an important public information platform to accelerate studies on N. rileyi microsclerotia.

Small RNA Deep Sequencing and the Effects of microRNA408 on Root Gravitropic Bending in Arabidopsis

NASA Astrophysics Data System (ADS)

Li, Huasheng; Lu, Jinying; Sun, Qiao; Chen, Yu; He, Dacheng; Liu, Min

2015-11-01

MicroRNA (miRNA) is a non-coding small RNA composed of 20 to 24 nucleotides that influences plant root development. This study analyzed the miRNA expression in Arabidopsis root tip cells using Illumina sequencing and real-time PCR before (sample 0) and 15 min after (sample 15) a 3-D clinostat rotational treatment was administered. After stimulation was performed, the expression levels of seven miRNA genes, including Arabidopsis miR160, miR161, miR394, miR402, miR403, miR408, and miR823, were significantly upregulated. Illumina sequencing results also revealed two novel miRNAsthat have not been previously reported, The target genes of these miRNAs included pentatricopeptide repeat-containing protein and diadenosine tetraphosphate hydrolase. An overexpression vector of Arabidopsis miR408 was constructed and transferred to Arabidopsis plant. The roots of plants over expressing miR408 exhibited a slower reorientation upon gravistimulation in comparison with those of wild-type. This result indicate that miR408 could play a role in root gravitropic response.

Simian virus 40 major late promoter: an upstream DNA sequence required for efficient in vitro transcription.

PubMed Central

Brady, J; Radonovich, M; Thoren, M; Das, G; Salzman, N P

1984-01-01

We have previously identified an 11-base DNA sequence, 5'-G-G-T-A-C-C-T-A-A-C-C-3' (simian virus 40 [SV40] map position 294 to 304), which is important in the control of SV40 late RNA expression in vitro and in vivo (Brady et al., Cell 31:625-633, 1982). We report here the identification of another domain of the SV40 late promoter. A series of mutants with deletions extending from SV40 map position 0 to 300 was prepared by nuclease BAL 31 treatment. The cloned templates were then analyzed for efficiency and accuracy of late SV40 RNA expression in the Manley in vitro transcription system. Our studies showed that, in addition to the promoter domain near map position 300, there are essential DNA sequences between nucleotide positions 74 and 95 that are required for efficient expression of late SV40 RNA. Included in this SV40 DNA sequence were two of the six GGGCGG SV40 repeat sequences and an 11-nucleotide segment which showed strong homology with the upstream sequences required for the efficient in vitro and in vivo expression of the histone H2A gene. This upstream promoter sequence supported transcription with the same efficiency even when it was moved 72 nucleotides closer to the major late cap site. In vitro promoter competition analysis demonstrated that the upstream promoter sequence, independent of the 294 to 304 promoter element, is capable of binding polymerase-transcription factors required for SV40 late gene transcription. Finally, we show that DNA sequences which control the specificity of RNA initiation at nucleotide 325 lie downstream of map position 294. Images PMID:6321950

Differential Expression of Three α-Galactosidase Genes and a Single β-Galactosidase Gene from Aspergillus niger

PubMed Central

de Vries, Ronald P.; van den Broeck, Hetty C.; Dekkers, Ester; Manzanares, Paloma; de Graaff, Leo H.; Visser, Jaap

1999-01-01

A gene encoding a third α-galactosidase (AglB) from Aspergillus niger has been cloned and sequenced. The gene consists of an open reading frame of 1,750 bp containing six introns. The gene encodes a protein of 443 amino acids which contains a eukaryotic signal sequence of 16 amino acids and seven putative N-glycosylation sites. The mature protein has a calculated molecular mass of 48,835 Da and a predicted pI of 4.6. An alignment of the AglB amino acid sequence with those of other α-galactosidases revealed that it belongs to a subfamily of α-galactosidases that also includes A. niger AglA. A. niger AglC belongs to a different subfamily that consists mainly of prokaryotic α-galactosidases. The expression of aglA, aglB, aglC, and lacA, the latter of which encodes an A. niger β-galactosidase, has been studied by using a number of monomeric, oligomeric, and polymeric compounds as growth substrates. Expression of aglA is only detected on galactose and galactose-containing oligomers and polymers. The aglB gene is expressed on all of the carbon sources tested, including glucose. Elevated expression was observed on xylan, which could be assigned to regulation via XlnR, the xylanolytic transcriptional activator. Expression of aglC was only observed on glucose, fructose, and combinations of glucose with xylose and galactose. High expression of lacA was detected on arabinose, xylose, xylan, and pectin. Similar to aglB, the expression on xylose and xylan can be assigned to regulation via XlnR. All four genes have distinct expression patterns which seem to mirror the natural substrates of the encoded proteins. PMID:10347026

Exploring the roles of DNA methylation in the metal-reducing bacterium Shewanella oneidensis MR-1

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

Bendall, Matthew L.; Luong, Khai; Wetmore, Kelly M.

2013-08-30

We performed whole genome analyses of DNA methylation in Shewanella 17 oneidensis MR-1 to examine its possible role in regulating gene expression and 18 other cellular processes. Single-Molecule Real Time (SMRT) sequencing 19 revealed extensive methylation of adenine (N6mA) throughout the 20 genome. These methylated bases were located in five sequence motifs, 21 including three novel targets for Type I restriction/modification enzymes. The 22 sequence motifs targeted by putative methyltranferases were determined via 23 SMRT sequencing of gene knockout mutants. In addition, we found S. 24 oneidensis MR-1 cultures grown under various culture conditions displayed 25 different DNA methylation patterns.more » However, the small number of differentially 26 methylated sites could not be directly linked to the much larger number of 27 differentially expressed genes in these conditions, suggesting DNA methylation is 28 not a major regulator of gene expression in S. oneidensis MR-1. The enrichment 29 of methylated GATC motifs in the origin of replication indicate DNA methylation 30 may regulate genome replication in a manner similar to that seen in Escherichia 31 coli. Furthermore, comparative analyses suggest that many 32 Gammaproteobacteria, including all members of the Shewanellaceae family, may 33 also utilize DNA methylation to regulate genome replication.« less

Reference genome sequence of the model plant Setaria

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

Bennetzen, Jeffrey L; Schmutz, Jeremy; Wang, Hao

We generated a high-quality reference genome sequence for foxtail millet (Setaria italica). The ~400-Mb assembly covers ~80% of the genome and >95% of the gene space. The assembly was anchored to a 992-locus genetic map and was annotated by comparison with >1.3 million expressed sequence tag reads. We produced more than 580 million RNA-Seq reads to facilitate expression analyses. We also sequenced Setaria viridis, the ancestral wild relative of S. italica, and identified regions of differential single-nucleotide polymorphism density, distribution of transposable elements, small RNA content, chromosomal rearrangement and segregation distortion. The genus Setaria includes natural and cultivated species thatmore » demonstrate a wide capacity for adaptation. The genetic basis of this adaptation was investigated by comparing five sequenced grass genomes. We also used the diploid Setaria genome to evaluate the ongoing genome assembly of a related polyploid, switchgrass (Panicum virgatum).« less

Reference genome sequence of the model plant Setaria.

PubMed

Bennetzen, Jeffrey L; Schmutz, Jeremy; Wang, Hao; Percifield, Ryan; Hawkins, Jennifer; Pontaroli, Ana C; Estep, Matt; Feng, Liang; Vaughn, Justin N; Grimwood, Jane; Jenkins, Jerry; Barry, Kerrie; Lindquist, Erika; Hellsten, Uffe; Deshpande, Shweta; Wang, Xuewen; Wu, Xiaomei; Mitros, Therese; Triplett, Jimmy; Yang, Xiaohan; Ye, Chu-Yu; Mauro-Herrera, Margarita; Wang, Lin; Li, Pinghua; Sharma, Manoj; Sharma, Rita; Ronald, Pamela C; Panaud, Olivier; Kellogg, Elizabeth A; Brutnell, Thomas P; Doust, Andrew N; Tuskan, Gerald A; Rokhsar, Daniel; Devos, Katrien M

2012-05-13

We generated a high-quality reference genome sequence for foxtail millet (Setaria italica). The ∼400-Mb assembly covers ∼80% of the genome and >95% of the gene space. The assembly was anchored to a 992-locus genetic map and was annotated by comparison with >1.3 million expressed sequence tag reads. We produced more than 580 million RNA-Seq reads to facilitate expression analyses. We also sequenced Setaria viridis, the ancestral wild relative of S. italica, and identified regions of differential single-nucleotide polymorphism density, distribution of transposable elements, small RNA content, chromosomal rearrangement and segregation distortion. The genus Setaria includes natural and cultivated species that demonstrate a wide capacity for adaptation. The genetic basis of this adaptation was investigated by comparing five sequenced grass genomes. We also used the diploid Setaria genome to evaluate the ongoing genome assembly of a related polyploid, switchgrass (Panicum virgatum).

Gene expression analysis of flax seed development

PubMed Central

2011-01-01

Background Flax, Linum usitatissimum L., is an important crop whose seed oil and stem fiber have multiple industrial applications. Flax seeds are also well-known for their nutritional attributes, viz., omega-3 fatty acids in the oil and lignans and mucilage from the seed coat. In spite of the importance of this crop, there are few molecular resources that can be utilized toward improving seed traits. Here, we describe flax embryo and seed development and generation of comprehensive genomic resources for the flax seed. Results We describe a large-scale generation and analysis of expressed sequences in various tissues. Collectively, the 13 libraries we have used provide a broad representation of genes active in developing embryos (globular, heart, torpedo, cotyledon and mature stages) seed coats (globular and torpedo stages) and endosperm (pooled globular to torpedo stages) and genes expressed in flowers, etiolated seedlings, leaves, and stem tissue. A total of 261,272 expressed sequence tags (EST) (GenBank accessions LIBEST_026995 to LIBEST_027011) were generated. These EST libraries included transcription factor genes that are typically expressed at low levels, indicating that the depth is adequate for in silico expression analysis. Assembly of the ESTs resulted in 30,640 unigenes and 82% of these could be identified on the basis of homology to known and hypothetical genes from other plants. When compared with fully sequenced plant genomes, the flax unigenes resembled poplar and castor bean more than grape, sorghum, rice or Arabidopsis. Nearly one-fifth of these (5,152) had no homologs in sequences reported for any organism, suggesting that this category represents genes that are likely unique to flax. Digital analyses revealed gene expression dynamics for the biosynthesis of a number of important seed constituents during seed development. Conclusions We have developed a foundational database of expressed sequences and collection of plasmid clones that comprise even low-expressed genes such as those encoding transcription factors. This has allowed us to delineate the spatio-temporal aspects of gene expression underlying the biosynthesis of a number of important seed constituents in flax. Flax belongs to a taxonomic group of diverse plants and the large sequence database will allow for evolutionary studies as well. PMID:21529361

Gene discovery in the hamster: a comparative genomics approach for gene annotation by sequencing of hamster testis cDNAs

PubMed Central

Oduru, Sreedhar; Campbell, Janee L; Karri, SriTulasi; Hendry, William J; Khan, Shafiq A; Williams, Simon C

2003-01-01

Background Complete genome annotation will likely be achieved through a combination of computer-based analysis of available genome sequences combined with direct experimental characterization of expressed regions of individual genomes. We have utilized a comparative genomics approach involving the sequencing of randomly selected hamster testis cDNAs to begin to identify genes not previously annotated on the human, mouse, rat and Fugu (pufferfish) genomes. Results 735 distinct sequences were analyzed for their relatedness to known sequences in public databases. Eight of these sequences were derived from previously unidentified genes and expression of these genes in testis was confirmed by Northern blotting. The genomic locations of each sequence were mapped in human, mouse, rat and pufferfish, where applicable, and the structure of their cognate genes was derived using computer-based predictions, genomic comparisons and analysis of uncharacterized cDNA sequences from human and macaque. Conclusion The use of a comparative genomics approach resulted in the identification of eight cDNAs that correspond to previously uncharacterized genes in the human genome. The proteins encoded by these genes included a new member of the kinesin superfamily, a SET/MYND-domain protein, and six proteins for which no specific function could be predicted. Each gene was expressed primarily in testis, suggesting that they may play roles in the development and/or function of testicular cells. PMID:12783626

Comparison of Five Major Trichome Regulatory Genes in Brassica villosa with Orthologues within the Brassicaceae

PubMed Central

Nayidu, Naghabushana K.; Kagale, Sateesh; Taheri, Ali; Withana-Gamage, Thushan S.; Parkin, Isobel A. P.; Sharpe, Andrew G.; Gruber, Margaret Y.

2014-01-01

Coding sequences for major trichome regulatory genes, including the positive regulators GLABRA 1(GL1), GLABRA 2 (GL2), ENHANCER OF GLABRA 3 (EGL3), and TRANSPARENT TESTA GLABRA 1 (TTG1) and the negative regulator TRIPTYCHON (TRY), were cloned from wild Brassica villosa, which is characterized by dense trichome coverage over most of the plant. Transcript (FPKM) levels from RNA sequencing indicated much higher expression of the GL2 and TTG1 regulatory genes in B. villosa leaves compared with expression levels of GL1 and EGL3 genes in either B. villosa or the reference genome species, glabrous B. oleracea; however, cotyledon TTG1 expression was high in both species. RNA sequencing and Q-PCR also revealed an unusual expression pattern for the negative regulators TRY and CPC, which were much more highly expressed in trichome-rich B. villosa leaves than in glabrous B. oleracea leaves and in glabrous cotyledons from both species. The B. villosa TRY expression pattern also contrasted with TRY expression patterns in two diploid Brassica species, and with the Arabidopsis model for expression of negative regulators of trichome development. Further unique sequence polymorphisms, protein characteristics, and gene evolution studies highlighted specific amino acids in GL1 and GL2 coding sequences that distinguished glabrous species from hairy species and several variants that were specific for each B. villosa gene. Positive selection was observed for GL1 between hairy and non-hairy plants, and as expected the origin of the four expressed positive trichome regulatory genes in B. villosa was predicted to be from B. oleracea. In particular the unpredicted expression patterns for TRY and CPC in B. villosa suggest additional characterization is needed to determine the function of the expanded families of trichome regulatory genes in more complex polyploid species within the Brassicaceae. PMID:24755905

OP17MICRORNA PROFILING USING SMALL RNA-SEQ IN PAEDIATRIC LOW GRADE GLIOMAS

PubMed Central

Jeyapalan, Jennie N.; Jones, Tania A.; Tatevossian, Ruth G.; Qaddoumi, Ibrahim; Ellison, David W.; Sheer, Denise

2014-01-01

INTRODUCTION: MicroRNAs regulate gene expression by targeting mRNAs for translational repression or degradation at the post-transcriptional level. In paediatric low-grade gliomas a few key genetic mutations have been identified, including BRAF fusions, FGFR1 duplications and MYB rearrangements. Our aim in the current study is to profile aberrant microRNA expression in paediatric low-grade gliomas and determine the role of epigenetic changes in the aetiology and behaviour of these tumours. METHOD: MicroRNA profiling of tumour samples (6 pilocytic, 2 diffuse, 2 pilomyxoid astrocytomas) and normal brain controls (4 adult normal brain samples and a primary glial progenitor cell-line) was performed using small RNA sequencing. Bioinformatic analysis included sequence alignment, analysis of the number of reads (CPM, counts per million) and differential expression. RESULTS: Sequence alignment identified 695 microRNAs, whose expression was compared in tumours v. normal brain. PCA and hierarchical clustering showed separate groups for tumours and normal brain. Computational analysis identified approximately 400 differentially expressed microRNAs in the tumours compared to matched location controls. Our findings will then be validated and integrated with extensive genetic and epigenetic information we have previously obtained for the full tumour cohort. CONCLUSION: We have identified microRNAs that are differentially expressed in paediatric low-grade gliomas. As microRNAs are known to target genes involved in the initiation and progression of cancer, they provide critical information on tumour pathogenesis and are an important class of biomarkers.

Identification of Differentially Expressed miRNAs between White and Black Hair Follicles by RNA-Sequencing in the Goat (Capra hircus)

PubMed Central

Wu, Zhenyang; Fu, Yuhua; Cao, Jianhua; Yu, Mei; Tang, Xiaohui; Zhao, Shuhong

2014-01-01

MicroRNAs (miRNAs) play a key role in many biological processes by regulating gene expression at the post-transcriptional level. A number of miRNAs have been identified from livestock species. However, compared with other animals, such as pigs and cows, the number of miRNAs identified in goats is quite low, particularly in hair follicles. In this study, to investigate the functional roles of miRNAs in goat hair follicles of goats with different coat colors, we sequenced miRNAs from two hair follicles samples (white and black) using Solexa sequencing. A total of 35,604,016 reads were obtained, which included 30,878,637 clean reads (86.73%). MiRDeep2 software identified 214 miRNAs. Among them, 205 were conserved among species and nine were novel miRNAs. Furthermore, DESeq software identified six differentially expressed miRNAs. Quantitative PCR confirmed differential expression of two miRNAs, miR-10b and miR-211. KEGG pathways were analyzed using the DAVID website for the predicted target genes of the differentially expressed miRNAs. Several signaling pathways including Notch and MAPK pathways may affect the process of coat color formation. Our study showed that the identified miRNAs might play an essential role in black and white follicle formation in goats. PMID:24879525

Linear Lepidopteran ambidensovirus 1 sequences drive random integration of a reporter gene in transfected Spodoptera frugiperda cells.

PubMed

Rizk, Francine; Laverdure, Sylvain; d'Alençon, Emmanuelle; Bossin, Hervé; Dupressoir, Thierry

2018-01-01

The Lepidopteran ambidensovirus 1 isolated from Junonia coenia (hereafter JcDV) is an invertebrate parvovirus considered as a viral transduction vector as well as a potential tool for the biological control of insect pests. Previous works showed that JcDV-based circular plasmids experimentally integrate into insect cells genomic DNA. In order to approach the natural conditions of infection and possible integration, we generated linear JcDV- gfp based molecules which were transfected into non permissive Spodoptera frugiperda ( Sf9 ) cultured cells. Cells were monitored for the expression of green fluorescent protein (GFP) and DNA was analyzed for integration of transduced viral sequences. Non-structural protein modulation of the VP-gene cassette promoter activity was additionally assayed. We show that linear JcDV-derived molecules are capable of long term genomic integration and sustained transgene expression in Sf9 cells. As expected, only the deletion of both inverted terminal repeats (ITR) or the polyadenylation signals of NS and VP genes dramatically impairs the global transduction/expression efficiency. However, all the integrated viral sequences we characterized appear "scrambled" whatever the viral content of the transfected vector. Despite a strong GFP expression, we were unable to recover any full sequence of the original constructs and found rearranged viral and non-viral sequences as well. Cellular flanking sequences were identified as non-coding ones. On the other hand, the kinetics of GFP expression over time led us to investigate the apparent down-regulation by non-structural proteins of the VP-gene cassette promoter. Altogether, our results show that JcDV-derived sequences included in linear DNA molecules are able to drive efficiently the integration and expression of a foreign gene into the genome of insect cells, whatever their composition, provided that at least one ITR is present. However, the transfected sequences were extensively rearranged with cellular DNA during or after random integration in the host cell genome. Lastly, the non-structural proteins seem to participate in the regulation of p9 promoter activity rather than to the integration of viral sequences.

Molecular mechanisms of pathogenesis in hepatocellular carcinoma revealed by RNA‑sequencing.

PubMed

Liu, Yao; Yang, Zhe; Du, Feng; Yang, Qiao; Hou, Jie; Yan, Xiaohong; Geng, Yi; Zhao, Yaning; Wang, Hua

2017-11-01

The present study aimed to explore the underlying molecular mechanisms of hepatocellular carcinoma (HCC). RNA‑sequencing profiles GSM629264 and GSM629265, from the GSE25599 data set, were downloaded from the Gene Expression Omnibus database and processed by quality evaluation. GSM629264 and GSM629265 were from HCC and adjacent non‑cancerous tissues, respectively. TopHat software was used for alignment analysis, followed by the detection of novel splicing sites. In addition, the Cufflinks software package was used to analyze gene expressions, and the Cuffdiff program was used to screen for differently expressed genes (DEGs) and differentially expressed splicing variants. Gene ontology functional enrichment and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses of DEGs were also performed. Transcription factors (TFs) and microRNAs (miRNAs) that regulate DEGs were identified, and a protein‑protein interaction (PPI) network was constructed. The hub node in the PPI network was obtained, and the TFs and miRNAs that regulated the hub node were further predicted. The quality of the sequencing data met the standards for analysis, and the clean reads were ~65%. Most sequencing reads mapped into coding sequence exons (CDS_exons), whereas other reads mapped into exon 3' untranslated regions (UTR_Exons), 5'UTR_Exons and Introns. Upregulated and downregulated DEGs between HCC and adjacent non‑cancerous tissues were screened. Genes of differentially expressed splicing variants were identified, including vesicle‑associated membrane protein 4, phosphatidylinositol glycan anchor biosynthesis class C, protein disulfide isomerase family A member 4 and growth arrest specific 5. Screened DEGs were enriched in the complement pathway. In the PPI network, ubiquitin C (UBC) was the hub node. UBC was predicted to be regulated by several TFs, including specificity protein 1 (SP1), FBJ murine osteosarcoma viral oncogene homolog (FOS), proto‑oncogene c‑JUN (JUN), FOS‑like antigen 2 (FOSL2) and SWI/SNF‑related, matrix‑associated, actin‑dependent regulator of chromatin, subfamily A, member 4 (SMARCA4), and several miRNAs, including miR‑30 and miR‑181. Results from the present study demonstrated that UBC, SP1, FOS, JUN, FOSL2, SMARCA4, miR‑30 and miR‑181 may participate in the development of HCC.

Investigation of mRNA quadruplex formation in Escherichia coli.

PubMed

Wieland, Markus; Hartig, Jörg S

2009-01-01

The protocol presented here allows for the investigation of the formation of unusual nucleic acid structures in the 5'-untranslated region (UTR) of bacteria by correlating gene expression levels to the in vitro stability of the respective structure. In particular, we describe the introduction of G-quadruplex forming sequences close to the ribosome-binding site (RBS) on the mRNA of a reporter gene and the subsequent read-out of the expression levels. Insertion of a stable secondary structure results in the cloaking of RBS and eventually reduced gene expression levels. The structures and stability of the introduced sequences are further characterized by circular dichroism (CD) spectroscopy and thermal melting experiments. The extent of inhibition is then correlated to the stability of the respective quadruplex structure, allowing judgement of whether factors other than thermodynamic stability affect the formation of a given quadruplex sequence in vivo. Measuring gene expression levels takes 2 d including cloning; CD experiments take 5 hours per experiment.

De novo Transcriptome Assembly of Common Wild Rice (Oryza rufipogon Griff.) and Discovery of Drought-Response Genes in Root Tissue Based on Transcriptomic Data.

PubMed

Tian, Xin-Jie; Long, Yan; Wang, Jiao; Zhang, Jing-Wen; Wang, Yan-Yan; Li, Wei-Min; Peng, Yu-Fa; Yuan, Qian-Hua; Pei, Xin-Wu

2015-01-01

The perennial O. rufipogon (common wild rice), which is considered to be the ancestor of Asian cultivated rice species, contains many useful genetic resources, including drought resistance genes. However, few studies have identified the drought resistance and tissue-specific genes in common wild rice. In this study, transcriptome sequencing libraries were constructed, including drought-treated roots (DR) and control leaves (CL) and roots (CR). Using Illumina sequencing technology, we generated 16.75 million bases of high-quality sequence data for common wild rice and conducted de novo assembly and annotation of genes without prior genome information. These reads were assembled into 119,332 unigenes with an average length of 715 bp. A total of 88,813 distinct sequences (74.42% of unigenes) significantly matched known genes in the NCBI NT database. Differentially expressed gene (DEG) analysis showed that 3617 genes were up-regulated and 4171 genes were down-regulated in the CR library compared with the CL library. Among the DEGs, 535 genes were expressed in roots but not in shoots. A similar comparison between the DR and CR libraries showed that 1393 genes were up-regulated and 315 genes were down-regulated in the DR library compared with the CR library. Finally, 37 genes that were specifically expressed in roots were screened after comparing the DEGs identified in the above-described analyses. This study provides a transcriptome sequence resource for common wild rice plants and establishes a digital gene expression profile of wild rice plants under drought conditions using the assembled transcriptome data as a reference. Several tissue-specific and drought-stress-related candidate genes were identified, representing a fully characterized transcriptome and providing a valuable resource for genetic and genomic studies in plants.

Genes encoding calmodulin-binding proteins in the Arabidopsis genome

NASA Technical Reports Server (NTRS)

Reddy, Vaka S.; Ali, Gul S.; Reddy, Anireddy S N.

2002-01-01

Analysis of the recently completed Arabidopsis genome sequence indicates that approximately 31% of the predicted genes could not be assigned to functional categories, as they do not show any sequence similarity with proteins of known function from other organisms. Calmodulin (CaM), a ubiquitous and multifunctional Ca(2+) sensor, interacts with a wide variety of cellular proteins and modulates their activity/function in regulating diverse cellular processes. However, the primary amino acid sequence of the CaM-binding domain in different CaM-binding proteins (CBPs) is not conserved. One way to identify most of the CBPs in the Arabidopsis genome is by protein-protein interaction-based screening of expression libraries with CaM. Here, using a mixture of radiolabeled CaM isoforms from Arabidopsis, we screened several expression libraries prepared from flower meristem, seedlings, or tissues treated with hormones, an elicitor, or a pathogen. Sequence analysis of 77 positive clones that interact with CaM in a Ca(2+)-dependent manner revealed 20 CBPs, including 14 previously unknown CBPs. In addition, by searching the Arabidopsis genome sequence with the newly identified and known plant or animal CBPs, we identified a total of 27 CBPs. Among these, 16 CBPs are represented by families with 2-20 members in each family. Gene expression analysis revealed that CBPs and CBP paralogs are expressed differentially. Our data suggest that Arabidopsis has a large number of CBPs including several plant-specific ones. Although CaM is highly conserved between plants and animals, only a few CBPs are common to both plants and animals. Analysis of Arabidopsis CBPs revealed the presence of a variety of interesting domains. Our analyses identified several hypothetical proteins in the Arabidopsis genome as CaM targets, suggesting their involvement in Ca(2+)-mediated signaling networks.

A Multiplexed Amplicon Approach for Detecting Gene Fusions by Next-Generation Sequencing.

PubMed

Beadling, Carol; Wald, Abigail I; Warrick, Andrea; Neff, Tanaya L; Zhong, Shan; Nikiforov, Yuri E; Corless, Christopher L; Nikiforova, Marina N

2016-03-01

Chromosomal rearrangements that result in oncogenic gene fusions are clinically important drivers of many cancer types. Rapid and sensitive methods are therefore needed to detect a broad range of gene fusions in clinical specimens that are often of limited quantity and quality. We describe a next-generation sequencing approach that uses a multiplex PCR-based amplicon panel to interrogate fusion transcripts that involve 19 driver genes and 94 partners implicated in solid tumors. The panel also includes control assays that evaluate the 3'/5' expression ratios of 12 oncogenic kinases, which might be used to infer gene fusion events when the partner is unknown or not included on the panel. There was good concordance between the solid tumor fusion gene panel and other methods, including fluorescence in situ hybridization, real-time PCR, Sanger sequencing, and other next-generation sequencing panels, because 40 specimens known to harbor gene fusions were correctly identified. No specific fusion reads were observed in 59 fusion-negative specimens. The 3'/5' expression ratio was informative for fusions that involved ALK, RET, and NTRK1 but not for BRAF or ROS1 fusions. However, among 37 ALK or RET fusion-negative specimens, four exhibited elevated 3'/5' expression ratios, indicating that fusions predicted solely by 3'/5' read ratios require confirmatory testing. Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Identification and temporal expression of putative circadian clock transcripts in the amphipod crustacean Talitrus saltator

PubMed Central

O’Grady, Joseph F.; Hoelters, Laura S.; Swain, Martin T.

2016-01-01

Background Talitrus saltator is an amphipod crustacean that inhabits the supralittoral zone on sandy beaches in the Northeast Atlantic and Mediterranean. T. saltator exhibits endogenous locomotor activity rhythms and time-compensated sun and moon orientation, both of which necessitate at least one chronometric mechanism. Whilst their behaviour is well studied, currently there are no descriptions of the underlying molecular components of a biological clock in this animal, and very few in other crustacean species. Methods We harvested brain tissue from animals expressing robust circadian activity rhythms and used homology cloning and Illumina RNAseq approaches to sequence and identify the core circadian clock and clock-related genes in these samples. We assessed the temporal expression of these genes in time-course samples from rhythmic animals using RNAseq. Results We identified a comprehensive suite of circadian clock gene homologues in T. saltator including the ‘core’ clock genes period (Talper), cryptochrome 2 (Talcry2), timeless (Taltim), clock (Talclk), and bmal1 (Talbmal1). In addition we describe the sequence and putative structures of 23 clock-associated genes including two unusual, extended isoforms of pigment dispersing hormone (Talpdh). We examined time-course RNAseq expression data, derived from tissues harvested from behaviourally rhythmic animals, to reveal rhythmic expression of these genes with approximately circadian period in Talper and Talbmal1. Of the clock-related genes, casein kinase IIβ (TalckIIβ), ebony (Talebony), jetlag (Taljetlag), pigment dispensing hormone (Talpdh), protein phosphatase 1 (Talpp1), shaggy (Talshaggy), sirt1 (Talsirt1), sirt7 (Talsirt7) and supernumerary limbs (Talslimb) show temporal changes in expression. Discussion We report the sequences of principle genes that comprise the circadian clock of T. saltator and highlight the conserved structural and functional domains of their deduced cognate proteins. Our sequencing data contribute to the growing inventory of described comparative clocks. Expression profiling of the identified clock genes illuminates tantalising targets for experimental manipulation to elucidate the molecular and cellular control of clock-driven phenotypes in this crustacean. PMID:27761341

Identification of microRNAs differentially expressed involved in male flower development.

PubMed

Wang, Zhengjia; Huang, Jianqin; Sun, Zhichao; Zheng, Bingsong

2015-03-01

Hickory (Carya cathayensis Sarg.) is one of the most economically important woody trees in eastern China, but its long flowering phase delays yield. Our understanding of the regulatory roles of microRNAs (miRNAs) in male flower development in hickory remains poor. Using high-throughput sequencing technology, we have pyrosequenced two small RNA libraries from two male flower differentiation stages in hickory. Analysis of the sequencing data identified 114 conserved miRNAs that belonged to 23 miRNA families, five novel miRNAs including their corresponding miRNA*s, and 22 plausible miRNA candidates. Differential expression analysis revealed 12 miRNA sequences that were upregulated in the later (reproductive) stage of male flower development. Quantitative real-time PCR showed similar expression trends as that of the deep sequencing. Novel miRNAs and plausible miRNA candidates were predicted using bioinformatic analysis methods. The miRNAs newly identified in this study have increased the number of known miRNAs in hickory, and the identification of differentially expressed miRNAs will provide new avenues for studies into miRNAs involved in the process of male flower development in hickory and other related trees.

Mouse Mesenchyme forkhead 2 (Mf2): expression, DNA binding and induction by sonic hedgehog during somitogenesis.

PubMed

Wu, S C; Grindley, J; Winnier, G E; Hargett, L; Hogan, B L

1998-01-01

Cloning and sequencing of mouse Mf2 (mesoderm/mesenchyme forkhead 2) cDNAs revealed an open reading frame encoding a putative protein of 492 amino acids which, after in vitro translation, binds to a DNA consensus sequence. Mf2 is expressed at high levels in the ventral region of newly formed somites, in sclerotomal derivatives, in lateral plate and cephalic mesoderm and in the first and second branchial arches. Other regions of mesodermal expression include the developing tongue, meninges, nose, whiskers, kidney, genital tubercule and limb joints. In the nervous system Mf2 is transcribed in restricted regions of the mid- and forebrain. In several tissues, including the early somite, Mf2 is expressed in cell populations adjacent to regions expressing sonic hedgehog (Shh) and in explant cultures of presomitic mesoderm Mf2 is induced by Shh secreted by COS cells. These results suggest that Mf2, like other murine forkhead genes, has multiple roles in embryogenesis, possibly mediating the response of cells to signaling molecules such as SHH.

Determination of multidrug resistance mechanisms in Clostridium perfringens type A isolates using RNA sequencing and 2D-electrophoresis.

PubMed

Ma, Yu-Hua; Ye, Gui-Sheng

2018-06-11

In this study, we screened differentially expressed genes in a multidrug-resistant isolate strain of Clostridium perfringens by RNA sequencing. We also separated and identified differentially expressed proteins (DEPs) in the isolate strain by two-dimensional electrophoresis (2-DE) and mass spectrometry (MS). The RNA sequencing results showed that, compared with the control strain, 1128 genes were differentially expressed in the isolate strain, and these included 227 up-regulated genes and 901 down-regulated genes. Bioinformatics analysis identified the following genes and gene categories that are potentially involved in multidrug resistance (MDR) in the isolate strain: drug transport, drug response, hydrolase activity, transmembrane transporter, transferase activity, amidase transmembrane transporter, efflux transmembrane transporter, bacterial chemotaxis, ABC transporter, and others. The results of the 2-DE showed that 70 proteins were differentially expressed in the isolate strain, 45 of which were up-regulated and 25 down-regulated. Twenty-seven DEPs were identified by MS and these included the following protein categories: ribosome, antimicrobial peptide resistance, and ABC transporter, all of which may be involved in MDR in the isolate strain of C. perfringens. The results provide reference data for further investigations on the drug resistant molecular mechanisms of C. perfringens.

Genes are differentially expressed at transcriptional level of Neocaridina denticulata following short-term exposure to nonylphenol.

PubMed

Liu, Chang-Lun; Sung, Hung-Hung

2011-09-01

To assess the toxicity of nonylphenol towards aquatic crustaceans, Neocaridina denticulata were exposed short-term to sublethal concentration (0.001-0.5 mg/L). Following treatment, differentially expressed genes were identified using suppression subtractive hybridization on samples prepared from whole specimens. There were 20 differentially expressed sequence tags that corresponded to known genes and could be divided into six functional classes: defence, translation, metabolism, ribosomal gene expression, respiration, and genes involved in the stress response. Using semi-quantitative RT-PCR, we found that 14 of the differentially expressed sequence tags significantly responded to nonylphenol, including six at a nominal concentration of 0.01 mg/L; among them, 12 genes were down-regulated. These results suggest that under non-lethal concentrations of nonylphenol, the polluted aquatic environment may still present a potential risk to N. denticulata.

Deep sequencing reveals cell-type-specific patterns of single-cell transcriptome variation.

PubMed

Dueck, Hannah; Khaladkar, Mugdha; Kim, Tae Kyung; Spaethling, Jennifer M; Francis, Chantal; Suresh, Sangita; Fisher, Stephen A; Seale, Patrick; Beck, Sheryl G; Bartfai, Tamas; Kuhn, Bernhard; Eberwine, James; Kim, Junhyong

2015-06-09

Differentiation of metazoan cells requires execution of different gene expression programs but recent single-cell transcriptome profiling has revealed considerable variation within cells of seeming identical phenotype. This brings into question the relationship between transcriptome states and cell phenotypes. Additionally, single-cell transcriptomics presents unique analysis challenges that need to be addressed to answer this question. We present high quality deep read-depth single-cell RNA sequencing for 91 cells from five mouse tissues and 18 cells from two rat tissues, along with 30 control samples of bulk RNA diluted to single-cell levels. We find that transcriptomes differ globally across tissues with regard to the number of genes expressed, the average expression patterns, and within-cell-type variation patterns. We develop methods to filter genes for reliable quantification and to calibrate biological variation. All cell types include genes with high variability in expression, in a tissue-specific manner. We also find evidence that single-cell variability of neuronal genes in mice is correlated with that in rats consistent with the hypothesis that levels of variation may be conserved. Single-cell RNA-sequencing data provide a unique view of transcriptome function; however, careful analysis is required in order to use single-cell RNA-sequencing measurements for this purpose. Technical variation must be considered in single-cell RNA-sequencing studies of expression variation. For a subset of genes, biological variability within each cell type appears to be regulated in order to perform dynamic functions, rather than solely molecular noise.

Multiple regulatory mechanisms of hepatocyte growth factor expression in malignant cells with a short poly(dA) sequence in the HGF gene promoter.

PubMed

Sakai, Kazuko; Takeda, Masayuki; Okamoto, Isamu; Nakagawa, Kazuhiko; Nishio, Kazuto

2015-01-01

Hepatocyte growth factor (HGF) expression is a poor prognostic factor in various types of cancer. Expression levels of HGF have been reported to be regulated by shorter poly(dA) sequences in the promoter region. In the present study, the poly(dA) mononucleotide tract in various types of human cancer cell lines was examined and compared with the HGF expression levels in those cells. Short deoxyadenosine repeat sequences were detected in five of the 55 cell lines used in the present study. The H69, IM95, CCK-81, Sui73 and H28 cells exhibited a truncated poly(dA) sequence in which the number of poly(dA) repeats was reduced by ≥5 bp. Two of the cell lines exhibited high HGF expression, determined by reverse transcription quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. The CCK-81, Sui73 and H28 cells with shorter poly(dA) sequences exhibited low HGF expression. The cause of the suppression of HGF expression in the CCK-81, Sui73 and H28 cells was clarified by two approaches, suppression by methylation and single nucleotide polymorphisms in the HGF gene. Exposure to 5-Aza-dC, an inhibitor of DNA methyltransferase 1, induced an increased expression of HGF in the CCK-81 cells, but not in the other cells. Single-nucleotide polymorphism (SNP) rs72525097 in intron 1 was detected in the Sui73 and H28 cells. Taken together, it was found that the defect of poly(dA) in the HGF promoter was present in various types of cancer, including lung, stomach, colorectal, pancreas and mesothelioma. The present study proposes the negative regulation mechanisms by methylation and SNP in intron 1 of HGF for HGF expression in cancer cells with short poly(dA).

Isolation, sequence identification and tissue expression profiles of 3 novel porcine genes: ASPA, NAGA, and HEXA.

PubMed

Shu, Xianghua; Liu, Yonggang; Yang, Liangyu; Song, Chunlian; Hou, Jiafa

2008-01-01

The complete coding sequences of 3 porcine genes - ASPA, NAGA, and HEXA - were amplified by the reverse transcriptase polymerase chain reaction (RT-PCR) based on the conserved sequence information of the mouse or other mammals and referenced pig ESTs. These 3 novel porcine genes were then deposited in the NCBI database and assigned GeneIDs: 100142661, 100142664 and 100142667. The phylogenetic tree analysis revealed that the porcine ASPA, NAGA, and HEXA all have closer genetic relationships with the ASPA, NAGA, and HEXA of cattle. Tissue expression profile analysis was also carried out and results revealed that swine ASPA, NAGA, and HEXA genes were differentially expressed in various organs, including skeletal muscle, the heart, liver, fat, kidney, lung, and small and large intestines. Our experiment is the first one to establish the foundation for further research on these 3 swine genes.

Conifer R2R3-MYB transcription factors: sequence analyses and gene expression in wood-forming tissues of white spruce (Picea glauca)

PubMed Central

Bedon, Frank; Grima-Pettenati, Jacqueline; Mackay, John

2007-01-01

Background Several members of the R2R3-MYB family of transcription factors act as regulators of lignin and phenylpropanoid metabolism during wood formation in angiosperm and gymnosperm plants. The angiosperm Arabidopsis has over one hundred R2R3-MYBs genes; however, only a few members of this family have been discovered in gymnosperms. Results We isolated and characterised full-length cDNAs encoding R2R3-MYB genes from the gymnosperms white spruce, Picea glauca (13 sequences), and loblolly pine, Pinus taeda L. (five sequences). Sequence similarities and phylogenetic analyses placed the spruce and pine sequences in diverse subgroups of the large R2R3-MYB family, although several of the sequences clustered closely together. We searched the highly variable C-terminal region of diverse plant MYBs for conserved amino acid sequences and identified 20 motifs in the spruce MYBs, nine of which have not previously been reported and three of which are specific to conifers. The number and length of the introns in spruce MYB genes varied significantly, but their positions were well conserved relative to angiosperm MYB genes. Quantitative RTPCR of MYB genes transcript abundance in root and stem tissues revealed diverse expression patterns; three MYB genes were preferentially expressed in secondary xylem, whereas others were preferentially expressed in phloem or were ubiquitous. The MYB genes expressed in xylem, and three others, were up-regulated in the compression wood of leaning trees within 76 hours of induction. Conclusion Our survey of 18 conifer R2R3-MYB genes clearly showed a gene family structure similar to that of Arabidopsis. Three of the sequences are likely to play a role in lignin metabolism and/or wood formation in gymnosperm trees, including a close homolog of the loblolly pine PtMYB4, shown to regulate lignin biosynthesis in transgenic tobacco. PMID:17397551

The genomes of many yam species contain transcriptionally active endogenous geminiviral sequences that may be functionally expressed

PubMed Central

Filloux, Denis; Murrell, Sasha; Koohapitagtam, Maneerat; Golden, Michael; Julian, Charlotte; Galzi, Serge; Uzest, Marilyne; Rodier-Goud, Marguerite; D’Hont, Angélique; Vernerey, Marie Stephanie; Wilkin, Paul; Peterschmitt, Michel; Winter, Stephan; Murrell, Ben; Martin, Darren P.; Roumagnac, Philippe

2015-01-01

Endogenous viral sequences are essentially ‘fossil records’ that can sometimes reveal the genomic features of long extinct virus species. Although numerous known instances exist of single-stranded DNA (ssDNA) genomes becoming stably integrated within the genomes of bacteria and animals, there remain very few examples of such integration events in plants. The best studied of these events are those which yielded the geminivirus-related DNA elements found within the nuclear genomes of various Nicotiana species. Although other ssDNA virus-like sequences are included within the draft genomes of various plant species, it is not entirely certain that these are not contaminants. The Nicotiana geminivirus-related DNA elements therefore remain the only definitively proven instances of endogenous plant ssDNA virus sequences. Here, we characterize two new classes of endogenous plant virus sequence that are also apparently derived from ancient geminiviruses in the genus Begomovirus. These two endogenous geminivirus-like elements (EGV1 and EGV2) are present in the Dioscorea spp. of the Enantiophyllum clade. We used fluorescence in situ hybridization to confirm that the EGV1 sequences are integrated in the D. alata genome and showed that one or two ancestral EGV sequences likely became integrated more than 1.4 million years ago during or before the diversification of the Asian and African Enantiophyllum Dioscorea spp. Unexpectedly, we found evidence of natural selection actively favouring the maintenance of EGV-expressed replication-associated protein (Rep) amino acid sequences, which clearly indicates that functional EGV Rep proteins were probably expressed for prolonged periods following endogenization. Further, the detection in D. alata of EGV gene transcripts, small 21–24 nt RNAs that are apparently derived from these transcripts, and expressed Rep proteins, provides evidence that some EGV genes are possibly still functionally expressed in at least some of the Enantiophyllum clade species. PMID:27774276

eRNA: a graphic user interface-based tool optimized for large data analysis from high-throughput RNA sequencing

PubMed Central

2014-01-01

Background RNA sequencing (RNA-seq) is emerging as a critical approach in biological research. However, its high-throughput advantage is significantly limited by the capacity of bioinformatics tools. The research community urgently needs user-friendly tools to efficiently analyze the complicated data generated by high throughput sequencers. Results We developed a standalone tool with graphic user interface (GUI)-based analytic modules, known as eRNA. The capacity of performing parallel processing and sample management facilitates large data analyses by maximizing hardware usage and freeing users from tediously handling sequencing data. The module miRNA identification” includes GUIs for raw data reading, adapter removal, sequence alignment, and read counting. The module “mRNA identification” includes GUIs for reference sequences, genome mapping, transcript assembling, and differential expression. The module “Target screening” provides expression profiling analyses and graphic visualization. The module “Self-testing” offers the directory setups, sample management, and a check for third-party package dependency. Integration of other GUIs including Bowtie, miRDeep2, and miRspring extend the program’s functionality. Conclusions eRNA focuses on the common tools required for the mapping and quantification analysis of miRNA-seq and mRNA-seq data. The software package provides an additional choice for scientists who require a user-friendly computing environment and high-throughput capacity for large data analysis. eRNA is available for free download at https://sourceforge.net/projects/erna/?source=directory. PMID:24593312

eRNA: a graphic user interface-based tool optimized for large data analysis from high-throughput RNA sequencing.

PubMed

Yuan, Tiezheng; Huang, Xiaoyi; Dittmar, Rachel L; Du, Meijun; Kohli, Manish; Boardman, Lisa; Thibodeau, Stephen N; Wang, Liang

2014-03-05

RNA sequencing (RNA-seq) is emerging as a critical approach in biological research. However, its high-throughput advantage is significantly limited by the capacity of bioinformatics tools. The research community urgently needs user-friendly tools to efficiently analyze the complicated data generated by high throughput sequencers. We developed a standalone tool with graphic user interface (GUI)-based analytic modules, known as eRNA. The capacity of performing parallel processing and sample management facilitates large data analyses by maximizing hardware usage and freeing users from tediously handling sequencing data. The module miRNA identification" includes GUIs for raw data reading, adapter removal, sequence alignment, and read counting. The module "mRNA identification" includes GUIs for reference sequences, genome mapping, transcript assembling, and differential expression. The module "Target screening" provides expression profiling analyses and graphic visualization. The module "Self-testing" offers the directory setups, sample management, and a check for third-party package dependency. Integration of other GUIs including Bowtie, miRDeep2, and miRspring extend the program's functionality. eRNA focuses on the common tools required for the mapping and quantification analysis of miRNA-seq and mRNA-seq data. The software package provides an additional choice for scientists who require a user-friendly computing environment and high-throughput capacity for large data analysis. eRNA is available for free download at https://sourceforge.net/projects/erna/?source=directory.

Identification of tissue-specific, abiotic stress-responsive gene expression patterns in wine grape (Vitis vinifera L.) based on curation and mining of large-scale EST data sets

PubMed Central

2011-01-01

Background Abiotic stresses, such as water deficit and soil salinity, result in changes in physiology, nutrient use, and vegetative growth in vines, and ultimately, yield and flavor in berries of wine grape, Vitis vinifera L. Large-scale expressed sequence tags (ESTs) were generated, curated, and analyzed to identify major genetic determinants responsible for stress-adaptive responses. Although roots serve as the first site of perception and/or injury for many types of abiotic stress, EST sequencing in root tissues of wine grape exposed to abiotic stresses has been extremely limited to date. To overcome this limitation, large-scale EST sequencing was conducted from root tissues exposed to multiple abiotic stresses. Results A total of 62,236 expressed sequence tags (ESTs) were generated from leaf, berry, and root tissues from vines subjected to abiotic stresses and compared with 32,286 ESTs sequenced from 20 public cDNA libraries. Curation to correct annotation errors, clustering and assembly of the berry and leaf ESTs with currently available V. vinifera full-length transcripts and ESTs yielded a total of 13,278 unique sequences, with 2302 singletons and 10,976 mapped to V. vinifera gene models. Of these, 739 transcripts were found to have significant differential expression in stressed leaves and berries including 250 genes not described previously as being abiotic stress responsive. In a second analysis of 16,452 ESTs from a normalized root cDNA library derived from roots exposed to multiple, short-term, abiotic stresses, 135 genes with root-enriched expression patterns were identified on the basis of their relative EST abundance in roots relative to other tissues. Conclusions The large-scale analysis of relative EST frequency counts among a diverse collection of 23 different cDNA libraries from leaf, berry, and root tissues of wine grape exposed to a variety of abiotic stress conditions revealed distinct, tissue-specific expression patterns, previously unrecognized stress-induced genes, and many novel genes with root-enriched mRNA expression for improving our understanding of root biology and manipulation of rootstock traits in wine grape. mRNA abundance estimates based on EST library-enriched expression patterns showed only modest correlations between microarray and quantitative, real-time reverse transcription-polymerase chain reaction (qRT-PCR) methods highlighting the need for deep-sequencing expression profiling methods. PMID:21592389

Expressed sequence tag analysis of human RPE/choroid for the NEIBank Project: over 6000 non-redundant transcripts, novel genes and splice variants.

PubMed

Wistow, Graeme; Bernstein, Steven L; Wyatt, M Keith; Fariss, Robert N; Behal, Amita; Touchman, Jeffrey W; Bouffard, Gerald; Smith, Don; Peterson, Katherine

2002-06-15

The retinal pigment epithelium (RPE) and choroid comprise a functional unit of the eye that is essential to normal retinal health and function. Here we describe expressed sequence tag (EST) analysis of human RPE/choroid as part of a project for ocular bioinformatics. A cDNA library (cs) was made from human RPE/choroid and sequenced. Data were analyzed and assembled using the program GRIST (GRouping and Identification of Sequence Tags). Complete sequencing, Northern and Western blots, RH mapping, peptide antibody synthesis and immunofluorescence (IF) have been used to examine expression patterns and genome location for selected transcripts and proteins. Ten thousand individual sequence reads yield over 6300 unique gene clusters of which almost half have no matches with named genes. One of the most abundant transcripts is from a gene (named "alpha") that maps to the BBS1 region of chromosome 11. A number of tissue preferred transcripts are common to both RPE/choroid and iris. These include oculoglycan/opticin, for which an alternative splice form is detected in RPE/choroid, and "oculospanin" (Ocsp), a novel tetraspanin that maps to chromosome 17q. Antiserum to Ocsp detects expression in RPE, iris, ciliary body, and retinal ganglion cells by IF. A newly identified gene for a zinc-finger protein (TIRC) maps to 19q13.4. Variant transcripts of several genes were also detected. Most notably, the predominant form of Bestrophin represented in cs contains a longer open reading frame as a result of splice junction skipping. The unamplified cs library gives a view of the transcriptional repertoire of the adult RPE/choroid. A large number of potentially novel genes and splice forms and candidates for genetic diseases are revealed. Clones from this collection are being included in a large, nonredundant set for cDNA microarray construction.

Using small RNA (sRNA) deep sequencing to understand global virus distribution in plants

USDA-ARS?s Scientific Manuscript database

Small RNAs (sRNAs), a class of regulatory RNAs, have been used to serve as the specificity determinants of suppressing gene expression in plants and animals. Next generation sequencing (NGS) uncovered the sRNA landscape in most organisms including their associated microbes. In the current study, w...

Evidence of function for conserved noncoding sequences in Arabidopsis thaliana.

PubMed

Spangler, Jacob B; Subramaniam, Sabarinath; Freeling, Michael; Feltus, F Alex

2012-01-01

• Whole genome duplication events provide a lineage with a large reservoir of genes that can be molded by evolutionary forces into phenotypes that fit alternative environments. A well-studied whole genome duplication, the α-event, occurred in an ancestor of the model plant Arabidopsis thaliana. Retained segments of the α-event have been defined in recent years in the form of duplicate protein coding sequences (α-pairs) and associated conserved noncoding DNA sequences (CNSs). Our aim was to identify any association between CNSs and α-pair co-functionality at the gene expression level. • Here, we tested for correlation between CNS counts and α-pair co-expression and expression intensity across nine expression datasets: aerial tissue, flowers, leaves, roots, rosettes, seedlings, seeds, shoots and whole plants. • We provide evidence for a putative regulatory role of the CNSs. The association of CNSs with α-pair co-expression and expression intensity varied by gene function, subgene position and the presence of transcription factor binding motifs. A range of possible CNS regulatory mechanisms, including intron-mediated enhancement, messenger RNA fold stability and transcriptional regulation, are discussed. • This study provides a framework to understand how CNS motifs are involved in the maintenance of gene expression after a whole genome duplication event. © 2011 The Authors. New Phytologist © 2011 New Phytologist Trust.

Transcriptome Sequencing of Gracilariopsis lemaneiformis to Analyze the Genes Related to Optically Active Phycoerythrin Synthesis.

PubMed

Huang, Xiaoyun; Zang, Xiaonan; Wu, Fei; Jin, Yuming; Wang, Haitao; Liu, Chang; Ding, Yating; He, Bangxiang; Xiao, Dongfang; Song, Xinwei; Liu, Zhu

2017-01-01

Gracilariopsis lemaneiformis (aka Gracilaria lemaneiformis) is a red macroalga rich in phycoerythrin, which can capture light efficiently and transfer it to photosystemⅡ. However, little is known about the synthesis of optically active phycoerythrinin in G. lemaneiformis at the molecular level. With the advent of high-throughput sequencing technology, analysis of genetic information for G. lemaneiformis by transcriptome sequencing is an effective means to get a deeper insight into the molecular mechanism of phycoerythrin synthesis. Illumina technology was employed to sequence the transcriptome of two strains of G. lemaneiformis- the wild type and a green-pigmented mutant. We obtained a total of 86915 assembled unigenes as a reference gene set, and 42884 unigenes were annotated in at least one public database. Taking the above transcriptome sequencing as a reference gene set, 4041 differentially expressed genes were screened to analyze and compare the gene expression profiles of the wild type and green mutant. By GO and KEGG pathway analysis, we concluded that three factors, including a reduction in the expression level of apo-phycoerythrin, an increase of chlorophyll light-harvesting complex synthesis, and reduction of phycoerythrobilin by competitive inhibition, caused the reduction of optically active phycoerythrin in the green-pigmented mutant.

An Ambystoma mexicanum EST sequencing project: analysis of 17,352 expressed sequence tags from embryonic and regenerating blastema cDNA libraries

PubMed Central

Habermann, Bianca; Bebin, Anne-Gaelle; Herklotz, Stephan; Volkmer, Michael; Eckelt, Kay; Pehlke, Kerstin; Epperlein, Hans Henning; Schackert, Hans Konrad; Wiebe, Glenis; Tanaka, Elly M

2004-01-01

Background The ambystomatid salamander, Ambystoma mexicanum (axolotl), is an important model organism in evolutionary and regeneration research but relatively little sequence information has so far been available. This is a major limitation for molecular studies on caudate development, regeneration and evolution. To address this lack of sequence information we have generated an expressed sequence tag (EST) database for A. mexicanum. Results Two cDNA libraries, one made from stage 18-22 embryos and the other from day-6 regenerating tail blastemas, generated 17,352 sequences. From the sequenced ESTs, 6,377 contigs were assembled that probably represent 25% of the expressed genes in this organism. Sequence comparison revealed significant homology to entries in the NCBI non-redundant database. Further examination of this gene set revealed the presence of genes involved in important cell and developmental processes, including cell proliferation, cell differentiation and cell-cell communication. On the basis of these data, we have performed phylogenetic analysis of key cell-cycle regulators. Interestingly, while cell-cycle proteins such as the cyclin B family display expected evolutionary relationships, the cyclin-dependent kinase inhibitor 1 gene family shows an unusual evolutionary behavior among the amphibians. Conclusions Our analysis reveals the importance of a comprehensive sequence set from a representative of the Caudata and illustrates that the EST sequence database is a rich source of molecular, developmental and regeneration studies. To aid in data mining, the ESTs have been organized into an easily searchable database that is freely available online. PMID:15345051

VaDiR: an integrated approach to Variant Detection in RNA.

PubMed

Neums, Lisa; Suenaga, Seiji; Beyerlein, Peter; Anders, Sara; Koestler, Devin; Mariani, Andrea; Chien, Jeremy

2018-02-01

Advances in next-generation DNA sequencing technologies are now enabling detailed characterization of sequence variations in cancer genomes. With whole-genome sequencing, variations in coding and non-coding sequences can be discovered. But the cost associated with it is currently limiting its general use in research. Whole-exome sequencing is used to characterize sequence variations in coding regions, but the cost associated with capture reagents and biases in capture rate limit its full use in research. Additional limitations include uncertainty in assigning the functional significance of the mutations when these mutations are observed in the non-coding region or in genes that are not expressed in cancer tissue. We investigated the feasibility of uncovering mutations from expressed genes using RNA sequencing datasets with a method called Variant Detection in RNA(VaDiR) that integrates 3 variant callers, namely: SNPiR, RVBoost, and MuTect2. The combination of all 3 methods, which we called Tier 1 variants, produced the highest precision with true positive mutations from RNA-seq that could be validated at the DNA level. We also found that the integration of Tier 1 variants with those called by MuTect2 and SNPiR produced the highest recall with acceptable precision. Finally, we observed a higher rate of mutation discovery in genes that are expressed at higher levels. Our method, VaDiR, provides a possibility of uncovering mutations from RNA sequencing datasets that could be useful in further functional analysis. In addition, our approach allows orthogonal validation of DNA-based mutation discovery by providing complementary sequence variation analysis from paired RNA/DNA sequencing datasets.

Single-cell RNA-sequencing reveals a distinct population of proglucagon-expressing cells specific to the mouse upper small intestine.

PubMed

Glass, Leslie L; Calero-Nieto, Fernando J; Jawaid, Wajid; Larraufie, Pierre; Kay, Richard G; Göttgens, Berthold; Reimann, Frank; Gribble, Fiona M

2017-10-01

To identify sub-populations of intestinal preproglucagon-expressing (PPG) cells producing Glucagon-like Peptide-1, and their associated expression profiles of sensory receptors, thereby enabling the discovery of therapeutic strategies that target these cell populations for the treatment of diabetes and obesity. We performed single cell RNA sequencing of PPG-cells purified by flow cytometry from the upper small intestine of 3 GLU-Venus mice. Cells from 2 mice were sequenced at low depth, and from the third mouse at high depth. High quality sequencing data from 234 PPG-cells were used to identify clusters by tSNE analysis. qPCR was performed to compare the longitudinal and crypt/villus locations of cluster-specific genes. Immunofluorescence and mass spectrometry were used to confirm protein expression. PPG-cells formed 3 major clusters: a group with typical characteristics of classical L-cells, including high expression of Gcg and Pyy (comprising 51% of all PPG-cells); a cell type overlapping with Gip-expressing K-cells (14%); and a unique cluster expressing Tph1 and Pzp that was predominantly located in proximal small intestine villi and co-produced 5-HT (35%). Expression of G-protein coupled receptors differed between clusters, suggesting the cell types are differentially regulated and would be differentially targetable. Our findings support the emerging concept that many enteroendocrine cell populations are highly overlapping, with individual cells producing a range of peptides previously assigned to distinct cell types. Different receptor expression profiles across the clusters highlight potential drug targets to increase gut hormone secretion for the treatment of diabetes and obesity. Copyright © 2017 The Authors. Published by Elsevier GmbH.. All rights reserved.

Differential gene expression in the siphonophore Nanomia bijuga (Cnidaria) assessed with multiple next-generation sequencing workflows.

PubMed

Siebert, Stefan; Robinson, Mark D; Tintori, Sophia C; Goetz, Freya; Helm, Rebecca R; Smith, Stephen A; Shaner, Nathan; Haddock, Steven H D; Dunn, Casey W

2011-01-01

We investigated differential gene expression between functionally specialized feeding polyps and swimming medusae in the siphonophore Nanomia bijuga (Cnidaria) with a hybrid long-read/short-read sequencing strategy. We assembled a set of partial gene reference sequences from long-read data (Roche 454), and generated short-read sequences from replicated tissue samples that were mapped to the references to quantify expression. We collected and compared expression data with three short-read expression workflows that differ in sample preparation, sequencing technology, and mapping tools. These workflows were Illumina mRNA-Seq, which generates sequence reads from random locations along each transcript, and two tag-based approaches, SOLiD SAGE and Helicos DGE, which generate reads from particular tag sites. Differences in expression results across workflows were mostly due to the differential impact of missing data in the partial reference sequences. When all 454-derived gene reference sequences were considered, Illumina mRNA-Seq detected more than twice as many differentially expressed (DE) reference sequences as the tag-based workflows. This discrepancy was largely due to missing tag sites in the partial reference that led to false negatives in the tag-based workflows. When only the subset of reference sequences that unambiguously have tag sites was considered, we found broad congruence across workflows, and they all identified a similar set of DE sequences. Our results are promising in several regards for gene expression studies in non-model organisms. First, we demonstrate that a hybrid long-read/short-read sequencing strategy is an effective way to collect gene expression data when an annotated genome sequence is not available. Second, our replicated sampling indicates that expression profiles are highly consistent across field-collected animals in this case. Third, the impacts of partial reference sequences on the ability to detect DE can be mitigated through workflow choice and deeper reference sequencing.

Differential Gene Expression in the Siphonophore Nanomia bijuga (Cnidaria) Assessed with Multiple Next-Generation Sequencing Workflows

PubMed Central

Siebert, Stefan; Robinson, Mark D.; Tintori, Sophia C.; Goetz, Freya; Helm, Rebecca R.; Smith, Stephen A.; Shaner, Nathan; Haddock, Steven H. D.; Dunn, Casey W.

2011-01-01

We investigated differential gene expression between functionally specialized feeding polyps and swimming medusae in the siphonophore Nanomia bijuga (Cnidaria) with a hybrid long-read/short-read sequencing strategy. We assembled a set of partial gene reference sequences from long-read data (Roche 454), and generated short-read sequences from replicated tissue samples that were mapped to the references to quantify expression. We collected and compared expression data with three short-read expression workflows that differ in sample preparation, sequencing technology, and mapping tools. These workflows were Illumina mRNA-Seq, which generates sequence reads from random locations along each transcript, and two tag-based approaches, SOLiD SAGE and Helicos DGE, which generate reads from particular tag sites. Differences in expression results across workflows were mostly due to the differential impact of missing data in the partial reference sequences. When all 454-derived gene reference sequences were considered, Illumina mRNA-Seq detected more than twice as many differentially expressed (DE) reference sequences as the tag-based workflows. This discrepancy was largely due to missing tag sites in the partial reference that led to false negatives in the tag-based workflows. When only the subset of reference sequences that unambiguously have tag sites was considered, we found broad congruence across workflows, and they all identified a similar set of DE sequences. Our results are promising in several regards for gene expression studies in non-model organisms. First, we demonstrate that a hybrid long-read/short-read sequencing strategy is an effective way to collect gene expression data when an annotated genome sequence is not available. Second, our replicated sampling indicates that expression profiles are highly consistent across field-collected animals in this case. Third, the impacts of partial reference sequences on the ability to detect DE can be mitigated through workflow choice and deeper reference sequencing. PMID:21829563

Delimiting regulatory sequences of the Drosophila melanogaster Ddc gene.

PubMed Central

Hirsh, J; Morgan, B A; Scholnick, S B

1986-01-01

We delimited sequences necessary for in vivo expression of the Drosophila melanogaster dopa decarboxylase gene Ddc. The expression of in vitro-altered genes was assayed following germ line integration via P-element vectors. Sequences between -209 and -24 were necessary for normally regulated expression, although genes lacking these sequences could be expressed at 10 to 50% of wild-type levels at specific developmental times. These genes showed components of normal developmental expression, which suggests that they retain some regulatory elements. All Ddc genes lacking the normal immediate 5'-flanking sequences were grossly deficient in larval central nervous system expression. Thus, this upstream region must contain at least one element necessary for this expression. A mutated Ddc gene without a normal TATA boxlike sequence used the normal RNA start points, indicating that this sequences is not required for start point specificity. Images PMID:3099170

Genome-wide identification of conserved intronic non-coding sequences using a Bayesian segmentation approach.

PubMed

Algama, Manjula; Tasker, Edward; Williams, Caitlin; Parslow, Adam C; Bryson-Richardson, Robert J; Keith, Jonathan M

2017-03-27

Computational identification of non-coding RNAs (ncRNAs) is a challenging problem. We describe a genome-wide analysis using Bayesian segmentation to identify intronic elements highly conserved between three evolutionarily distant vertebrate species: human, mouse and zebrafish. We investigate the extent to which these elements include ncRNAs (or conserved domains of ncRNAs) and regulatory sequences. We identified 655 deeply conserved intronic sequences in a genome-wide analysis. We also performed a pathway-focussed analysis on genes involved in muscle development, detecting 27 intronic elements, of which 22 were not detected in the genome-wide analysis. At least 87% of the genome-wide and 70% of the pathway-focussed elements have existing annotations indicative of conserved RNA secondary structure. The expression of 26 of the pathway-focused elements was examined using RT-PCR, providing confirmation that they include expressed ncRNAs. Consistent with previous studies, these elements are significantly over-represented in the introns of transcription factors. This study demonstrates a novel, highly effective, Bayesian approach to identifying conserved non-coding sequences. Our results complement previous findings that these sequences are enriched in transcription factors. However, in contrast to previous studies which suggest the majority of conserved sequences are regulatory factor binding sites, the majority of conserved sequences identified using our approach contain evidence of conserved RNA secondary structures, and our laboratory results suggest most are expressed. Functional roles at DNA and RNA levels are not mutually exclusive, and many of our elements possess evidence of both. Moreover, ncRNAs play roles in transcriptional and post-transcriptional regulation, and this may contribute to the over-representation of these elements in introns of transcription factors. We attribute the higher sensitivity of the pathway-focussed analysis compared to the genome-wide analysis to improved alignment quality, suggesting that enhanced genomic alignments may reveal many more conserved intronic sequences.

Pyruvate metabolism: A therapeutic opportunity in radiation-induced skin injury

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

Yoo, Hyun; Kang, Jeong Wook; Lee, Dong Won

Ionizing radiation is used to treat a range of cancers. Despite recent technological progress, radiation therapy can damage the skin at the administration site. The specific molecular mechanisms involved in this effect have not been fully characterized. In this study, the effects of pyruvate, on radiation-induced skin injury were investigated, including the role of the pyruvate dehydrogenase kinase 2 (PDK2) signaling pathway. Next generation sequencing (NGS) identified a wide range of gene expression differences between the control and irradiated mice, including reduced expression of PDK2. This was confirmed using Q-PCR. Cell culture studies demonstrated that PDK2 overexpression and a highmore » cellular pyruvate concentration inhibited radiation-induced cytokine expression. Immunohistochemical studies demonstrated radiation-induced skin thickening and gene expression changes. Oral pyruvate treatment markedly downregulated radiation-induced changes in skin thickness and inflammatory cytokine expression. These findings indicated that regulation of the pyruvate metabolic pathway could provide an effective approach to the control of radiation-induced skin damage. - Highlights: • The effects of radiation on skin thickness in mice. • Next generation sequencing revealed that radiation inhibited pyruvate dehydrogenase kinase 2 expression. • PDK2 inhibited irradiation-induced cytokine gene expression. • Oral pyruvate treatment markedly downregulated radiation-induced changes in skin thickness.« less

Characterization and improvement of RNA-Seq precision in quantitative transcript expression profiling.

PubMed

Łabaj, Paweł P; Leparc, Germán G; Linggi, Bryan E; Markillie, Lye Meng; Wiley, H Steven; Kreil, David P

2011-07-01

Measurement precision determines the power of any analysis to reliably identify significant signals, such as in screens for differential expression, independent of whether the experimental design incorporates replicates or not. With the compilation of large-scale RNA-Seq datasets with technical replicate samples, however, we can now, for the first time, perform a systematic analysis of the precision of expression level estimates from massively parallel sequencing technology. This then allows considerations for its improvement by computational or experimental means. We report on a comprehensive study of target identification and measurement precision, including their dependence on transcript expression levels, read depth and other parameters. In particular, an impressive recall of 84% of the estimated true transcript population could be achieved with 331 million 50 bp reads, with diminishing returns from longer read lengths and even less gains from increased sequencing depths. Most of the measurement power (75%) is spent on only 7% of the known transcriptome, however, making less strongly expressed transcripts harder to measure. Consequently, <30% of all transcripts could be quantified reliably with a relative error<20%. Based on established tools, we then introduce a new approach for mapping and analysing sequencing reads that yields substantially improved performance in gene expression profiling, increasing the number of transcripts that can reliably be quantified to over 40%. Extrapolations to higher sequencing depths highlight the need for efficient complementary steps. In discussion we outline possible experimental and computational strategies for further improvements in quantification precision. rnaseq10@boku.ac.at

Genome-wide localization and expression profiling establish Sp2 as a sequence-specific transcription factor regulating vitally important genes

PubMed Central

Terrados, Gloria; Finkernagel, Florian; Stielow, Bastian; Sadic, Dennis; Neubert, Juliane; Herdt, Olga; Krause, Michael; Scharfe, Maren; Jarek, Michael; Suske, Guntram

2012-01-01

The transcription factor Sp2 is essential for early mouse development and for proliferation of mouse embryonic fibroblasts in culture. Yet its mechanisms of action and its target genes are largely unknown. In this study, we have combined RNA interference, in vitro DNA binding, chromatin immunoprecipitation sequencing and global gene-expression profiling to investigate the role of Sp2 for cellular functions, to define target sites and to identify genes regulated by Sp2. We show that Sp2 is important for cellular proliferation that it binds to GC-boxes and occupies proximal promoters of genes essential for vital cellular processes including gene expression, replication, metabolism and signalling. Moreover, we identified important key target genes and cellular pathways that are directly regulated by Sp2. Most significantly, Sp2 binds and activates numerous sequence-specific transcription factor and co-activator genes, and represses the whole battery of cholesterol synthesis genes. Our results establish Sp2 as a sequence-specific regulator of vitally important genes. PMID:22684502

Deep sequencing-based transcriptome analysis of Plutella xylostella larvae parasitized by Diadegma semiclausum

PubMed Central

2011-01-01

Background Parasitoid insects manipulate their hosts' physiology by injecting various factors into their host upon parasitization. Transcriptomic approaches provide a powerful approach to study insect host-parasitoid interactions at the molecular level. In order to investigate the effects of parasitization by an ichneumonid wasp (Diadegma semiclausum) on the host (Plutella xylostella), the larval transcriptome profile was analyzed using a short-read deep sequencing method (Illumina). Symbiotic polydnaviruses (PDVs) associated with ichneumonid parasitoids, known as ichnoviruses, play significant roles in host immune suppression and developmental regulation. In the current study, D. semiclausum ichnovirus (DsIV) genes expressed in P. xylostella were identified and their sequences compared with other reported PDVs. Five of these genes encode proteins of unknown identity, that have not previously been reported. Results De novo assembly of cDNA sequence data generated 172,660 contigs between 100 and 10000 bp in length; with 35% of > 200 bp in length. Parasitization had significant impacts on expression levels of 928 identified insect host transcripts. Gene ontology data illustrated that the majority of the differentially expressed genes are involved in binding, catalytic activity, and metabolic and cellular processes. In addition, the results show that transcription levels of antimicrobial peptides, such as gloverin, cecropin E and lysozyme, were up-regulated after parasitism. Expression of ichnovirus genes were detected in parasitized larvae with 19 unique sequences identified from five PDV gene families including vankyrin, viral innexin, repeat elements, a cysteine-rich motif, and polar residue rich protein. Vankyrin 1 and repeat element 1 genes showed the highest transcription levels among the DsIV genes. Conclusion This study provides detailed information on differential expression of P. xylostella larval genes following parasitization, DsIV genes expressed in the host and also improves our current understanding of this host-parasitoid interaction. PMID:21906285

Genome and Transcriptome Sequencing of the Ostreid herpesvirus 1 From Tomales Bay, California

NASA Astrophysics Data System (ADS)

Burge, C. A.; Langevin, S.; Closek, C. J.; Roberts, S. B.; Friedman, C. S.

2016-02-01

Mass mortalities of larval and seed bivalve molluscs attributed to the Ostreid herpesvirus 1 (OsHV-1) occur globally. OsHV-1 was fully sequenced and characterized as a member of the Family Malacoherpesviridae. Multiple strains of OsHV-1 exist and may vary in virulence, i.e. OsHV-1 µvar. For most global variants of OsHV-1, sequence data is limited to PCR-based sequencing of segments, including two recent genomes. In the United States, OsHV-1 is limited to detection in adjacent embayments in California, Tomales and Drakes bays. Limited DNA sequence data of OsHV-1 infecting oysters in Tomales Bay indicates the virus detected in Tomales Bay is similar but not identical to any one global variant of OsHV-1. In order to better understand both strain variation and virulence of OsHV-1 infecting oysters in Tomales Bay, we used genomic and transcriptomic sequencing. Meta-genomic sequencing (Illumina MiSeq) was conducted from infected oysters (n=4 per year) collected in 2003, 2007, and 2014, where full OsHV-1 genome sequences and low overall microbial diversity were achieved from highly infected oysters. Increased microbial diversity was detected in three of four samples sequenced from 2003, where qPCR based genome copy numbers of OsHV-1 were lower. Expression analysis (SOLiD RNA sequencing) of OsHV-1 genes expressed in oyster larvae at 24 hours post exposure revealed a nearly complete transcriptome, with several highly expressed genes, which are similar to recent transcriptomic analyses of other OsHV-1 variants. Taken together, our results indicate that genome and transcriptome sequencing may be powerful tools in understanding both strain variation and virulence of non-culturable marine viruses.

PNMA family: Protein interaction network and cell signalling pathways implicated in cancer and apoptosis.

PubMed

Pang, Siew Wai; Lahiri, Chandrajit; Poh, Chit Laa; Tan, Kuan Onn

2018-05-01

Paraneoplastic Ma Family (PNMA) comprises a growing number of family members which share relatively conserved protein sequences encoded by the human genome and is localized to several human chromosomes, including the X-chromosome. Based on sequence analysis, PNMA family members share sequence homology to the Gag protein of LTR retrotransposon, and several family members with aberrant protein expressions have been reported to be closely associated with the human Paraneoplastic Disorder (PND). In addition, gene mutations of specific members of PNMA family are known to be associated with human mental retardation or 3-M syndrome consisting of restrictive post-natal growth or dwarfism, and development of skeletal abnormalities. Other than sequence homology, the physiological function of many members in this family remains unclear. However, several members of this family have been characterized, including cell signalling events mediated by these proteins that are associated with apoptosis, and cancer in different cell types. Furthermore, while certain PNMA family members show restricted gene expression in the human brain and testis, other PNMA family members exhibit broader gene expression or preferential and selective protein interaction profiles, suggesting functional divergence within the family. Functional analysis of some members of this family have identified protein domains that are required for subcellular localization, protein-protein interactions, and cell signalling events which are the focus of this review paper. Copyright © 2018 Elsevier Inc. All rights reserved.

Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks

PubMed Central

Trapnell, Cole; Roberts, Adam; Goff, Loyal; Pertea, Geo; Kim, Daehwan; Kelley, David R; Pimentel, Harold; Salzberg, Steven L; Rinn, John L; Pachter, Lior

2012-01-01

Recent advances in high-throughput cDNA sequencing (RNA-seq) can reveal new genes and splice variants and quantify expression genome-wide in a single assay. The volume and complexity of data from RNA-seq experiments necessitate scalable, fast and mathematically principled analysis software. TopHat and Cufflinks are free, open-source software tools for gene discovery and comprehensive expression analysis of high-throughput mRNA sequencing (RNA-seq) data. Together, they allow biologists to identify new genes and new splice variants of known ones, as well as compare gene and transcript expression under two or more conditions. This protocol describes in detail how to use TopHat and Cufflinks to perform such analyses. It also covers several accessory tools and utilities that aid in managing data, including CummeRbund, a tool for visualizing RNA-seq analysis results. Although the procedure assumes basic informatics skills, these tools assume little to no background with RNA-seq analysis and are meant for novices and experts alike. The protocol begins with raw sequencing reads and produces a transcriptome assembly, lists of differentially expressed and regulated genes and transcripts, and publication-quality visualizations of analysis results. The protocol's execution time depends on the volume of transcriptome sequencing data and available computing resources but takes less than 1 d of computer time for typical experiments and ~1 h of hands-on time. PMID:22383036

IDP-ASE: haplotyping and quantifying allele-specific expression at the gene and gene isoform level by hybrid sequencing

PubMed Central

Deonovic, Benjamin; Wang, Yunhao; Weirather, Jason; Wang, Xiu-Jie; Au, Kin Fai

2017-01-01

Abstract Allele-specific expression (ASE) is a fundamental problem in studying gene regulation and diploid transcriptome profiles, with two key challenges: (i) haplotyping and (ii) estimation of ASE at the gene isoform level. Existing ASE analysis methods are limited by a dependence on haplotyping from laborious experiments or extra genome/family trio data. In addition, there is a lack of methods for gene isoform level ASE analysis. We developed a tool, IDP-ASE, for full ASE analysis. By innovative integration of Third Generation Sequencing (TGS) long reads with Second Generation Sequencing (SGS) short reads, the accuracy of haplotyping and ASE quantification at the gene and gene isoform level was greatly improved as demonstrated by the gold standard data GM12878 data and semi-simulation data. In addition to methodology development, applications of IDP-ASE to human embryonic stem cells and breast cancer cells indicate that the imbalance of ASE and non-uniformity of gene isoform ASE is widespread, including tumorigenesis relevant genes and pluripotency markers. These results show that gene isoform expression and allele-specific expression cooperate to provide high diversity and complexity of gene regulation and expression, highlighting the importance of studying ASE at the gene isoform level. Our study provides a robust bioinformatics solution to understand ASE using RNA sequencing data only. PMID:27899656

Construction of Pará rubber tree genome and multi-transcriptome database accelerates rubber researches.

PubMed

Makita, Yuko; Kawashima, Mika; Lau, Nyok Sean; Othman, Ahmad Sofiman; Matsui, Minami

2018-01-19

Natural rubber is an economically important material. Currently the Pará rubber tree, Hevea brasiliensis is the main commercial source. Little is known about rubber biosynthesis at the molecular level. Next-generation sequencing (NGS) technologies brought draft genomes of three rubber cultivars and a variety of RNA sequencing (RNA-seq) data. However, no current genome or transcriptome databases (DB) are organized by gene. A gene-oriented database is a valuable support for rubber research. Based on our original draft genome sequence of H. brasiliensis RRIM600, we constructed a rubber tree genome and transcriptome DB. Our DB provides genome information including gene functional annotations and multi-transcriptome data of RNA-seq, full-length cDNAs including PacBio Isoform sequencing (Iso-Seq), ESTs and genome wide transcription start sites (TSSs) derived from CAGE technology. Using our original and publically available RNA-seq data, we calculated co-expressed genes for identifying functionally related gene sets and/or genes regulated by the same transcription factor (TF). Users can access multi-transcriptome data through both a gene-oriented web page and a genome browser. For the gene searching system, we provide keyword search, sequence homology search and gene expression search; users can also select their expression threshold easily. The rubber genome and transcriptome DB provides rubber tree genome sequence and multi-transcriptomics data. This DB is useful for comprehensive understanding of the rubber transcriptome. This will assist both industrial and academic researchers for rubber and economically important close relatives such as R. communis, M. esculenta and J. curcas. The Rubber Transcriptome DB release 2017.03 is accessible at http://matsui-lab.riken.jp/rubber/ .

Enhanced green fluorescent protein (egfp) gene expression in Tetraselmis subcordiformis chloroplast with endogenous regulators.

PubMed

Cui, Yulin; Zhao, Jialin; Hou, Shichang; Qin, Song

2016-05-01

On the basis of fundamental genetic transformation technologies, the goal of this study was to optimize Tetraselmis subcordiformis chloroplast transformation through the use of endogenous regulators. The genes rrn16S, rbcL, psbA, and psbC are commonly highly expressed in chloroplasts, and the regulators of these genes are often used in chloroplast transformation. For lack of a known chloroplast genome sequence, the genome-walking method was used here to obtain full sequences of T. subcordiformis endogenous regulators. The resulting regulators, including three promoters, two terminators, and a ribosome combination sequence, were inserted into the previously constructed plasmid pPSC-R, with the egfp gene included as a reporter gene, and five chloroplast expression vectors prepared. These vectors were successfully transformed into T. subcordiformis by particle bombardment and the efficiency of each vector tested by assessing EGFP fluorescence via microscopy. The results showed that these vectors exhibited higher efficiency than the former vector pPSC-G carrying exogenous regulators, and the vector pRFA with Prrn, psbA-5'RE, and TpsbA showed the highest efficiency. This research provides a set of effective endogenous regulators for T. subcordiformis and will facilitate future fundamental studies of this alga.

Expression profiling during arabidopsis/downy mildew interaction reveals a highly-expressed effector that attenuates responses to salicylic acid.

PubMed

Asai, Shuta; Rallapalli, Ghanasyam; Piquerez, Sophie J M; Caillaud, Marie-Cécile; Furzer, Oliver J; Ishaque, Naveed; Wirthmueller, Lennart; Fabro, Georgina; Shirasu, Ken; Jones, Jonathan D G

2014-10-01

Plants have evolved strong innate immunity mechanisms, but successful pathogens evade or suppress plant immunity via effectors delivered into the plant cell. Hyaloperonospora arabidopsidis (Hpa) causes downy mildew on Arabidopsis thaliana, and a genome sequence is available for isolate Emoy2. Here, we exploit the availability of genome sequences for Hpa and Arabidopsis to measure gene-expression changes in both Hpa and Arabidopsis simultaneously during infection. Using a high-throughput cDNA tag sequencing method, we reveal expression patterns of Hpa predicted effectors and Arabidopsis genes in compatible and incompatible interactions, and promoter elements associated with Hpa genes expressed during infection. By resequencing Hpa isolate Waco9, we found it evades Arabidopsis resistance gene RPP1 through deletion of the cognate recognized effector ATR1. Arabidopsis salicylic acid (SA)-responsive genes including PR1 were activated not only at early time points in the incompatible interaction but also at late time points in the compatible interaction. By histochemical analysis, we found that Hpa suppresses SA-inducible PR1 expression, specifically in the haustoriated cells into which host-translocated effectors are delivered, but not in non-haustoriated adjacent cells. Finally, we found a highly-expressed Hpa effector candidate that suppresses responsiveness to SA. As this approach can be easily applied to host-pathogen interactions for which both host and pathogen genome sequences are available, this work opens the door towards transcriptome studies in infection biology that should help unravel pathogen infection strategies and the mechanisms by which host defense responses are overcome.

Comparative transcriptome analysis reveals differentially expressed genes associated with sex expression in garden asparagus (Asparagus officinalis).

PubMed

Li, Shu-Fen; Zhang, Guo-Jun; Zhang, Xue-Jin; Yuan, Jin-Hong; Deng, Chuan-Liang; Gao, Wu-Jun

2017-08-22

Garden asparagus (Asparagus officinalis) is a highly valuable vegetable crop of commercial and nutritional interest. It is also commonly used to investigate the mechanisms of sex determination and differentiation in plants. However, the sex expression mechanisms in asparagus remain poorly understood. De novo transcriptome sequencing via Illumina paired-end sequencing revealed more than 26 billion bases of high-quality sequence data from male and female asparagus flower buds. A total of 72,626 unigenes with an average length of 979 bp were assembled. In comparative transcriptome analysis, 4876 differentially expressed genes (DEGs) were identified in the possible sex-determining stage of female and male/supermale flower buds. Of these DEGs, 433, including 285 male/supermale-biased and 149 female-biased genes, were annotated as flower related. Of the male/supermale-biased flower-related genes, 102 were probably involved in anther development. In addition, 43 DEGs implicated in hormone response and biosynthesis putatively associated with sex expression and reproduction were discovered. Moreover, 128 transcription factor (TF)-related genes belonging to various families were found to be differentially expressed, and this finding implied the essential roles of TF in sex determination or differentiation in asparagus. Correlation analysis indicated that miRNA-DEG pairs were also implicated in asparagus sexual development. Our study identified a large number of DEGs involved in the sex expression and reproduction of asparagus, including known genes participating in plant reproduction, plant hormone signaling, TF encoding, and genes with unclear functions. We also found that miRNAs might be involved in the sex differentiation process. Our study could provide a valuable basis for further investigations on the regulatory networks of sex determination and differentiation in asparagus and facilitate further genetic and genomic studies on this dioecious species.

Small RNA sequencing and functional characterization reveals microRNA-143 tumor suppressor activity in liposarcoma

PubMed Central

Ugras, Stacy; Brill, Elliott; Jacobsen, Anders; Hafner, Markus; Socci, Nicholas D.; DeCarolis, Penelope L.; Khanin, Raya; O'Connor, Rachael; Mihailovic, Aleksandra; Taylor, Barry S.; Sheridan, Robert; Gimble, Jeffrey M.; Viale, Agnes; Crago, Aimee; Antonescu, Cristina R.; Sander, Chris; Tuschl, Thomas; Singer, Samuel

2011-01-01

Liposarcoma remains the most common mesenchymal cancer, with a mortality rate of 60% among patients with this disease. To address the present lack of therapeutic options, we embarked upon a study of microRNA (miRNA) expression alterations associated with liposarcomagenesis with the goal of exploiting differentially expressed miRNAs and the gene products they regulate as potential therapeutic targets. MicroRNA expression was profiled in samples of normal adipose tissue, well-differentiated liposarcoma, and dedifferentiated liposarcoma by both deep sequencing of small RNA libraries and hybridization-based Agilent microarrays. The expression profiles discriminated liposarcoma from normal adipose tissue and well-differentiated from dedifferentiated disease. We defined over 40 miRNAs that were dysregulated in dedifferentiated liposarcomas in both the sequencing and the microarray analysis. The upregulated miRNAs included two cancer-associated species (miR-21, miR-26a), and the downregulated miRNAs included two species that were highly abundant in adipose tissue (miR-143, miR-145). Restoring miR-143 expression in dedifferentiated liposarcoma cells inhibited proliferation, induced apoptosis, and decreased expression of BCL2, TOP2A, PRC1, and PLK1. The downregulation of PRC1 and its docking partner PLK1 suggests that miR-143 inhibits cytokinesis in these cells. In support of this idea, treatment with a PLK1 inhibitor potently induced G2/M growth arrest and apoptosis in liposarcoma cells. Taken together, our findings suggest that miR-143 re-expression vectors or selective agents directed at miR-143 or its targets may have therapeutic value in dedifferentiated liposarcoma. PMID:21693658

Expression of NAD(P)H quinone dehydrogenase 1 (NQO1) is increased in the endometrium of women with endometrial cancer and women with polycystic ovary syndrome.

PubMed

Atiomo, William; Shafiee, Mohamad Nasir; Chapman, Caroline; Metzler, Veronika M; Abouzeid, Jad; Latif, Ayşe; Chadwick, Amy; Kitson, Sarah; Sivalingam, Vanitha N; Stratford, Ian J; Rutland, Catrin S; Persson, Jenny L; Ødum, Niels; Fuentes-Utrilla, Pablo; Jeyapalan, Jennie N; Heery, David M; Crosbie, Emma J; Mongan, Nigel P

2017-11-01

Women with a prior history of polycystic ovary syndrome (PCOS) have an increased risk of endometrial cancer (EC). To investigate whether the endometrium of women with PCOS possesses gene expression changes similar to those found in EC. Patients with EC, PCOS and control women unaffected by either PCOS or EC were recruited into a cross-sectional study at the Nottingham University Hospital, UK. For RNA sequencing, representative individual endometrial biopsies were obtained from women with EC, PCOS and a woman unaffected by PCOS or EC. Expression of a subset of differentially expressed genes identified by RNA sequencing, including NAD(P)H quinone dehydrogenase 1 (NQO1), was validated by quantitative reverse transcriptase PCR validation (n = 76) and in the cancer genome atlas UCEC (uterine corpus endometrioid carcinoma) RNA sequencing data set (n = 381). The expression of NQO1 was validated by immunohistochemistry in EC samples from a separate cohort (n = 91) comprised of consecutive patients who underwent hysterectomy at St Mary's Hospital, Manchester, between 2011 and 2013. A further 6 postmenopausal women with histologically normal endometrium who underwent hysterectomy for genital prolapse were also included. Informed consent and local ethics approval were obtained for the study. We show for the first that NQO1 expression is significantly increased in the endometrium of women with PCOS and EC. Immunohistochemistry confirms significantly increased NQO1 protein expression in EC relative to nonmalignant endometrial tissue (P < .0001). The results obtained here support a previously unrecognized molecular link between PCOS and EC involving NQO1. © 2017 The Authors. Clinical Endocrinology Published by John Wiley & Sons Ltd.

Cloning, analysis and functional annotation of expressed sequence tags from the Earthworm Eisenia fetida

PubMed Central

Pirooznia, Mehdi; Gong, Ping; Guan, Xin; Inouye, Laura S; Yang, Kuan; Perkins, Edward J; Deng, Youping

2007-01-01

Background Eisenia fetida, commonly known as red wiggler or compost worm, belongs to the Lumbricidae family of the Annelida phylum. Little is known about its genome sequence although it has been extensively used as a test organism in terrestrial ecotoxicology. In order to understand its gene expression response to environmental contaminants, we cloned 4032 cDNAs or expressed sequence tags (ESTs) from two E. fetida libraries enriched with genes responsive to ten ordnance related compounds using suppressive subtractive hybridization-PCR. Results A total of 3144 good quality ESTs (GenBank dbEST accession number EH669363–EH672369 and EL515444–EL515580) were obtained from the raw clone sequences after cleaning. Clustering analysis yielded 2231 unique sequences including 448 contigs (from 1361 ESTs) and 1783 singletons. Comparative genomic analysis showed that 743 or 33% of the unique sequences shared high similarity with existing genes in the GenBank nr database. Provisional function annotation assigned 830 Gene Ontology terms to 517 unique sequences based on their homology with the annotated genomes of four model organisms Drosophila melanogaster, Mus musculus, Saccharomyces cerevisiae, and Caenorhabditis elegans. Seven percent of the unique sequences were further mapped to 99 Kyoto Encyclopedia of Genes and Genomes pathways based on their matching Enzyme Commission numbers. All the information is stored and retrievable at a highly performed, web-based and user-friendly relational database called EST model database or ESTMD version 2. Conclusion The ESTMD containing the sequence and annotation information of 4032 E. fetida ESTs is publicly accessible at . PMID:18047730

Molecular phenotype of zebrafish ovarian follicle by serial analysis of gene expression and proteomic profiling, and comparison with the transcriptomes of other animals

PubMed Central

Knoll-Gellida, Anja; André, Michèle; Gattegno, Tamar; Forgue, Jean; Admon, Arie; Babin, Patrick J

2006-01-01

Background The ability of an oocyte to develop into a viable embryo depends on the accumulation of specific maternal information and molecules, such as RNAs and proteins. A serial analysis of gene expression (SAGE) was carried out in parallel with proteomic analysis on fully-grown ovarian follicles from zebrafish (Danio rerio). The data obtained were compared with ovary/follicle/egg molecular phenotypes of other animals, published or available in public sequence databases. Results Sequencing of 27,486 SAGE tags identified 11,399 different ones, including 3,329 tags with an occurrence superior to one. Fifty-eight genes were expressed at over 0.15% of the total population and represented 17.34% of the mRNA population identified. The three most expressed transcripts were a rhamnose-binding lectin, beta-actin 2, and a transcribed locus similar to the H2B histone family. Comparison with the large-scale expressed sequence tags sequencing approach revealed highly expressed transcripts that were not previously known to be expressed at high levels in fish ovaries, like the short-sized polarized metallothionein 2 transcript. A higher sensitivity for the detection of transcripts with a characterized maternal genetic contribution was also demonstrated compared to large-scale sequencing of cDNA libraries. Ferritin heavy polypeptide 1, heat shock protein 90-beta, lactate dehydrogenase B4, beta-actin isoforms, tubulin beta 2, ATP synthase subunit 9, together with 40 S ribosomal protein S27a, were common highly-expressed transcripts of vertebrate ovary/unfertilized egg. Comparison of transcriptome and proteome data revealed that transcript levels provide little predictive value with respect to the extent of protein abundance. All the proteins identified by proteomic analysis of fully-grown zebrafish follicles had at least one transcript counterpart, with two exceptions: eosinophil chemotactic cytokine and nothepsin. Conclusion This study provides a complete sequence data set of maternal mRNA stored in zebrafish germ cells at the end of oogenesis. This catalogue contains highly-expressed transcripts that are part of a vertebrate ovarian expressed gene signature. Comparison of transcriptome and proteome data identified downregulated transcripts or proteins potentially incorporated in the oocyte by endocytosis. The molecular phenotype described provides groundwork for future experimental approaches aimed at identifying functionally important stored maternal transcripts and proteins involved in oogenesis and early stages of embryo development. PMID:16526958

Young infants' generalization of emotional expressions: effects of familiarity.

PubMed

Walker-Andrews, Arlene S; Krogh-Jespersen, Sheila; Mayhew, Estelle M Y; Coffield, Caroline N

2011-08-01

From birth, infants are exposed to a wealth of emotional information in their interactions. Much research has been done to investigate the development of emotion perception, and factors influencing that development. The current study investigates the role of familiarity on 3.5-month-old infants' generalization of emotional expressions. Infants were assigned to one of two habituation sequences: in one sequence, infants were visually habituated to parental expressions of happy or sad. At test, infants viewed either a continuation of the habituation sequence, their mother depicting a novel expression, an unfamiliar female depicting the habituated expression, or an unfamiliar female depicting a novel expression. In the second sequence, a new sample of infants was matched to the infants in the first sequence. These infants viewed the same habituation and test sequences, but the actors were unfamiliar to them. Only those infants who viewed their own mothers and fathers during the habituation sequence increased looking. They dishabituated looking to maternal novel expressions, the unfamiliar female's novel expression, and the unfamiliar female depicting the habituated expression, especially when sad parental expressions were followed by an expression change to happy or to a change in person. Infants are guided in their recognition of emotional expressions by the familiarity of their parents, before generalizing to others. 2011 APA, all rights reserved

Global Gene Expression Patterns and Somatic Mutations in Sporadic Intracranial Aneurysms.

PubMed

Li, Zhili; Tan, Haibin; Shi, Yi; Huang, Guangfu; Wang, Zhenyu; Liu, Ling; Yin, Cheng; Wang, Qi

2017-04-01

High-throughput sequencing technologies can expand our understanding of the pathologic basis of intracranial aneurysms (IAs). Our study was aimed to decipher the gene expression signature and genetic factors associated with IAs. We determined the gene expression levels of 3 cases of IAs by RNA sequencing. Bioinformatics analysis was conducted to identify the differentially expressed genes (DEGs) and uncover their biological function. In addition, whole genome sequencing was performed on an additional 6 cases of IAs to detect the potential somatic alterations in DEGs. Compared with the normal arterial tissue, 1709 genes were differentially expressed in IAs arterial tissue. The most significantly up-regulated gene and down-regulated gene, H19 and HIST1H3J, may be essential for tumorigenesis of IAs. Hub protein of IKBKG in protein-protein interaction network was probably involved in the inflammation process in aneurysms. Another 2 hub proteins, ACTB and MKI67IP, as well as up-regulated genes, might be abnormally activated in aneurysms and involved in the pathogenesis of IAs. Further whole genome sequencing and filtering yielded 4 candidate somatic single nucleotide variants including MUC3B, and BLM may be involved in the pathogenesis of IAs. Even though, our results do not support the hypothesis of somatic mutations occurred in the DEGs. Two-dimensional genomic data from transcriptome and whole genome sequencing indicated that no somatic mutations occurred in DEGs. In addition, 3 DEGs (IKBKG, ACTB, and MKI67IP) and 2 mutant genes (MUC3B and BLM) were essential in IAs. Copyright © 2017 Elsevier Inc. All rights reserved.

COL1A1 transgene expression in stably transfected osteoblastic cells. Relative contributions of first intron, 3'-flanking sequences, and sequences derived from the body of the human COL1A1 minigene

NASA Technical Reports Server (NTRS)

Breault, D. T.; Lichtler, A. C.; Rowe, D. W.

1997-01-01

Collagen reporter gene constructs have be used to identify cell-specific sequences needed for transcriptional activation. The elements required for endogenous levels of COL1A1 expression, however, have not been elucidated. The human COL1A1 minigene is expressed at high levels and likely harbors sequence elements required for endogenous levels of activity. Using stably transfected osteoblastic Py1a cells, we studied a series of constructs (pOBColCAT) designed to characterize further the elements required for high level of expression. pOBColCAT, which contains the COL1A1 first intron, was expressed at 50-100-fold higher levels than ColCAT 3.6, which lacks the first intron. This difference is best explained by improved mRNA processing rather than a transcriptional effect. Furthermore, variation in activity observed with the intron deletion constructs is best explained by altered mRNA splicing. Two major regions of the human COL1A1 minigene, the 3'-flanking sequences and the minigene body, were introduced into pOBColCAT to assess both transcriptional enhancing activity and the effect on mRNA stability. Analysis of the minigene body, which includes the first five exons and introns fused with the terminal six introns and exons, revealed an orientation-independent 5-fold increase in CAT activity. In contrast the 3'-flanking sequences gave rise to a modest 61% increase in CAT activity. Neither region increased the mRNA half-life of the parent construct, suggesting that CAT-specific mRNA instability elements may serve as dominant negative regulators of stability. This study suggests that other sites within the body of the COL1A1 minigene are important for high expression, e.g. during periods of rapid extracellular matrix production.

Expression of a recombinant human sperm-agglutinating mini-antibody in tobacco (Nicotiana tabacum L.).

PubMed

Xu, Bingfang; Copolla, Michael; Herr, John C; Timko, Michael P

2007-01-01

The murine monoclonal antibody (mAB) S19 recognizes an N-linked carbohydrate antigen designated sperm agglutination antigen-1 (SAGA1) located on the membrane protein CD52. This antigen is added to the sperm surface during epididymal maturation. Binding of the S19 mAB to SAGA-1 causes the rapid agglutination of sperm and blocks pre-fertilization events. Previous studies indicated that the S19 mAB may be a potential specific spermicidal agent (termed a spermistatic) capable of replacing current spermicidal products that contain harsh detergents with harmful side effects. The nucleotide sequences encoding the heavy (H) and light (L) chains of the S19 antibody were cloned. A chimeric gene was constructed using the nucleotide sequences encoding the variable regions of both the H and L chains, and this gene (scFv1 9) was expressed in transgenic tobacco (Nicotiana tabacum L.) to produce a recombinant anti-sperm antibody (RASA). Highest levels of RASA expression were observed in BY-2 plant cell suspension cultures and regenerated N. tabacum cv. Xanthi plants transformant in which the RASA coding sequences were expressed under the control of the Cauliflower Mosaic Virus 35S promoter containing a double-enhancer sequence (2X CaMV 35S). Subsequent modifications of the transgene including the addition of a 5'-untranslated sequence from the tobacco etch virus (TEV leader sequence), N-terminal fusion of the coding region with an endoplasmic reticulum targeting signal of patatin (pat) and C-terminal fusion with the endoplasmic reticulum retention signal peptide KDEL showed further enhancement of RASA expression. The plant-expressed RASA formed intrachain disulfide bonds and was primarily soluble in the cytoplasmic fraction of the cells. Introduction of a poly-histidine (6xHIS) tag in the recombinant RASA protein allowed for rapid purification of the recombinant protein using Ni-NTA chromatography. Optimization of scale-up production and purification of this plant-derived recombinant protein should provide large quantities of an inexpensive spermistatic plantibody.

A small test of a sequence-based typing method: definition of the B*1520 allele.

PubMed

Domena, J D; Little, A M; Arnett, K L; Adams, E J; Marsh, S G; Parham, P

1994-10-01

Santamaria et al. (Human Immunology 1993 37: 39-50) describe a method of sequence-based typing (SBT) for HLA-A, B and C alleles said to give "unambiguous typing of any sample, heterozygous or homozygous, without requiring additional typing information". From SBT analysis, which involves determination of partial sequences of mixed alleles, these investigators reported that cell lines KT17 (HLA-B35,62) and OLGA (HLA-B62) from the reference panel of the 10th International Histocompatibility Workshop express novel variants of HLA-B15 (B1501-MN6) and HLA-B35 (B3501-MN7) respectively. To study further the novel alleles, we cloned and sequenced full-length HLA-B cDNA clones isolated from the KT17 and OLGA cell lines. We find that KT17 expresses B*3501, as assigned by SBT, and B*1501, the common allele encoding the B62 antigen. We were unable to confirm that KT17 expresses the novel B1501-MN6 variant identified by SBT. For OLGA our analysis confirms the partial sequences obtained by SBT. Thus OLGA expresses B*1501 and a novel HLA-B allele. The complete sequence of the latter shows it is a hybrid having exons 1 and 2 in common with B*1501 and other B15 subtypes and exons 3-7 in common with B*3501 and related molecules including B*5301 and B*5801. The novel allele has been designated B*1520 because of its sequence similarity with the B15 group; furthermore, serological analysis shows that the B*1520 product does not express epitopes in common with either B35, B53 or B58. The B*1520 heavy chain has a similar isoelectric point to A*3101; B*1520 was undetected by previous applications of isoelectric focusing because B*1520 and A31 are both expressed by OLGA. In conclusion, HLA-B typing of two cell lines by cDNA cloning and sequencing gives concordant results with SBT for three of the four alleles. The cause of the discrepancy for the fourth allele is unknown, however, this finding indicates that the novel HLA-A, B and C sequences emerging from SBT studies need independent verification.

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

Bennetzen, Jeffrey L; Yang, Xiaohan; Ye, Chuyu

We generated a high-quality reference genome sequence for foxtail millet (Setaria italica). The {approx}400-Mb assembly covers {approx}80% of the genome and >95% of the gene space. The assembly was anchored to a 992-locus genetic map and was annotated by comparison with >1.3 million expressed sequence tag reads. We produced more than 580 million RNA-Seq reads to facilitate expression analyses. We also sequenced Setaria viridis, the ancestral wild relative of S. italica, and identified regions of differential single-nucleotide polymorphism density, distribution of transposable elements, small RNA content, chromosomal rearrangement and segregation distortion. The genus Setaria includes natural and cultivated species thatmore » demonstrate a wide capacity for adaptation. The genetic basis of this adaptation was investigated by comparing five sequenced grass genomes. We also used the diploid Setaria genome to evaluate the ongoing genome assembly of a related polyploid, switchgrass (Panicum virgatum).« less

Issues with RNA-seq analysis in non-model organisms: A salmonid example.

PubMed

Sundaram, Arvind; Tengs, Torstein; Grimholt, Unni

2017-10-01

High throughput sequencing (HTS) is useful for many purposes as exemplified by the other topics included in this special issue. The purpose of this paper is to look into the unique challenges of using this technology in non-model organisms where resources such as genomes, functional genome annotations or genome complexity provide obstacles not met in model organisms. To describe these challenges, we narrow our scope to RNA sequencing used to study differential gene expression in response to pathogen challenge. As a demonstration species we chose Atlantic salmon, which has a sequenced genome with poor annotation and an added complexity due to many duplicated genes. We find that our RNA-seq analysis pipeline deciphers between duplicates despite high sequence identity. However, annotation issues provide problems in linking differentially expressed genes to pathways. Also, comparing results between approaches and species are complicated due to lack of standardized annotation. Copyright © 2017 Elsevier Ltd. All rights reserved.

Combined hairpin-antisense compositions and methods for modulating expression

DOEpatents

Shanklin, John; Nguyen, Tam

2014-08-05

A nucleotide construct comprising a nucleotide sequence that forms a stem and a loop, wherein the loop comprises a nucleotide sequence that modulates expression of a target, wherein the stem comprises a nucleotide sequence that modulates expression of a target, and wherein the target modulated by the nucleotide sequence in the loop and the target modulated by the nucleotide sequence in the stem may be the same or different. Vectors, methods of regulating target expression, methods of providing a cell, and methods of treating conditions comprising the nucleotide sequence are also disclosed.

Combined hairpin-antisense compositions and methods for modulating expression

DOEpatents

Shanklin, John; Nguyen, Tam Huu

2015-11-24

A nucleotide construct comprising a nucleotide sequence that forms a stem and a loop, wherein the loop comprises a nucleotide sequence that modulates expression of a target, wherein the stem comprises a nucleotide sequence that modulates expression of a target, and wherein the target modulated by the nucleotide sequence in the loop and the target modulated by the nucleotide sequence in the stem may be the same or different. Vectors, methods of regulating target expression, methods of providing a cell, and methods of treating conditions comprising the nucleotide sequence are also disclosed.

Comparative transcriptome analysis of microsclerotia development in Nomuraea rileyi

PubMed Central

2013-01-01

Background Nomuraea rileyi is used as an environmental-friendly biopesticide. However, mass production and commercialization of this organism are limited due to its fastidious growth and sporulation requirements. When cultured in amended medium, we found that N. rileyi could produce microsclerotia bodies, replacing conidiophores as the infectious agent. However, little is known about the genes involved in microsclerotia development. In the present study, the transcriptomes were analyzed using next-generation sequencing technology to find the genes involved in microsclerotia development. Results A total of 4.69 Gb of clean nucleotides comprising 32,061 sequences was obtained, and 20,919 sequences were annotated (about 65%). Among the annotated sequences, only 5928 were annotated with 34 gene ontology (GO) functional categories, and 12,778 sequences were mapped to 165 pathways by searching against the Kyoto Encyclopedia of Genes and Genomes pathway (KEGG) database. Furthermore, we assessed the transcriptomic differences between cultures grown in minimal and amended medium. In total, 4808 sequences were found to be differentially expressed; 719 differentially expressed unigenes were assigned to 25 GO classes and 1888 differentially expressed unigenes were assigned to 161 KEGG pathways, including 25 enrichment pathways. Subsequently, we examined the up-regulation or uniquely expressed genes following amended medium treatment, which were also expressed on the enrichment pathway, and found that most of them participated in mediating oxidative stress homeostasis. To elucidate the role of oxidative stress in microsclerotia development, we analyzed the diversification of unigenes using quantitative reverse transcription-PCR (RT-qPCR). Conclusion Our findings suggest that oxidative stress occurs during microsclerotia development, along with a broad metabolic activity change. Our data provide the most comprehensive sequence resource available for the study of N. rileyi. We believe that the transcriptome datasets will serve as an important public information platform to accelerate studies on N. rileyi microsclerotia. PMID:23777366

In vitro manipulation of gene expression in larval Schistosoma: a model for postgenomic approaches in Trematoda

PubMed Central

YOSHINO, TIMOTHY P.; DINGUIRARD, NATHALIE; DE MORAES MOURÃO, MARINA

2013-01-01

SUMMARY With rapid developments in DNA and protein sequencing technologies, combined with powerful bioinformatics tools, a continued acceleration of gene identification in parasitic helminths is predicted, potentially leading to discovery of new drug and vaccine targets, enhanced diagnostics and insights into the complex biology underlying host-parasite interactions. For the schistosome blood flukes, with the recent completion of genome sequencing and comprehensive transcriptomic datasets, there has accumulated massive amounts of gene sequence data, for which, in the vast majority of cases, little is known about actual functions within the intact organism. In this review we attempt to bring together traditional in vitro cultivation approaches and recent emergent technologies of molecular genomics, transcriptomics and genetic manipulation to illustrate the considerable progress made in our understanding of trematode gene expression and function during development of the intramolluscan larval stages. Using several prominent trematode families (Schistosomatidae, Fasciolidae, Echinostomatidae), we have focused on the current status of in vitro larval isolation/cultivation as a source of valuable raw material supporting gene discovery efforts in model digeneans that include whole genome sequencing, transcript and protein expression profiling during larval development, and progress made in the in vitro manipulation of genes and their expression in larval trematodes using transgenic and RNA interference (RNAi) approaches. PMID:19961646

EST Express: PHP/MySQL based automated annotation of ESTs from expression libraries

PubMed Central

Smith, Robin P; Buchser, William J; Lemmon, Marcus B; Pardinas, Jose R; Bixby, John L; Lemmon, Vance P

2008-01-01

Background Several biological techniques result in the acquisition of functional sets of cDNAs that must be sequenced and analyzed. The emergence of redundant databases such as UniGene and centralized annotation engines such as Entrez Gene has allowed the development of software that can analyze a great number of sequences in a matter of seconds. Results We have developed "EST Express", a suite of analytical tools that identify and annotate ESTs originating from specific mRNA populations. The software consists of a user-friendly GUI powered by PHP and MySQL that allows for online collaboration between researchers and continuity with UniGene, Entrez Gene and RefSeq. Two key features of the software include a novel, simplified Entrez Gene parser and tools to manage cDNA library sequencing projects. We have tested the software on a large data set (2,016 samples) produced by subtractive hybridization. Conclusion EST Express is an open-source, cross-platform web server application that imports sequences from cDNA libraries, such as those generated through subtractive hybridization or yeast two-hybrid screens. It then provides several layers of annotation based on Entrez Gene and RefSeq to allow the user to highlight useful genes and manage cDNA library projects. PMID:18402700

EST Express: PHP/MySQL based automated annotation of ESTs from expression libraries.

PubMed

Smith, Robin P; Buchser, William J; Lemmon, Marcus B; Pardinas, Jose R; Bixby, John L; Lemmon, Vance P

2008-04-10

Several biological techniques result in the acquisition of functional sets of cDNAs that must be sequenced and analyzed. The emergence of redundant databases such as UniGene and centralized annotation engines such as Entrez Gene has allowed the development of software that can analyze a great number of sequences in a matter of seconds. We have developed "EST Express", a suite of analytical tools that identify and annotate ESTs originating from specific mRNA populations. The software consists of a user-friendly GUI powered by PHP and MySQL that allows for online collaboration between researchers and continuity with UniGene, Entrez Gene and RefSeq. Two key features of the software include a novel, simplified Entrez Gene parser and tools to manage cDNA library sequencing projects. We have tested the software on a large data set (2,016 samples) produced by subtractive hybridization. EST Express is an open-source, cross-platform web server application that imports sequences from cDNA libraries, such as those generated through subtractive hybridization or yeast two-hybrid screens. It then provides several layers of annotation based on Entrez Gene and RefSeq to allow the user to highlight useful genes and manage cDNA library projects.

RNA sequencing uncovers antisense RNAs and novel small RNAs in Streptococcus pyogenes.

PubMed

Le Rhun, Anaïs; Beer, Yan Yan; Reimegård, Johan; Chylinski, Krzysztof; Charpentier, Emmanuelle

2016-01-01

Streptococcus pyogenes is a human pathogen responsible for a wide spectrum of diseases ranging from mild to life-threatening infections. During the infectious process, the temporal and spatial expression of pathogenicity factors is tightly controlled by a complex network of protein and RNA regulators acting in response to various environmental signals. Here, we focus on the class of small RNA regulators (sRNAs) and present the first complete analysis of sRNA sequencing data in S. pyogenes. In the SF370 clinical isolate (M1 serotype), we identified 197 and 428 putative regulatory RNAs by visual inspection and bioinformatics screening of the sequencing data, respectively. Only 35 from the 197 candidates identified by visual screening were assigned a predicted function (T-boxes, ribosomal protein leaders, characterized riboswitches or sRNAs), indicating how little is known about sRNA regulation in S. pyogenes. By comparing our list of predicted sRNAs with previous S. pyogenes sRNA screens using bioinformatics or microarrays, 92 novel sRNAs were revealed, including antisense RNAs that are for the first time shown to be expressed in this pathogen. We experimentally validated the expression of 30 novel sRNAs and antisense RNAs. We show that the expression profile of 9 sRNAs including 2 predicted regulatory elements is affected by the endoribonucleases RNase III and/or RNase Y, highlighting the critical role of these enzymes in sRNA regulation.

A draft of the genome and four transcriptomes of a medicinal and pesticidal angiosperm Azadirachta indica

PubMed Central

2012-01-01

Background The Azadirachta indica (neem) tree is a source of a wide number of natural products, including the potent biopesticide azadirachtin. In spite of its widespread applications in agriculture and medicine, the molecular aspects of the biosynthesis of neem terpenoids remain largely unexplored. The current report describes the draft genome and four transcriptomes of A. indica and attempts to contextualise the sequence information in terms of its molecular phylogeny, transcript expression and terpenoid biosynthesis pathways. A. indica is the first member of the family Meliaceae to be sequenced using next generation sequencing approach. Results The genome and transcriptomes of A. indica were sequenced using multiple sequencing platforms and libraries. The A. indica genome is AT-rich, bears few repetitive DNA elements and comprises about 20,000 genes. The molecular phylogenetic analyses grouped A. indica together with Citrus sinensis from the Rutaceae family validating its conventional taxonomic classification. Comparative transcript expression analysis showed either exclusive or enhanced expression of known genes involved in neem terpenoid biosynthesis pathways compared to other sequenced angiosperms. Genome and transcriptome analyses in A. indica led to the identification of repeat elements, nucleotide composition and expression profiles of genes in various organs. Conclusions This study on A. indica genome and transcriptomes will provide a model for characterization of metabolic pathways involved in synthesis of bioactive compounds, comparative evolutionary studies among various Meliaceae family members and help annotate their genomes. A better understanding of molecular pathways involved in the azadirachtin synthesis in A. indica will pave ways for bulk production of environment friendly biopesticides. PMID:22958331

Identification of Abundantly Expressed Novel and Conserved Genes from the Infective Larval Stage of Toxocara canis by an Expressed Sequence Tag Strategy

PubMed Central

Tetteh, Kevin K. A.; Loukas, Alex; Tripp, Cindy; Maizels, Rick M.

1999-01-01

Larvae of Toxocara canis, a nematode parasite of dogs, infect humans, causing visceral and ocular larva migrans. In noncanid hosts, larvae neither grow nor differentiate but endure in a state of arrested development. Reasoning that parasite protein production is orientated to immune evasion, we undertook a random sequencing project from a larval cDNA library to characterize the most highly expressed transcripts. In all, 266 clones were sequenced, most from both 3′ and 5′ ends, and similarity searches against GenBank protein and dbEST nucleotide databases were conducted. Cluster analyses showed that 128 distinct gene products had been found, all but 3 of which represented newly identified genes. Ninety-five genes were represented by a single clone, but seven transcripts were present at high frequencies, each composing >2% of all clones sequenced. These high-abundance transcripts include a mucin and a C-type lectin, which are both major excretory-secretory antigens released by parasites. Four highly expressed novel gene transcripts, termed ant (abundant novel transcript) genes, were found. Together, these four genes comprised 18% of all cDNA clones isolated, but no similar sequences occur in the Caenorhabditis elegans genome. While the coding regions of the four genes are dissimilar, their 3′ untranslated tracts have significant homology in nucleotide sequence. The discovery of these abundant, parasite-specific genes of newly identified lectins and mucins, as well as a range of conserved and novel proteins, provides defined candidates for future analysis of the molecular basis of immune evasion by T. canis. PMID:10456930

Generation and analysis of expressed sequence tags from the bone marrow of Chinese Sika deer.

PubMed

Yao, Baojin; Zhao, Yu; Zhang, Mei; Li, Juan

2012-03-01

Sika deer is one of the best-known and highly valued animals of China. Despite its economic, cultural, and biological importance, there has not been a large-scale sequencing project for Sika deer to date. With the ultimate goal of sequencing the complete genome of this organism, we first established a bone marrow cDNA library for Sika deer and generated a total of 2,025 reads. After processing the sequences, 2,017 high-quality expressed sequence tags (ESTs) were obtained. These ESTs were assembled into 1,157 unigenes, including 238 contigs and 919 singletons. Comparative analyses indicated that 888 (76.75%) of the unigenes had significant matches to sequences in the non-redundant protein database, In addition to highly expressed genes, such as stearoyl-CoA desaturase, cytochrome c oxidase, adipocyte-type fatty acid-binding protein, adiponectin and thymosin beta-4, we also obtained vascular endothelial growth factor-A and heparin-binding growth-associated molecule, both of which are of great importance for angiogenesis research. There were 244 (21.09%) unigenes with no significant match to any sequence in current protein or nucleotide databases, and these sequences may represent genes with unknown function in Sika deer. Open reading frame analysis of the sequences was performed using the getorf program. In addition, the sequences were functionally classified using the gene ontology hierarchy, clusters of orthologous groups of proteins and Kyoto encyclopedia of genes and genomes databases. Analysis of ESTs described in this paper provides an important resource for the transcriptome exploration of Sika deer, and will also facilitate further studies on functional genomics, gene discovery and genome annotation of Sika deer.

Chromosomal arrangement of leghemoglobin genes in soybean.

PubMed Central

Lee, J S; Brown, G G; Verma, D P

1983-01-01

A cluster of four different leghemoglobin (Lb) genes was isolated from AluI-HaeIII and EcoRI genomic libraries of soybean in a set of overlapping clones which together include 45 kilobases (kb) of contiguous DNA. These four genes, including a pseudogene, are present in the same orientation and are arranged in the order: 5'-Lba-Lbc1-Lb psi-Lbc3-3'. The intergenic regions average 2.5 kb. In addition to this main Lb locus, there are other Lb genes which do not appear to be contiguous to this locus. A sequence probably common to the 3' region of Lb loci was found flanking the Lbc3 gene. The 3' flanking region of the main Lb locus also contains a sequence that appears to be expressed more abundantly in root tissue. Another sequence which is primarily expressed in root and leaf is found 5' to two Lb loci. Overall, the main leghemoglobin locus is similar in structure to the mammalian globin gene loci. Images PMID:6310504

NEIBank: Genomics and bioinformatics resources for vision research

PubMed Central

Peterson, Katherine; Gao, James; Buchoff, Patee; Jaworski, Cynthia; Bowes-Rickman, Catherine; Ebright, Jessica N.; Hauser, Michael A.; Hoover, David

2008-01-01

NEIBank is an integrated resource for genomics and bioinformatics in vision research. It includes expressed sequence tag (EST) data and sequence-verified cDNA clones for multiple eye tissues of several species, web-based access to human eye-specific SAGE data through EyeSAGE, and comprehensive, annotated databases of known human eye disease genes and candidate disease gene loci. All expression- and disease-related data are integrated in EyeBrowse, an eye-centric genome browser. NEIBank provides a comprehensive overview of current knowledge of the transcriptional repertoires of eye tissues and their relation to pathology. PMID:18648525

Production of foot-and-mouth disease virus capsid proteins by the TEV protease.

PubMed

Puckette, Michael; Smith, Justin D; Gabbert, Lindsay; Schutta, Christopher; Barrera, José; Clark, Benjamin A; Neilan, John G; Rasmussen, Max

2018-06-10

Protective immunity to viral pathogens often includes production of neutralizing antibodies to virus capsid proteins. Many viruses produce capsid proteins by expressing a precursor polyprotein and related protease from a single open reading frame. The foot-and-mouth disease virus (FMDV) expresses a 3C protease (3Cpro) that cleaves a P1 polyprotein intermediate into individual capsid proteins, but the FMDV 3Cpro also degrades many host cell proteins and reduces the viability of host cells, including subunit vaccine production cells. To overcome the limitations of using the a wild-type 3Cpro in FMDV subunit vaccine expression systems, we altered the protease restriction sequences within a FMDV P1 polyprotein to enable production of FMDV capsid proteins by the Tobacco Etch Virus NIa protease (TEVpro). Separate TEVpro and modified FMDV P1 proteins were produced from a single open reading frame by an intervening FMDV 2A sequence. The modified FMDV P1 polyprotein was successfully processed by the TEVpro in both mammalian and bacterial cells. More broadly, this method of polyprotein production and processing may be adapted to other recombinant expression systems, especially plant-based expression. Published by Elsevier B.V.

The gene for fibroblast activation protein {alpha} (FAP), a putative cell surface-bound serine protease expressed in cancer stroma and wound healing, maps to chromosome band 2q23

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

Mathew, S.; Murty, V.V.V.S.; Chaganti, R.S.K.

The human fibroblast activation protein {alpha} (FAP{alpha}) is an inducible cell surface glycoprotein of M{sub r} 95,000 recognized by a number of monoclonal antibodies (mAbs), including the prototype mAb F19. Immunohistochemical studies have shown that FAP{alpha} expression in vivo is tightly regulated, with transient expression in some fetal mesenchymal tissues but absence of expression in most normal adult tissues. Reexpression of FAP{alpha} is observed in the reactive stromal fibroblasts of several common types of epithelial cancers, including >90% of breast, colorectal, and lung carcinomas and healing wounds. Cloning and sequence analysis of an FAP{alpha}-specific cDNA has revealed that the moleculemore » is encoded by a novel gene, FAP, which shows sequence similarity to members of the serine protease family of integral membrane proteins, namely dipeptidyl peptidase IV (DPPIV, also known as lymphocyte activation antigen, CD26, or adenosine dearoinase binding protein) and DPPX, a DPPIV-related molecule of unknown function. 15 refs., 1 fig.« less

Discovery and profiling of novel and conserved microRNAs during flower development in Carya cathayensis via deep sequencing.

PubMed

Wang, Zheng Jia; Huang, Jian Qin; Huang, You Jun; Li, Zheng; Zheng, Bing Song

2012-08-01

Hickory (Carya cathayensis Sarg.) is an economically important woody plant in China, but its long juvenile phase delays yield. MicroRNAs (miRNAs) are critical regulators of genes and important for normal plant development and physiology, including flower development. We used Solexa technology to sequence two small RNA libraries from two floral differentiation stages in hickory to identify miRNAs related to flower development. We identified 39 conserved miRNA sequences from 114 loci belonging to 23 families as well as two novel and ten potential novel miRNAs belonging to nine families. Moreover, 35 conserved miRNA*s and two novel miRNA*s were detected. Twenty miRNA sequences from 49 loci belonging to 11 families were differentially expressed; all were up-regulated at the later stage of flower development in hickory. Quantitative real-time PCR of 12 conserved miRNA sequences, five novel miRNA families, and two novel miRNA*s validated that all were expressed during hickory flower development, and the expression patterns were similar to those detected with Solexa sequencing. Finally, a total of 146 targets of the novel and conserved miRNAs were predicted. This study identified a diverse set of miRNAs that were closely related to hickory flower development and that could help in plant floral induction.

Methods and materials relating to IMPDH and GMP production

DOEpatents

Collart, Frank R.; Huberman, Eliezer

1997-01-01

Disclosed are purified and isolated DNA sequences encoding eukaryotic proteins possessing biological properties of inosine 5'-monophosphate dehydrogenase ("IMPDH"). Illustratively, mammalian (e.g., human) IMPDH-encoding DNA sequences are useful in transformation or transfection of host cells for the large scale recombinant production of the enzymatically active expression products and/or products (e.g., GMP) resulting from IMPDH catalyzed synthesis in cells. Vectors including IMPDH-encoding DNA sequences are useful in gene amplification procedures. Recombinant proteins and synthetic peptides provided by the invention are useful as immunological reagents and in the preparation of antibodies (including polyclonal and monoclonal antibodies) for quantitative detection of IMPDH.

Use of a protein engineering strategy to overcome limitations in the production of "Difficult to Express" recombinant proteins.

PubMed

Hussain, Hirra; Fisher, David I; Abbott, W Mark; Roth, Robert G; Dickson, Alan J

2017-10-01

Certain recombinant proteins are deemed "difficult to express" in mammalian expression systems requiring significant cell and/or process engineering to abrogate expression bottlenecks. With increasing demand for the production of recombinant proteins in mammalian cells, low protein yields can have significant consequences for industrial processes. To investigate the molecular mechanisms that restrict expression of recombinant proteins, naturally secreted model proteins were analyzed from the tissue inhibitors of metalloproteinase (TIMP) protein family. In particular, TIMP-2 and TIMP-3 were subjected to detailed study. TIMP proteins share significant sequence homology (∼50% identity and ∼70% similarity in amino acid sequence). However, they show marked differences in secretion in mammalian expression systems despite this extensive sequence homology. Using these two proteins as models, this study characterized the molecular mechanisms responsible for poor recombinant protein production. Our results reveal that both TIMP-2 and TIMP-3 are detectable at mRNA and protein level within the cell but only TIMP-2 is secreted effectively into the extracellular medium. Analysis of protein localization and the nature of intracellular protein suggest TIMP-3 is severely limited in its post-translational processing. To overcome this challenge, modification of the TIMP-3 sequence to include a furin protease-cleavable pro-sequence resulted in secretion of the modified TIMP-3 protein, however, incomplete processing was observed. Based on the TIMP-3 data, the protein engineering approach was optimized and successfully applied in combination with cell engineering, the overexpression of furin, to another member of the TIMP protein family (the poorly expressed TIMP-4). Use of the described protein engineering strategy resulted in successful secretion of poorly (TIMP-4) and non-secreted (TIMP-3) targets, and presents a novel strategy to enhance the production of "difficult" recombinant targets. Biotechnol. Bioeng. 2017;114: 2348-2359. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

A molecular analysis of desiccation tolerance mechanisms in the anhydrobiotic nematode Panagrolaimus superbus using expressed sequenced tags

PubMed Central

2012-01-01

Background Some organisms can survive extreme desiccation by entering into a state of suspended animation known as anhydrobiosis. Panagrolaimus superbus is a free-living anhydrobiotic nematode that can survive rapid environmental desiccation. The mechanisms that P. superbus uses to combat the potentially lethal effects of cellular dehydration may include the constitutive and inducible expression of protective molecules, along with behavioural and/or morphological adaptations that slow the rate of cellular water loss. In addition, inducible repair and revival programmes may also be required for successful rehydration and recovery from anhydrobiosis. Results To identify constitutively expressed candidate anhydrobiotic genes we obtained 9,216 ESTs from an unstressed mixed stage population of P. superbus. We derived 4,009 unigenes from these ESTs. These unigene annotations and sequences can be accessed at http://www.nematodes.org/nembase4/species_info.php?species=PSC. We manually annotated a set of 187 constitutively expressed candidate anhydrobiotic genes from P. superbus. Notable among those is a putative lineage expansion of the lea (late embryogenesis abundant) gene family. The most abundantly expressed sequence was a member of the nematode specific sxp/ral-2 family that is highly expressed in parasitic nematodes and secreted onto the surface of the nematodes' cuticles. There were 2,059 novel unigenes (51.7% of the total), 149 of which are predicted to encode intrinsically disordered proteins lacking a fixed tertiary structure. One unigene may encode an exo-β-1,3-glucanase (GHF5 family), most similar to a sequence from Phytophthora infestans. GHF5 enzymes have been reported from several species of plant parasitic nematodes, with horizontal gene transfer (HGT) from bacteria proposed to explain their evolutionary origin. This P. superbus sequence represents another possible HGT event within the Nematoda. The expression of five of the 19 putative stress response genes tested was upregulated in response to desiccation. These were the antioxidants glutathione peroxidase, dj-1 and 1-Cys peroxiredoxin, an shsp sequence and an lea gene. Conclusions P. superbus appears to utilise a strategy of combined constitutive and inducible gene expression in preparation for entry into anhydrobiosis. The apparent lineage expansion of lea genes, together with their constitutive and inducible expression, suggests that LEA3 proteins are important components of the anhydrobiotic protection repertoire of P. superbus. PMID:22281184

BNU-LSVED: a multimodal spontaneous expression database in educational environment

NASA Astrophysics Data System (ADS)

Sun, Bo; Wei, Qinglan; He, Jun; Yu, Lejun; Zhu, Xiaoming

2016-09-01

In the field of pedagogy or educational psychology, emotions are treated as very important factors, which are closely associated with cognitive processes. Hence, it is meaningful for teachers to analyze students' emotions in classrooms, thus adjusting their teaching activities and improving students ' individual development. To provide a benchmark for different expression recognition algorithms, a large collection of training and test data in classroom environment has become an acute problem that needs to be resolved. In this paper, we present a multimodal spontaneous database in real learning environment. To collect the data, students watched seven kinds of teaching videos and were simultaneously filmed by a camera. Trained coders made one of the five learning expression labels for each image sequence extracted from the captured videos. This subset consists of 554 multimodal spontaneous expression image sequences (22,160 frames) recorded in real classrooms. There are four main advantages in this database. 1) Due to recorded in the real classroom environment, viewer's distance from the camera and the lighting of the database varies considerably between image sequences. 2) All the data presented are natural spontaneous responses to teaching videos. 3) The multimodal database also contains nonverbal behavior including eye movement, head posture and gestures to infer a student ' s affective state during the courses. 4) In the video sequences, there are different kinds of temporal activation patterns. In addition, we have demonstrated the labels for the image sequences are in high reliability through Cronbach's alpha method.

Insights into rubber biosynthesis from transcriptome analysis of Hevea brasiliensis latex.

PubMed

Chow, Keng-See; Wan, Kiew-Lian; Isa, Mohd Noor Mat; Bahari, Azlina; Tan, Siang-Hee; Harikrishna, K; Yeang, Hoong-Yeet

2007-01-01

Hevea brasiliensis is the most widely cultivated species for commercial production of natural rubber (cis-polyisoprene). In this study, 10,040 expressed sequence tags (ESTs) were generated from the latex of the rubber tree, which represents the cytoplasmic content of a single cell type, in order to analyse the latex transcription profile with emphasis on rubber biosynthesis-related genes. A total of 3,441 unique transcripts (UTs) were obtained after quality editing and assembly of EST sequences. Functional classification of UTs according to the Gene Ontology convention showed that 73.8% were related to genes of unknown function. Among highly expressed ESTs, a significant proportion encoded proteins related to rubber biosynthesis and stress or defence responses. Sequences encoding rubber particle membrane proteins (RPMPs) belonging to three protein families accounted for 12% of the ESTs. Characterization of these ESTs revealed nine RPMP variants (7.9-27 kDa) including the 14 kDa REF (rubber elongation factor) and 22 kDa SRPP (small rubber particle protein). The expression of multiple RPMP isoforms in latex was shown using antibodies against REF and SRPP. Both EST and quantitative reverse transcription-PCR (QRT-PCR) analyses demonstrated REF and SRPP to be the most abundant transcripts in latex. Besides rubber biosynthesis, comparative sequence analysis showed that the RPMPs are highly similar to sequences in the plant kingdom having stress-related functions. Implications of the RPMP function in cis-polyisoprene biosynthesis in the context of transcript abundance and differential gene expression are discussed.

Transcriptional insulation of the human keratin 18 gene in transgenic mice.

PubMed Central

Neznanov, N; Thorey, I S; Ceceña, G; Oshima, R G

1993-01-01

Expression of the 10-kb human keratin 18 (K18) gene in transgenic mice results in efficient and appropriate tissue-specific expression in a variety of internal epithelial organs, including liver, lung, intestine, kidney, and the ependymal epithelium of brain, but not in spleen, heart, or skeletal muscle. Expression at the RNA level is directly proportional to the number of integrated K18 transgenes. These results indicate that the K18 gene is able to insulate itself both from the commonly observed cis-acting effects of the sites of integration and from the potential complications of duplicated copies of the gene arranged in head-to-tail fashion. To begin to identify the K18 gene sequences responsible for this property of transcriptional insulation, additional transgenic mouse lines containing deletions of either the 5' or 3' distal end of the K18 gene have been characterized. Deletion of 1.5 kb of the distal 5' flanking sequence has no effect upon either the tissue specificity or the copy number-dependent behavior of the transgene. In contrast, deletion of the 3.5-kb 3' flanking sequence of the gene results in the loss of the copy number-dependent behavior of the gene in liver and intestine. However, expression in kidney, lung, and brain remains efficient and copy number dependent in these transgenic mice. Furthermore, herpes simplex virus thymidine kinase gene expression is copy number dependent in transgenic mice when the gene is located between the distal 5'- and 3'-flanking sequences of the K18 gene. Each adult transgenic male expressed the thymidine kinase gene in testes and brain and proportionally to the number of integrated transgenes. We conclude that the characteristic of copy number-dependent expression of the K18 gene is tissue specific because the sequence requirements for transcriptional insulation in adult liver and intestine are different from those for lung and kidney. In addition, the behavior of the transgenic thymidine kinase gene in testes and brain suggests that the property of transcriptional insulation of the K18 gene may be conferred by the distal flanking sequences of the K18 gene and, additionally, may function for other genes. Images PMID:7681143

Molecular Cloning and Expression of Three Polygalacturonase cDNAs from the Tarnished Plant Bug, Lygus lineolaris

PubMed Central

Allen, Margaret L.; Mertens, Jeffrey A.

2008-01-01

Three unique cDNAs encoding putative polygalacturonase enzymes were isolated from the tarnished plant bug, Lygus lineolaris (Palisot de Beauvois) (Hemiptera: Miridae). The three nucleotide sequences were dissimilar to one another, but the deduced amino acid sequences were similar to each other and to other polygalacturonases from insects, fungi, plants, and bacteria. Four conserved segments characteristic of polygalacturonases were present, but with some notable semiconservative substitutions. Two of four expected disulfide bridge—forming cysteine pairs were present. All three inferred protein translations included predicted signal sequences of 17 to 20 amino acids. Amplification of genomic DNA identified an intron in one of the genes, Llpg1, in the 5′ untranslated region. Semiquantitative RT-PCR revealed expression in all stages of the insect except the eggs. Expression in adults, male and female, was highly variable, indicating a family of highly inducible and diverse enzymes adapted to the generalist polyphagous nature of this important pest. PMID:20233096

Method of increasing conversion of a fatty acid to its corresponding dicarboxylic acid

DOEpatents

Craft, David L.; Wilson, C. Ron; Eirich, Dudley; Zhang, Yeyan

2004-09-14

A nucleic acid sequence including a CYP promoter operably linked to nucleic acid encoding a heterologous protein is provided to increase transcription of the nucleic acid. Expression vectors and host cells containing the nucleic acid sequence are also provided. The methods and compositions described herein are especially useful in the production of polycarboxylic acids by yeast cells.

Hybrid Sequencing of Full-Length cDNA Transcripts of Stems and Leaves in Dendrobium officinale

PubMed Central

He, Liu; Fu, Shuhua; Xu, Zhichao; Yan, Jun; Xu, Jiang; Zhou, Hong; Zhou, Jianguo; Chen, Xinlian; Li, Ying; Au, Kin Fai; Yao, Hui

2017-01-01

Dendrobium officinale is an extremely valuable orchid used in traditional Chinese medicine, so sought after that it has a higher market value than gold. Although the expression profiles of some genes involved in the polysaccharide synthesis have previously been investigated, little research has been carried out on their alternatively spliced isoforms in D. officinale. In addition, information regarding the translocation of sugars from leaves to stems in D. officinale also remains limited. We analyzed the polysaccharide content of D. officinale leaves and stems, and completed in-depth transcriptome sequencing of these two diverse tissue types using second-generation sequencing (SGS) and single-molecule real-time (SMRT) sequencing technology. The results of this study yielded a digital inventory of gene and mRNA isoform expressions. A comparative analysis of both transcriptomes uncovered a total of 1414 differentially expressed genes, including 844 that were up-regulated and 570 that were down-regulated in stems. Of these genes, one sugars will eventually be exported transporter (SWEET) and one sucrose transporter (SUT) are expressed to a greater extent in D. officinale stems than in leaves. Two glycosyltransferase (GT) and four cellulose synthase (Ces) genes undergo a distinct degree of alternative splicing. In the stems, the content of polysaccharides is twice as much as that in the leaves. The differentially expressed GT and transcription factor (TF) genes will be the focus of further study. The genes DoSWEET4 and DoSUT1 are significantly expressed in the stem, and are likely to be involved in sugar loading in the phloem. PMID:28981454

Sample sequencing of vascular plants demonstrates widespread conservation and divergence of microRNAs.

PubMed

Chávez Montes, Ricardo A; de Fátima Rosas-Cárdenas, Flor; De Paoli, Emanuele; Accerbi, Monica; Rymarquis, Linda A; Mahalingam, Gayathri; Marsch-Martínez, Nayelli; Meyers, Blake C; Green, Pamela J; de Folter, Stefan

2014-04-23

Small RNAs are pivotal regulators of gene expression that guide transcriptional and post-transcriptional silencing mechanisms in eukaryotes, including plants. Here we report a comprehensive atlas of sRNA and miRNA from 3 species of algae and 31 representative species across vascular plants, including non-model plants. We sequence and quantify sRNAs from 99 different tissues or treatments across species, resulting in a data set of over 132 million distinct sequences. Using miRBase mature sequences as a reference, we identify the miRNA sequences present in these libraries. We apply diverse profiling methods to examine critical sRNA and miRNA features, such as size distribution, tissue-specific regulation and sequence conservation between species, as well as to predict putative new miRNA sequences. We also develop database resources, computational analysis tools and a dedicated website, http://smallrna.udel.edu/. This study provides new insights on plant sRNAs and miRNAs, and a foundation for future studies.

Sequences Of Amino Acids For Human Serum Albumin

NASA Technical Reports Server (NTRS)

Carter, Daniel C.

1992-01-01

Sequences of amino acids defined for use in making polypeptides one-third to one-sixth as large as parent human serum albumin molecule. Smaller, chemically stable peptides have diverse applications including service as artificial human serum and as active components of biosensors and chromatographic matrices. In applications involving production of artificial sera from new sequences, little or no concern about viral contaminants. Smaller genetically engineered polypeptides more easily expressed and produced in large quantities, making commercial isolation and production more feasible and profitable.

Characterization of Toll-like receptor 3 gene in large yellow croaker, Pseudosciaena crocea.

PubMed

Huang, Xue-Na; Wang, Zhi-Yong; Yao, Cui-Luan

2011-07-01

Toll-like receptor 3 (TLR3) plays an important role in innate immune responses. In this report, the full-length cDNA sequence and genomic structure of Pseudosciaena crocea TLR3 (PcTLR3) were identified and characterized. The full-length cDNA of PcTLR3 was of 3384 bp, including a 5'-terminal untranslated region (UTR) of 65 bp, a 3'-terminal UTR of 589 bp and an open reading frame (ORF) of 2730 bp encoding a polypeptide of 909 amino acid residues. The full-length genome sequence of PcTLR3 was composed of 5721 nucleotides, including five exons and four introns. The putative PcTLR3 protein contained a signal peptide sequence, 16 leucine-rich repeat (LRR) motifs, a transmembrane region and a Toll/interleukin-1 receptor (TIR) domain. Quantitative real-time reverse transcription PCR analysis revealed a broad expression of PcTLR3 in most tissues, with the predominant expression in liver, then intestine, and the weakest expression in blood cells. The expression of PcTLR3 after injection with poly inosinic:cytidylic (I:C) and Vibrio parahemolyticus was tested in spleen, blood cells and liver. The results indicated that PcTLR3 transcripts could be induced in the three tissues by injection with poly I:C. The highest expression was in the blood cells with 43.5 times (at 6h) greater expression than in the control (p<0.05). In addition, after V. parahemolyticus challenge, a moderate up-regulation and down-regulation of PcTLR3 was found in blood cells and liver, respectively. Our results suggested that PcTLR3 might play an important role in fish's defense against both viral and bacterial infection. Copyright © 2011 Elsevier Ltd. All rights reserved.

Comparative Analysis of Expressed Genes from Cacao Meristems Infected by Moniliophthora perniciosa

PubMed Central

Gesteira, Abelmon S.; Micheli, Fabienne; Carels, Nicolas; Da Silva, Aline C.; Gramacho, Karina P.; Schuster, Ivan; Macêdo, Joci N.; Pereira, Gonçalo A. G.; Cascardo, Júlio C. M.

2007-01-01

Background and Aims Witches' broom disease is caused by the hemibiotrophic basidiomycete Moniliophthora perniciosa, and is one of the most important diseases of cacao in the western hemisphere. Because very little is known about the global process of such disease development, expressed sequence tags (ESTs) were used to identify genes expressed during the Theobroma cacao–Moniliophthora perniciosa interaction. Methods Two cDNA libraries corresponding to the resistant (RT) and susceptible (SP) cacao–M. perniciosa interactions were constructed from total RNA, using the DB SMART Creator cDNA library kit (Clontech). Clones were randomly selected, sequenced from the 5′ end and analysed using bioinformatics tools including in silico analysis of the differential gene expression. Key Results A total of 6884 ESTs were generated from the RT and SP cDNA libraries. These ESTs were composed of 2585 singlets and 341 contigs for a total of 2926 non-redundant sequences. The redundancy of the libraries was low and their specificity high when compared with the few other cacao libraries already published. Sequence analysis allowed the assignment of a putative functional category for 54 % of sequences, whereas approx. 22 % of sequences corresponded to unknown function and approx. 24 % of sequences did not show any significant similarity with other proteins present in the database. Despite the similar overall distribution of the sequences in functional categories between the two libraries, qualitative differences were observed. Genes involved during the defence response to pathogen infection or in programmed cell death were identified, such as pathogenesis related-proteins, trypsin inhibitor or oxalate oxidase, and some of them showed an in silico differential expression between the resistant and the susceptible interactions. Conclusions As far as is known this is the first EST resource from the cacao–M. perniciosa interaction and it is believed that it will provide a significant contribution to the understanding of the molecular mechanisms of the resistance and susceptibility of cacao to M. perniciosa, to develop strategies to control witches broom, and as a source of polymorphism for molecular marker development and marker-assisted selection. PMID:17557832

Activation of the mitogen-activated protein kinase pathway by bone sialoprotein regulates osteoblast differentiation.

PubMed

Gordon, Jonathan A R; Hunter, Graeme K; Goldberg, Harvey A

2009-01-01

Bone sialoprotein (BSP) is an abundant protein in the extracellular matrix of bone that has been suggested to have several different physiological functions, including the nucleation of hydroxyapatite (HA), promotion of cell attachment and binding of collagen. Studies in our lab have demonstrated that increased expression of BSP in osteoblast cells can increase expression of the osteoblast-related genes Runx2 and Osx as well as alkaline phosphatase and osteocalcin and increase matrix mineralization. To determine the molecular mechanisms responsible for the BSP-mediated increase in osteoblastic differentiation, several functional domain mutants of BSP were expressed in primary rat bone osteoblastic cells, including the contiguous glutamic acid sequences (polyGlu) and the arginine-glycine-aspartic acid (RGD) motif. Markers of osteoblast differentiation, including matrix mineralization and alkaline phosphatase staining, were increased in cells expressing BSP mutants of the polyGlu sequences but not in cells expressing RGD-mutated BSP. We also determined the dependence on integrin-associated pathways in promoting BSP-mediated differentiation responses in osteoblasts by demonstrating the activation of focal adhesion kinase, MAP kinase-associated proteins ERK1/2, ribosomal s6 kinase 2 and the AP-1 protein cFos. Thus, the mechanism regulating osteoblast differentiation by BSP was determined to be dependent on integrin-mediated intracellular signaling pathways. Copyright 2008 S. Karger AG, Basel.

Expressed sequence tag analysis of adult human lens for the NEIBank Project: over 2000 non-redundant transcripts, novel genes and splice variants.

PubMed

Wistow, Graeme; Bernstein, Steven L; Wyatt, M Keith; Behal, Amita; Touchman, Jeffrey W; Bouffard, Gerald; Smith, Don; Peterson, Katherine

2002-06-15

To explore the expression profile of the human lens and to provide a resource for microarray studies, expressed sequence tag (EST) analysis has been performed on cDNA libraries from adult lenses. A cDNA library was constructed from two adult (40 year old) human lenses. Over two thousand clones were sequenced from the unamplified, un-normalized library. The library was then normalized and a further 2200 sequences were obtained. All the data were analyzed using GRIST (GRouping and Identification of Sequence Tags), a procedure for gene identification and clustering. The lens library (by) contains a low percentage of non-mRNA contaminants and a high fraction (over 75%) of apparently full length cDNA clones. Approximately 2000 reads from the unamplified library yields 810 clusters, potentially representing individual genes expressed in the lens. After normalization, the content of crystallins and other abundant cDNAs is markedly reduced and a similar number of reads from this library (fs) yields 1455 unique groups of which only two thirds correspond to named genes in GenBank. Among the most abundant cDNAs is one for a novel gene related to glutamine synthetase, which was designated "lengsin" (LGS). Analyses of ESTs also reveal examples of alternative transcripts, including a major alternative splice form for the lens specific membrane protein MP19. Variant forms for other transcripts, including those encoding the apoptosis inhibitor Livin and the armadillo repeat protein ARVCF, are also described. The lens cDNA libraries are a resource for gene discovery, full length cDNAs for functional studies and microarrays. The discovery of an abundant, novel transcript, lengsin, and a major novel splice form of MP19 reflect the utility of unamplified libraries constructed from dissected tissue. Many novel transcripts and splice forms are represented, some of which may be candidates for genetic diseases.

Identification of the Regulator Gene Responsible for the Acetone-Responsive Expression of the Binuclear Iron Monooxygenase Gene Cluster in Mycobacteria ▿

PubMed Central

Furuya, Toshiki; Hirose, Satomi; Semba, Hisashi; Kino, Kuniki

2011-01-01

The mimABCD gene cluster encodes the binuclear iron monooxygenase that oxidizes propane and phenol in Mycobacterium smegmatis strain MC2 155 and Mycobacterium goodii strain 12523. Interestingly, expression of the mimABCD gene cluster is induced by acetone. In this study, we investigated the regulator gene responsible for this acetone-responsive expression. In the genome sequence of M. smegmatis strain MC2 155, the mimABCD gene cluster is preceded by a gene designated mimR, which is divergently transcribed. Sequence analysis revealed that MimR exhibits amino acid similarity with the NtrC family of transcriptional activators, including AcxR and AcoR, which are involved in acetone and acetoin metabolism, respectively. Unexpectedly, many homologs of the mimR gene were also found in the sequenced genomes of actinomycetes. A plasmid carrying a transcriptional fusion of the intergenic region between the mimR and mimA genes with a promoterless green fluorescent protein (GFP) gene was constructed and introduced into M. smegmatis strain MC2 155. Using a GFP reporter system, we confirmed by deletion and complementation analyses that the mimR gene product is the positive regulator of the mimABCD gene cluster expression that is responsive to acetone. M. goodii strain 12523 also utilized the same regulatory system as M. smegmatis strain MC2 155. Although transcriptional activators of the NtrC family generally control transcription using the σ54 factor, a gene encoding the σ54 factor was absent from the genome sequence of M. smegmatis strain MC2 155. These results suggest the presence of a novel regulatory system in actinomycetes, including mycobacteria. PMID:21856847

Grouping of multicopper oxidases in Lentinula edodes by sequence similarities and expression patterns.

PubMed

Sakamoto, Yuichi; Nakade, Keiko; Yoshida, Kentaro; Natsume, Satoshi; Miyazaki, Kazuhiro; Sato, Shiho; van Peer, Arend F; Konno, Naotake

2015-12-01

The edible white rot fungus Lentinula edodes possesses a variety of lignin degrading enzymes such as manganese peroxidases and laccases. Laccases belong to the multicopper oxidases, which have a wide range of catalytic activities including polyphenol degradation and synthesis, lignin degradation, and melanin formation. The exact number of laccases in L. edodes is unknown, as are their complete properties and biological functions. We analyzed the draft genome sequence of L. edodes D703PP-9 and identified 13 multicopper oxidase-encoding genes; 11 laccases in sensu stricto, of which three are new, and two ferroxidases. lcc8, a laccase previously reported in L. edodes, was not identified in D703PP-9 genome. Phylogenetic analysis showed that the 13 multicopper oxidases can be classified into laccase sensu stricto subfamily 1, laccase sensu stricto subfamily 2 and ferroxidases. From sequence similarities and expression patterns, laccase sensu stricto subfamily 1 can be divided into two subgroups. Laccase sensu stricto subfamily 1 group A members are mainly secreted from mycelia, while laccase sensu stricto subfamily 1 group B members are expressed mainly in fruiting bodies during growth or after harvesting but are lowly expressed in mycelia. Laccase sensu stricto subfamily 2 members are mainly expressed in mycelia, and two ferroxidases are mainly expressed in the fruiting body during growth or after harvesting, and are expressed at very low levels in mycelium. Our data suggests that L. edodes laccases in same group share expression patterns and would have common biological functions.

Clinical polyomavirus BK variants with agnogene deletion are non-functional but rescued by trans-complementation

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

Myhre, Marit Renee; Olsen, Gunn-Hege; Gosert, Rainer

High-level replication of polyomavirus BK (BKV) in kidney transplant recipients is associated with the emergence of BKV variants with rearranged (rr) non-coding control region (NCCR) increasing viral early gene expression and cytopathology. Cloning and sequencing revealed the presence of a BKV quasispecies which included non-functional variants when assayed in a recombinant virus assay. Here we report that the rr-NCCR of BKV variants RH-3 and RH-12, both bearing a NCCR deletion including the 5' end of the agnoprotein coding sequence, mediated early and late viral reporter gene expression in kidney cells. However, in a recombinant virus they failed to produce infectiousmore » progeny despite large T-antigen and VP1 expression and the formation of nuclear virus-like particles. Infectious progeny was generated when the agnogene was reconstructed in cis or agnoprotein provided in trans from a co-existing BKV rr-NCCR variant. We conclude that complementation can rescue non-functional BKV variants in vitro and possibly in vivo.« less

RNA-seq Data: Challenges in and Recommendations for Experimental Design and Analysis.

PubMed

Williams, Alexander G; Thomas, Sean; Wyman, Stacia K; Holloway, Alisha K

2014-10-01

RNA-seq is widely used to determine differential expression of genes or transcripts as well as identify novel transcripts, identify allele-specific expression, and precisely measure translation of transcripts. Thoughtful experimental design and choice of analysis tools are critical to ensure high-quality data and interpretable results. Important considerations for experimental design include number of replicates, whether to collect paired-end or single-end reads, sequence length, and sequencing depth. Common analysis steps in all RNA-seq experiments include quality control, read alignment, assigning reads to genes or transcripts, and estimating gene or transcript abundance. Our aims are two-fold: to make recommendations for common components of experimental design and assess tool capabilities for each of these steps. We also test tools designed to detect differential expression, since this is the most widespread application of RNA-seq. We hope that these analyses will help guide those who are new to RNA-seq and will generate discussion about remaining needs for tool improvement and development. Copyright © 2014 John Wiley & Sons, Inc.

Genome-wide identification, classification, and expression analysis of the arabinogalactan protein gene family in rice (Oryza sativa L.)

PubMed Central

Zhao, Jie

2010-01-01

Arabinogalactan proteins (AGPs) comprise a family of hydroxyproline-rich glycoproteins that are implicated in plant growth and development. In this study, 69 AGPs are identified from the rice genome, including 13 classical AGPs, 15 arabinogalactan (AG) peptides, three non-classical AGPs, three early nodulin-like AGPs (eNod-like AGPs), eight non-specific lipid transfer protein-like AGPs (nsLTP-like AGPs), and 27 fasciclin-like AGPs (FLAs). The results from expressed sequence tags, microarrays, and massively parallel signature sequencing tags are used to analyse the expression of AGP-encoding genes, which is confirmed by real-time PCR. The results reveal that several rice AGP-encoding genes are predominantly expressed in anthers and display differential expression patterns in response to abscisic acid, gibberellic acid, and abiotic stresses. Based on the results obtained from this analysis, an attempt has been made to link the protein structures and expression patterns of rice AGP-encoding genes to their functions. Taken together, the genome-wide identification and expression analysis of the rice AGP gene family might facilitate further functional studies of rice AGPs. PMID:20423940

Integrating transcriptome and genome re-sequencing data to identify key genes and mutations affecting chicken eggshell qualities.

PubMed

Zhang, Quan; Zhu, Feng; Liu, Long; Zheng, Chuan Wei; Wang, De He; Hou, Zhuo Cheng; Ning, Zhong Hua

2015-01-01

Eggshell damages lead to economic losses in the egg production industry and are a threat to human health. We examined 49-wk-old Rhode Island White hens (Gallus gallus) that laid eggs having shells with significantly different strengths and thicknesses. We used HiSeq 2000 (Illumina) sequencing to characterize the chicken transcriptome and whole genome to identify the key genes and genetic mutations associated with eggshell calcification. We identified a total of 14,234 genes expressed in the chicken uterus, representing 89% of all annotated chicken genes. A total of 889 differentially expressed genes were identified by comparing low eggshell strength (LES) and normal eggshell strength (NES) genomes. The DEGs are enriched in calcification-related processes, including calcium ion transport and calcium signaling pathways as revealed by gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis. Some important matrix proteins, such as OC-116, LTF and SPP1, were also expressed differentially between two groups. A total of 3,671,919 single-nucleotide polymorphisms (SNPs) and 508,035 Indels were detected in protein coding genes by whole-genome re-sequencing, including 1775 non-synonymous variations and 19 frame-shift Indels in DEGs. SNPs and Indels found in this study could be further investigated for eggshell traits. This is the first report to integrate the transcriptome and genome re-sequencing to target the genetic variations which decreased the eggshell qualities. These findings further advance our understanding of eggshell calcification in the chicken uterus.

The Vigna unguiculata Gene Expression Atlas (VuGEA) from de novo assembly and quantification of RNA-seq data provides insights into seed maturation mechanisms.

PubMed

Yao, Shaolun; Jiang, Chuan; Huang, Ziyue; Torres-Jerez, Ivone; Chang, Junil; Zhang, Heng; Udvardi, Michael; Liu, Renyi; Verdier, Jerome

2016-10-01

Legume research and cultivar development are important for sustainable food production, especially of high-protein seed. Thanks to the development of deep-sequencing technologies, crop species have been taken to the front line, even without completion of their genome sequences. Black-eyed pea (Vigna unguiculata) is a legume species widely grown in semi-arid regions, which has high potential to provide stable seed protein production in a broad range of environments, including drought conditions. The black-eyed pea reference genotype has been used to generate a gene expression atlas of the major plant tissues (i.e. leaf, root, stem, flower, pod and seed), with a developmental time series for pods and seeds. From these various organs, 27 cDNA libraries were generated and sequenced, resulting in more than one billion reads. Following filtering, these reads were de novo assembled into 36 529 transcript sequences that were annotated and quantified across the different tissues. A set of 24 866 unique transcript sequences, called Unigenes, was identified. All the information related to transcript identification, annotation and quantification were stored into a gene expression atlas webserver (http://vugea.noble.org), providing a user-friendly interface and necessary tools to analyse transcript expression in black-eyed pea organs and to compare data with other legume species. Using this gene expression atlas, we inferred details of molecular processes that are active during seed development, and identified key putative regulators of seed maturation. Additionally, we found evidence for conservation of regulatory mechanisms involving miRNA in plant tissues subjected to drought and seeds undergoing desiccation. © 2016 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.

Construction of a cDNA library from female adult of Toxocara canis, and analysis of EST and immune-related genes expressions.

PubMed

Zhou, Rongqiong; Xia, Qingyou; Huang, Hancheng; Lai, Min; Wang, Zhenxin

2011-10-01

Toxocara canis is a widespread intestinal nematode parasite of dogs, which can also cause disease in humans. We employed an expressed sequence tag (EST) strategy in order to study gene-expression including development, digestion and reproduction of T. canis. ESTs provided a rapid way to identify genes, particularly in organisms for which we have very little molecular information. In this study, a cDNA library was constructed from a female adult of T. canis and 215 high-quality ESTs from 5'-ends of the cDNA clones representing 79 unigenes were obtained. The titer of the primary cDNA library was 1.83×10(6)pfu/mL with a recombination rate of 99.33%. Most of the sequences ranged from 300 to 900bp with an average length of 656bp. Cluster analysis of these ESTs allowed identification of 79 unique sequences containing 28 contigs and 51 singletons. BLASTX searches revealed that 18 unigenes (22.78% of the total) or 70 ESTs (32.56% of the total) were novel genes that had no significant matches to any protein sequences in the public databases. The rest of the 61 unigenes (77.22% of the total) or 145 ESTs (67.44% of the total) were closely matched to the known genes or sequences deposited in the public databases. These genes were classified into seven groups based on their known or putative biological functions. We also confirmed the gene expression patterns of several immune-related genes using RT-PCR examination. This work will provide a valuable resource for the further investigations in the stage-, sex- and tissue-specific gene transcription or expression. Copyright © 2011. Published by Elsevier Inc.

Identification of expressed resistance gene analogs (RGA.) from peanut expressed sequence tage (EST.) and development of RGA-SSR markers

USDA-ARS?s Scientific Manuscript database

Cultivated peanut (Arachis hypogaea L.) is an important food and oil crop grown in more than 100 countries for providing edible oil and protein. A wide variety of pathogens including fungi, bacteria, viruses, and nematodes severely constrain peanut yield and quality. Therefore, it is very imp...

Expression of the adhesion G protein-coupled receptor A2 (adgra2) during Xenopus laevis development.

PubMed

Seigfried, Franziska A; Dietmann, Petra; Kühl, Michael; Kühl, Susanne J

2018-06-01

The adhesion G protein-coupled receptor A2 (Adgra2) is a seven transmembrane receptor that has been described to be a regulator for angiogenesis in mice. Furthermore, the zebrafish ouchless mutant is unable to develop dorsal root ganglia through a disrupted trafficking of Adgra2. Besides RNA sequencing data, nothing is reported about Adgra2 in the south African crawled frog Xenopus laevis. In this study, we investigated for the first time the spatio-temporal expression of adgra2 during early Xenopus embryogenesis in detail. In silico approaches showed that the genomic adgra2 region as well as the Adgra2 protein sequence is highly conserved among different species including Xenopus. RT-PCR experiments confirmed that embryonic adgra2 expression is primarily detected at the beginning of neurulation and is then present throughout the whole Xenopus embryogenesis until stage 42. Whole mount in situ hybridization approaches visualized adgra2 expression in many tissues during Xenopus embryogenesis such as the cardiovascular system including the heart, the migrating neural crest cells and the developing eye including the periocular mesenchyme. Our results indicate a role of Adgra2 for embryogenesis and are a good starting point for further functional studies during early vertebrate development. Copyright © 2018 Elsevier B.V. All rights reserved.

Intrinsic and extrinsic approaches for detecting genes in a bacterial genome.

PubMed Central

Borodovsky, M; Rudd, K E; Koonin, E V

1994-01-01

The unannotated regions of the Escherichia coli genome DNA sequence from the EcoSeq6 database, totaling 1,278 'intergenic' sequences of the combined length of 359,279 basepairs, were analyzed using computer-assisted methods with the aim of identifying putative unknown genes. The proposed strategy for finding new genes includes two key elements: i) prediction of expressed open reading frames (ORFs) using the GeneMark method based on Markov chain models for coding and non-coding regions of Escherichia coli DNA, and ii) search for protein sequence similarities using programs based on the BLAST algorithm and programs for motif identification. A total of 354 putative expressed ORFs were predicted by GeneMark. Using the BLASTX and TBLASTN programs, it was shown that 208 ORFs located in the unannotated regions of the E. coli chromosome are significantly similar to other protein sequences. Identification of 182 ORFs as probable genes was supported by GeneMark and BLAST, comprising 51.4% of the GeneMark 'hits' and 87.5% of the BLAST 'hits'. 73 putative new genes, comprising 20.6% of the GeneMark predictions, belong to ancient conserved protein families that include both eubacterial and eukaryotic members. This value is close to the overall proportion of highly conserved sequences among eubacterial proteins, indicating that the majority of the putative expressed ORFs that are predicted by GeneMark, but have no significant BLAST hits, nevertheless are likely to be real genes. The majority of the putative genes identified by BLAST search have been described since the release of the EcoSeq6 database, but about 70 genes have not been detected so far. Among these new identifications are genes encoding proteins with a variety of predicted functions including dehydrogenases, kinases, several other metabolic enzymes, ATPases, rRNA methyltransferases, membrane proteins, and different types of regulatory proteins. Images PMID:7984428

Global Transcriptome Analysis of the Tentacle of the Jellyfish Cyanea capillata Using Deep Sequencing and Expressed Sequence Tags: Insight into the Toxin- and Degenerative Disease-Related Transcripts

PubMed Central

Liu, Dan; Wang, Qianqian; Ruan, Zengliang; He, Qian; Zhang, Liming

2015-01-01

Background Jellyfish contain diverse toxins and other bioactive components. However, large-scale identification of novel toxins and bioactive components from jellyfish has been hampered by the low efficiency of traditional isolation and purification methods. Results We performed de novo transcriptome sequencing of the tentacle tissue of the jellyfish Cyanea capillata. A total of 51,304,108 reads were obtained and assembled into 50,536 unigenes. Of these, 21,357 unigenes had homologues in public databases, but the remaining unigenes had no significant matches due to the limited sequence information available and species-specific novel sequences. Functional annotation of the unigenes also revealed general gene expression profile characteristics in the tentacle of C. capillata. A primary goal of this study was to identify putative toxin transcripts. As expected, we screened many transcripts encoding proteins similar to several well-known toxin families including phospholipases, metalloproteases, serine proteases and serine protease inhibitors. In addition, some transcripts also resembled molecules with potential toxic activities, including cnidarian CfTX-like toxins with hemolytic activity, plancitoxin-1, venom toxin-like peptide-6, histamine-releasing factor, neprilysin, dipeptidyl peptidase 4, vascular endothelial growth factor A, angiotensin-converting enzyme-like and endothelin-converting enzyme 1-like proteins. Most of these molecules have not been previously reported in jellyfish. Interestingly, we also characterized a number of transcripts with similarities to proteins relevant to several degenerative diseases, including Huntington’s, Alzheimer’s and Parkinson’s diseases. This is the first description of degenerative disease-associated genes in jellyfish. Conclusion We obtained a well-categorized and annotated transcriptome of C. capillata tentacle that will be an important and valuable resource for further understanding of jellyfish at the molecular level and information on the underlying molecular mechanisms of jellyfish stinging. The findings of this study may also be used in comparative studies of gene expression profiling among different jellyfish species. PMID:26551022

Global Transcriptome Analysis of the Tentacle of the Jellyfish Cyanea capillata Using Deep Sequencing and Expressed Sequence Tags: Insight into the Toxin- and Degenerative Disease-Related Transcripts.

PubMed

Liu, Guoyan; Zhou, Yonghong; Liu, Dan; Wang, Qianqian; Ruan, Zengliang; He, Qian; Zhang, Liming

2015-01-01

Jellyfish contain diverse toxins and other bioactive components. However, large-scale identification of novel toxins and bioactive components from jellyfish has been hampered by the low efficiency of traditional isolation and purification methods. We performed de novo transcriptome sequencing of the tentacle tissue of the jellyfish Cyanea capillata. A total of 51,304,108 reads were obtained and assembled into 50,536 unigenes. Of these, 21,357 unigenes had homologues in public databases, but the remaining unigenes had no significant matches due to the limited sequence information available and species-specific novel sequences. Functional annotation of the unigenes also revealed general gene expression profile characteristics in the tentacle of C. capillata. A primary goal of this study was to identify putative toxin transcripts. As expected, we screened many transcripts encoding proteins similar to several well-known toxin families including phospholipases, metalloproteases, serine proteases and serine protease inhibitors. In addition, some transcripts also resembled molecules with potential toxic activities, including cnidarian CfTX-like toxins with hemolytic activity, plancitoxin-1, venom toxin-like peptide-6, histamine-releasing factor, neprilysin, dipeptidyl peptidase 4, vascular endothelial growth factor A, angiotensin-converting enzyme-like and endothelin-converting enzyme 1-like proteins. Most of these molecules have not been previously reported in jellyfish. Interestingly, we also characterized a number of transcripts with similarities to proteins relevant to several degenerative diseases, including Huntington's, Alzheimer's and Parkinson's diseases. This is the first description of degenerative disease-associated genes in jellyfish. We obtained a well-categorized and annotated transcriptome of C. capillata tentacle that will be an important and valuable resource for further understanding of jellyfish at the molecular level and information on the underlying molecular mechanisms of jellyfish stinging. The findings of this study may also be used in comparative studies of gene expression profiling among different jellyfish species.

First comparative characterization of three distinct ferritin subunits from a teleost: Evidence for immune-responsive mRNA expression and iron depriving activity of seahorse (Hippocampus abdominalis) ferritins.

PubMed

Oh, Minyoung; Umasuthan, Navaneethaiyer; Elvitigala, Don Anushka Sandaruwan; Wan, Qiang; Jo, Eunyoung; Ko, Jiyeon; Noh, Gyeong Eon; Shin, Sangok; Rho, Sum; Lee, Jehee

2016-02-01

Ferritins play an indispensable role in iron homeostasis through their iron-withholding function in living beings. In the current study, cDNA sequences of three distinct ferritin subunits, including a ferritin H, a ferritin M, and a ferritin L, were identified from big belly seahorse, Hippocampus abdominalis, and molecularly characterized. Complete coding sequences (CDS) of seahorse ferritin H (HaFerH), ferritin M (HaFerM), and ferritin L (HaFerL) subunits were comprised of 531, 528, and 522 base pairs (bp), respectively, which encode polypeptides of 177, 176, and 174 amino acids, respectively, with molecular masses of ∼20-21 kDa. Our in silico analyses demonstrate that these three ferritin subunits exhibit the typical characteristics of ferritin superfamily members including iron regulatory elements, domain signatures, and reactive centers. The coding sequences of HaFerH, M, and L were cloned and the corresponding proteins were overexpressed in a bacterial system. Recombinantly expressed HaFer proteins demonstrated detectable in vivo iron sequestrating (ferroxidase) activity, consistent with their putative iron binding capability. Quantification of the basal expression of these three HaFer sequences in selected tissues demonstrated a gene-specific ubiquitous spatial distribution pattern, with abundance of mRNA in HaFerM in the liver and predominant expression of HaFerH and HaFerL in blood. Interestingly, the basal expression of all three ferritin genes was found to be significantly modulated against pathogenic stress mounted by lipopolysaccharides (LPS), poly I:C, Streptococcus iniae, and Edwardsiella tarda. Collectively, our findings suggest that the three HaFer subunits may be involved in iron (II) homeostasis in big belly seahorse and that they are important in its host defense mechanisms. Copyright © 2016 Elsevier Ltd. All rights reserved.

The glucose transporter 1 -GLUT1- from the white shrimp Litopenaeus vannamei is up-regulated during hypoxia.

PubMed

Martínez-Quintana, José A; Peregrino-Uriarte, Alma B; Gollas-Galván, Teresa; Gómez-Jiménez, Silvia; Yepiz-Plascencia, Gloria

2014-12-01

During hypoxia the shrimp Litopenaeus vannamei accelerates anaerobic glycolysis to obtain energy; therefore, a correct supply of glucose to the cells is needed. Facilitated glucose transport across the cells is mediated by a group of membrane embedded integral proteins called GLUT; being GLUT1 the most ubiquitous form. In this work, we report the first cDNA nucleotide and deduced amino acid sequences of a glucose transporter 1 from L. vannamei. A 1619 bp sequence was obtained by RT-PCR and RACE approaches. The 5´ UTR is 161 bp and the poly A tail is exactly after the stop codon in the mRNA. The ORF is 1485 bp and codes for 485 amino acids. The deduced protein sequence has high identity to GLUT1 proteins from several species and contains all the main features of glucose transporter proteins, including twelve transmembrane domains, the conserved motives and amino acids involved in transport activity, ligands binding and membrane anchor. Therefore, we decided to name this sequence, glucose transporter 1 of L. vannamei (LvGLUT1). A partial gene sequence of 8.87 Kbp was also obtained; it contains the complete coding sequence divided in 10 exons. LvGlut1 expression was detected in hemocytes, hepatopancreas, intestine gills, muscle and pleopods. The higher relative expression was found in gills and the lower in hemocytes. This indicates that LvGlut1 is ubiquitously expressed but its levels are tissue-specific and upon short-term hypoxia, the GLUT1 transcripts increase 3.7-fold in hepatopancreas and gills. To our knowledge, this is the first evidence of expression of GLUT1 in crustaceans.

Cloning of human prourokinase cDNA without the signal peptide and expression in Escherichia coli.

PubMed

Hu, B; Li, J; Yu, W; Fang, J

1993-01-01

Human prourokinase (pro-UK) cDNA without the signal peptide was obtained using synthetic oligonucleotide and DNA recombination techniques and was successfully expressed in E. coli. The plasmid pMMUK which contained pro-UK cDNA (including both the entire coding sequence and the sequence for signal peptide) was digested with Hind III and PstI, so that the N-terminal 371-bp fragment could be recovered. A 304-bp fragment was collected from the 371-bp fragment after partial digestion with Fnu4HI in order to remove the signal peptide sequence. An intermediate plasmid was formed after this 304-bp fragment and the synthetic oligonucleotide was ligated with pUC18. Correctness of the ligation was confirmed by enzyme digestion and sequencing. By joining the PstI-PstI fragment of pro-UK to the plasmid we obtained the final plasmid which contained the entire coding sequence of pro-UK without the signal peptide. The coding sequence with correct orientation was inserted into pBV220 under the control of the temperature-induced promoter PRPL, and mature pro-UK was expressed in E. coli at 42 degrees C. Both sonicated supernatant and inclusion bodies of the bacterial host JM101 showed positive results by ELISA and FAPA assays. After renaturation, the biological activity of the expressed product was increased from 500-1000IU/L to about 60,000IU/L. The bacterial pro-UK showed a molecular weight of about 47,000 daltons by Western blot analysis. It can be completely inhibited by UK antiserum but not by t-PA antiserum nor by normal rabbit serum.

Orthologs in Arabidopsis thaliana of the Hsp70 interacting protein Hip

PubMed Central

Webb, Mary Alice; Cavaletto, John M.; Klanrit, Preekamol; Thompson, Gary A.

2001-01-01

The Hsp70-interacting protein Hip binds to the adenosine triphosphatase domain of Hsp70, stabilizing it in the adenosine 5′-diphosphate–ligated conformation and promoting binding of target polypeptides. In mammalian cells, Hip is a component of the cytoplasmic chaperone heterocomplex that regulates signal transduction via interaction with hormone receptors and protein kinases. Analysis of the complete genome sequence of the model flowering plant Arabidopsis thaliana revealed 2 genes encoding Hip orthologs. The deduced sequence of AtHip-1 consists of 441 amino acid residues and is 42% identical to human Hip. AtHip-1 contains the same functional domains characterized in mammalian Hip, including an N-terminal dimerization domain, an acidic domain, 3 tetratricopeptide repeats flanked by a highly charged region, a series of degenerate GGMP repeats, and a C-terminal region similar to the Sti1/Hop/p60 protein. The deduced amino acid sequence of AtHip-2 consists of 380 amino acid residues. AtHip-2 consists of a truncated Hip-like domain that is 46% identical to human Hip, followed by a C-terminal domain related to thioredoxin. AtHip-2 is 63% identical to another Hip-thioredoxin protein recently identified in Vitis labrusca (grape). The truncated Hip domain in AtHip-2 includes the amino terminus, the acidic domain, and tetratricopeptide repeats with flanking charged region. Analyses of expressed sequence tag databases indicate that both AtHip-1 and AtHip-2 are expressed in A thaliana and that orthologs of Hip are also expressed widely in other plants. The similarity between AtHip-1 and its mammalian orthologs is consistent with a similar role in plant cells. The sequence of AtHip-2 suggests the possibility of additional unique chaperone functions. PMID:11599566

Identification, Characterization, and Recombinant Expression of Epidermicin NI01, a Novel Unmodified Bacteriocin Produced by Staphylococcus epidermidis That Displays Potent Activity against Staphylococci

PubMed Central

Sandiford, Stephanie

2012-01-01

We describe the discovery, purification, characterization, and expression of an antimicrobial peptide, epidermicin NI01, which is an unmodified bacteriocin produced by Staphylococcus epidermidis strain 224. It is a highly cationic, hydrophobic, plasmid-encoded peptide that exhibits potent antimicrobial activity toward a wide range of pathogenic Gram-positive bacteria including methicillin-resistant Staphylococcus aureus (MRSA), enterococci, and biofilm-forming S. epidermidis strains. Purification of the peptide was achieved using a combination of hydrophobic interaction, cation exchange, and high-performance liquid chromatography (HPLC). Matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) analysis yielded a molecular mass of 6,074 Da, and partial sequence data of the peptide were elucidated using a combination of tandem mass spectrometry (MS/MS) and de novo sequencing. The draft genome sequence of the producing strain was obtained using 454 pyrosequencing technology, thus enabling the identification of the structural gene using the de novo peptide sequence data previously obtained. Epidermicin NI01 contains 51 residues with four tryptophan and nine lysine residues, and the sequence showed approximately 50% identity to peptides lacticin Z, lacticin Q, and aureocin A53, all of which belong to a new family of unmodified type II-like bacteriocins. The peptide is active in the nanomolar range against S. epidermidis, MRSA isolates, and vancomycin-resistant enterococci. Other unique features displayed by epidermicin include a high degree of protease stability and the ability to retain antimicrobial activity over a pH range of 2 to 10, and exposure to the peptide does not result in development of resistance in susceptible isolates. In this study we also show the structural gene alone can be cloned into Escherichia coli strain BL21(DE3), and expression yields active peptide. PMID:22155816

miR-ID: A novel, circularization-based platform for detection of microRNAs

PubMed Central

Kumar, Pavan; Johnston, Brian H.; Kazakov, Sergei A.

2011-01-01

MicroRNAs (miRNAs) are important regulators of gene expression and have great potential as biomarkers, prognostic indicators, and therapeutic targets. Determining the expression patterns of these molecules is essential for elucidating their biogenesis, regulation, relation to disease, and response to therapy. Although PCR-based assays are commonly used for expression profiling of miRNAs, the small size, sequence heterogeneity, and (in some cases) end modifications of miRNAs constrain the performance of existing PCR methods. Here we introduce miR-ID, a novel method that avoids these constraints while providing superior sensitivity and sequence specificity at a lower cost. It also has the unique ability to differentiate unmodified small RNAs from those carrying 2′-OMe groups at their 3′-ends while detecting both forms. miR-ID is comprised of the following steps: (1) circularization of the miRNA by a ligase; (2) reverse transcription of the circularized miRNA (RTC), producing tandem repeats of a DNA sequence complementary to the miRNA; and (3) qPCR amplification of segments of this multimeric cDNA using 5′-overlapping primers and a nonspecific dye such as SYBR Green. No chemically modified probes (e.g., TaqMan) or primers (e.g., LNA) are required. The circular RNA and multimeric cDNA templates provide unmatched flexibility in the positioning of primers, which may include straddling the boundaries between these repetitive miRNA sequences. miR-ID is based on new findings that are themselves of general interest, including reverse transcription of small RNA circles and the use of 5′-overlapping primers for detection of repetitive sequences by qPCR. PMID:21169480

From reads to genes to pathways: differential expression analysis of RNA-Seq experiments using Rsubread and the edgeR quasi-likelihood pipeline.

PubMed

Chen, Yunshun; Lun, Aaron T L; Smyth, Gordon K

2016-01-01

In recent years, RNA sequencing (RNA-seq) has become a very widely used technology for profiling gene expression. One of the most common aims of RNA-seq profiling is to identify genes or molecular pathways that are differentially expressed (DE) between two or more biological conditions. This article demonstrates a computational workflow for the detection of DE genes and pathways from RNA-seq data by providing a complete analysis of an RNA-seq experiment profiling epithelial cell subsets in the mouse mammary gland. The workflow uses R software packages from the open-source Bioconductor project and covers all steps of the analysis pipeline, including alignment of read sequences, data exploration, differential expression analysis, visualization and pathway analysis. Read alignment and count quantification is conducted using the Rsubread package and the statistical analyses are performed using the edgeR package. The differential expression analysis uses the quasi-likelihood functionality of edgeR.

The Plasmodium falciparum transcriptome in severe malaria reveals altered expression of genes involved in important processes including surface antigen–encoding var genes

PubMed Central

Tonkin-Hill, Gerry Q.; Trianty, Leily; Noviyanti, Rintis; Nguyen, Hanh H. T.; Sebayang, Boni F.; Lampah, Daniel A.; Marfurt, Jutta; Cobbold, Simon A.; Rambhatla, Janavi S.; McConville, Malcolm J.; Rogerson, Stephen J.; Brown, Graham V.; Day, Karen P.; Price, Ric N.; Anstey, Nicholas M.

2018-01-01

Within the human host, the malaria parasite Plasmodium falciparum is exposed to multiple selection pressures. The host environment changes dramatically in severe malaria, but the extent to which the parasite responds to—or is selected by—this environment remains unclear. From previous studies, the parasites that cause severe malaria appear to increase expression of a restricted but poorly defined subset of the PfEMP1 variant, surface antigens. PfEMP1s are major targets of protective immunity. Here, we used RNA sequencing (RNAseq) to analyse gene expression in 44 parasite isolates that caused severe and uncomplicated malaria in Papuan patients. The transcriptomes of 19 parasite isolates associated with severe malaria indicated that these parasites had decreased glycolysis without activation of compensatory pathways; altered chromatin structure and probably transcriptional regulation through decreased histone methylation; reduced surface expression of PfEMP1; and down-regulated expression of multiple chaperone proteins. Our RNAseq also identified novel associations between disease severity and PfEMP1 transcripts, domains, and smaller sequence segments and also confirmed all previously reported associations between expressed PfEMP1 sequences and severe disease. These findings will inform efforts to identify vaccine targets for severe malaria and also indicate how parasites adapt to—or are selected by—the host environment in severe malaria. PMID:29529020

Age-related regulation of genes: slow homeostatic changes and age-dimension technology

NASA Astrophysics Data System (ADS)

Kurachi, Kotoku; Zhang, Kezhong; Huo, Jeffrey; Ameri, Afshin; Kuwahara, Mitsuhiro; Fontaine, Jean-Marc; Yamamoto, Kei; Kurachi, Sumiko

2002-11-01

Through systematic studies of pro- and anti-blood coagulation factors, we have determined molecular mechanisms involving two genetic elements, age-related stability element (ASE), GAGGAAG and age-related increase element (AIE), a unique stretch of dinucleotide repeats (AIE). ASE and AIE are essential for age-related patterns of stable and increased gene expression patterns, respectively. Such age-related gene regulatory mechanisms are also critical for explaining homeostasis in various physiological reactions as well as slow homeostatic changes in them. The age-related increase expression of the human factor IX (hFIX) gene requires the presence of both ASE and AIE, which apparently function additively. The anti-coagulant factor protein C (hPC) gene uses an ASE (CAGGAG) to produce age-related stable expression. Both ASE sequences (G/CAGAAG) share consensus sequence of the transcriptional factor PEA-3 element. No other similar sequences, including another PEA-3 consensus sequence, GAGGATG, function in conferring age-related gene regulation. The age-regulatory mechanisms involving ASE and AIE apparently function universally with different genes and across different animal species. These findings have led us to develop a new field of research and applications, which we named “age-dimension technology (ADT)”. ADT has exciting potential for modifying age-related expression of genes as well as associated physiological processes, and developing novel, more effective prophylaxis or treatments for age-related diseases.

Reduced expression of APC-1B but not APC-1A by the deletion of promoter 1B is responsible for familial adenomatous polyposis.

PubMed

Yamaguchi, Kiyoshi; Nagayama, Satoshi; Shimizu, Eigo; Komura, Mitsuhiro; Yamaguchi, Rui; Shibuya, Tetsuo; Arai, Masami; Hatakeyama, Seira; Ikenoue, Tsuneo; Ueno, Masashi; Miyano, Satoru; Imoto, Seiya; Furukawa, Yoichi

2016-05-24

Germline mutations in the tumor suppressor gene APC are associated with familial adenomatous polyposis (FAP). Here we applied whole-genome sequencing (WGS) to the DNA of a sporadic FAP patient in which we did not find any pathological APC mutations by direct sequencing. WGS identified a promoter deletion of approximately 10 kb encompassing promoter 1B and exon1B of APC. Additional allele-specific expression analysis by deep cDNA sequencing revealed that the deletion reduced the expression of the mutated APC allele to as low as 11.2% in the total APC transcripts, suggesting that the residual mutant transcripts were driven by other promoter(s). Furthermore, cap analysis of gene expression (CAGE) demonstrated that the deleted promoter 1B region is responsible for the great majority of APC transcription in many tissues except the brain. The deletion decreased the transcripts of APC-1B to 39-45% in the patient compared to the healthy controls, but it did not decrease those of APC-1A. Different deletions including promoter 1B have been reported in FAP patients. Taken together, our results strengthen the evidence that analysis of structural variations in promoter 1B should be considered for the FAP patients whose pathological mutations are not identified by conventional direct sequencing.

Expression of a polyubiquitin promoter isolated from Gladiolus.

PubMed

Joung, Young Hee; Kamo, Kathryn

2006-10-01

A polyubiquitin promoter (GUBQ1) including its 5'UTR and intron was isolated from the floral monocot Gladiolus because high levels of expression could not be obtained using publicly available promoters isolated from either cereals or dicots. Sequencing of the promoter revealed highly conserved 5' and 3' intron splicing sites for the 1.234 kb intron. The coding sequence of the first two ubiquitin genes showed the highest homology (87 and 86%, respectively) to the ubiquitin genes of Nicotiana tabacum and Oryza sativa RUBQ2. Transient expression following gene gun bombardment showed that relative levels of GUS activity with the GUBQ1 promoter were comparable to the CaMV 35S promoter in gladiolus, tobacco, rose, rice, and the floral monocot freesia. The highest levels of GUS expression with GUBQ1 were attained with Gladiolus. The full-length GUBQ1 promoter including 5'UTR and intron were necessary for maximum GUS expression in Gladiolus. The relative GUS activity for the promoter only was 9%, and the activity for the promoter with 5'UTR and 399 bp of the full-length 1.234 kb intron was 41%. Arabidopsis plants transformed with uidA under GUBQ1 showed moderate GUS expression throughout young leaves and in the vasculature of older leaves. The highest levels of transient GUS expression in Gladiolus have been achieved using the GUBQ1 promoter. This promoter should be useful for genetic engineering of disease resistance in Gladiolus, rose, and freesia, where high levels of gene expression are important.

Development of a set of SNP markers present in expressed genes of the apple.

PubMed

Chagné, David; Gasic, Ksenija; Crowhurst, Ross N; Han, Yuepeng; Bassett, Heather C; Bowatte, Deepa R; Lawrence, Timothy J; Rikkerink, Erik H A; Gardiner, Susan E; Korban, Schuyler S

2008-11-01

Molecular markers associated with gene coding regions are useful tools for bridging functional and structural genomics. Due to their high abundance in plant genomes, single nucleotide polymorphisms (SNPs) are present within virtually all genomic regions, including most coding sequences. The objective of this study was to develop a set of SNPs for the apple by taking advantage of the wealth of genomics resources available for the apple, including a large collection of expressed sequenced tags (ESTs). Using bioinformatics tools, a search for SNPs within an EST database of approximately 350,000 sequences developed from a variety of apple accessions was conducted. This resulted in the identification of a total of 71,482 putative SNPs. As the apple genome is reported to be an ancient polyploid, attempts were made to verify whether those SNPs detected in silico were attributable either to allelic polymorphisms or to gene duplication or paralogous or homeologous sequence variations. To this end, a set of 464 PCR primer pairs was designed, PCR was amplified using two subsets of plants, and the PCR products were sequenced. The SNPs retrieved from these sequences were then mapped onto apple genetic maps, including a newly constructed map of a Royal Gala x A689-24 cross and a Malling 9 x Robusta 5, map using a bin mapping strategy. The SNP genotyping was performed using the high-resolution melting (HRM) technique. A total of 93 new markers containing 210 coding SNPs were successfully mapped. This new set of SNP markers for the apple offers new opportunities for understanding the genetic control of important horticultural traits using quantitative trait loci (QTL) or linkage disequilibrium analysis. These also serve as useful markers for aligning physical and genetic maps, and as potential transferable markers across the Rosaceae family.

Expressed sequence tag (EST) analysis of two subspecies of Metarhizium anisopliae reveals a plethora of secreted proteins with potential activity in insect hosts.

PubMed

Freimoser, Florian M; Screen, Steven; Bagga, Savita; Hu, Gang; St Leger, Raymond J

2003-01-01

Expressed sequence tag (EST) libraries for Metarhizium anisopliae, the causative agent of green muscardine disease, were developed from the broad host-range pathogen Metarhizium anisopliae sf. anisopliae and the specific grasshopper pathogen, M. anisopliae sf. acridum. Approximately 1,700 5' end sequences from each subspecies were generated from cDNA libraries representing fungi grown under conditions that maximize secretion of cuticle-degrading enzymes. Both subspecies had ESTs for virtually all pathogenicity-related genes cloned to date from M. anisopliae, but many novel genes encoding potential virulence factors were also tagged. Enzymes with potential targets in the insect host included proteases, chitinases, phospholipases, lipases, esterases, phosphatases and enzymes producing toxic secondary metabolites. A diverse array of proteases composed 36 % of all M. anisopliae sf. anisopliae ESTs. Eighty percent of the ESTs that could be clustered into functional groups had significant matches (E<10(-5)) in other ascomycete fungi. These included genes reported to have specific roles in pathogens with plant or vertebrate hosts. Many of the remaining ESTs had their best BLAST match among animal, plant and bacterial sequences. These include genes with plant and microbial counterparts that produce potent antimicrobials. The abundance of transcripts discovered for different functional groups varied between the two subspecies of M. anisopliae in a manner consistent with ecological adaptations of the two pathogens. By hastening gene discovery this project has enhanced development of improved mycoinsecticides. In addition, the M. anisopliae ESTs represent a significant contribution to the extensive database of sequences from ascomycetes that are saprophytes or plant and vertebrate pathogens. Comparative analyses of these sequences is providing important information about the biology and evolutionary history of this clade.

Comprehensive Molecular Characterization of Urothelial Bladder Carcinoma

PubMed Central

2014-01-01

Urothelial carcinoma of the bladder is a common malignancy that causes approximately 150,000 deaths per year worldwide. To date, no molecularly targeted agents have been approved for the disease. As part of The Cancer Genome Atlas project, we report here an integrated analysis of 131 urothelial carcinomas to provide a comprehensive landscape of molecular alterations. There were statistically significant recurrent mutations in 32 genes, including multiple genes involved in cell cycle regulation, chromatin regulation, and kinase signaling pathways, as well as 9 genes not previously reported as significantly mutated in any cancer. RNA sequencing revealed four expression subtypes, two of which (papillary-like and basal/squamous-like) were also evident in miRNA sequencing and protein data. Whole-genome and RNA sequencing identified recurrent in-frame activating FGFR3-TACC3 fusions and expression or integration of several viruses (including HPV16) that are associated with gene inactivation. Our analyses identified potential therapeutic targets in 69% of the tumours, including 42% with targets in the PI3K/AKT/mTOR pathway and 45% with targets (including ERBB2) in the RTK/MAPK pathway. Chromatin regulatory genes were more frequently mutated in urothelial carcinoma than in any common cancer studied to date, suggesting the future possibility of targeted therapy for chromatin abnormalities. PMID:24476821

RNA sequencing uncovers antisense RNAs and novel small RNAs in Streptococcus pyogenes

PubMed Central

Le Rhun, Anaïs; Beer, Yan Yan; Reimegård, Johan; Chylinski, Krzysztof; Charpentier, Emmanuelle

2016-01-01

ABSTRACT Streptococcus pyogenes is a human pathogen responsible for a wide spectrum of diseases ranging from mild to life-threatening infections. During the infectious process, the temporal and spatial expression of pathogenicity factors is tightly controlled by a complex network of protein and RNA regulators acting in response to various environmental signals. Here, we focus on the class of small RNA regulators (sRNAs) and present the first complete analysis of sRNA sequencing data in S. pyogenes. In the SF370 clinical isolate (M1 serotype), we identified 197 and 428 putative regulatory RNAs by visual inspection and bioinformatics screening of the sequencing data, respectively. Only 35 from the 197 candidates identified by visual screening were assigned a predicted function (T-boxes, ribosomal protein leaders, characterized riboswitches or sRNAs), indicating how little is known about sRNA regulation in S. pyogenes. By comparing our list of predicted sRNAs with previous S. pyogenes sRNA screens using bioinformatics or microarrays, 92 novel sRNAs were revealed, including antisense RNAs that are for the first time shown to be expressed in this pathogen. We experimentally validated the expression of 30 novel sRNAs and antisense RNAs. We show that the expression profile of 9 sRNAs including 2 predicted regulatory elements is affected by the endoribonucleases RNase III and/or RNase Y, highlighting the critical role of these enzymes in sRNA regulation. PMID:26580233

Phylogenomic analysis of UDP glycosyltransferase 1 multigene family in Linum usitatissimum identified genes with varied expression patterns.

PubMed

Barvkar, Vitthal T; Pardeshi, Varsha C; Kale, Sandip M; Kadoo, Narendra Y; Gupta, Vidya S

2012-05-08

The glycosylation process, catalyzed by ubiquitous glycosyltransferase (GT) family enzymes, is a prevalent modification of plant secondary metabolites that regulates various functions such as hormone homeostasis, detoxification of xenobiotics and biosynthesis and storage of secondary metabolites. Flax (Linum usitatissimum L.) is a commercially grown oilseed crop, important because of its essential fatty acids and health promoting lignans. Identification and characterization of UDP glycosyltransferase (UGT) genes from flax could provide valuable basic information about this important gene family and help to explain the seed specific glycosylated metabolite accumulation and other processes in plants. Plant genome sequencing projects are useful to discover complexity within this gene family and also pave way for the development of functional genomics approaches. Taking advantage of the newly assembled draft genome sequence of flax, we identified 137 UDP glycosyltransferase (UGT) genes from flax using a conserved signature motif. Phylogenetic analysis of these protein sequences clustered them into 14 major groups (A-N). Expression patterns of these genes were investigated using publicly available expressed sequence tag (EST), microarray data and reverse transcription quantitative real time PCR (RT-qPCR). Seventy-three per cent of these genes (100 out of 137) showed expression evidence in 15 tissues examined and indicated varied expression profiles. The RT-qPCR results of 10 selected genes were also coherent with the digital expression analysis. Interestingly, five duplicated UGT genes were identified, which showed differential expression in various tissues. Of the seven intron loss/gain positions detected, two intron positions were conserved among most of the UGTs, although a clear relationship about the evolution of these genes could not be established. Comparison of the flax UGTs with orthologs from four other sequenced dicot genomes indicated that seven UGTs were flax diverged. Flax has a large number of UGT genes including few flax diverged ones. Phylogenetic analysis and expression profiles of these genes identified tissue and condition specific repertoire of UGT genes from this crop. This study would facilitate precise selection of candidate genes and their further characterization of substrate specificities and in planta functions.

Phylogenomic analysis of UDP glycosyltransferase 1 multigene family in Linum usitatissimum identified genes with varied expression patterns

PubMed Central

2012-01-01

Background The glycosylation process, catalyzed by ubiquitous glycosyltransferase (GT) family enzymes, is a prevalent modification of plant secondary metabolites that regulates various functions such as hormone homeostasis, detoxification of xenobiotics and biosynthesis and storage of secondary metabolites. Flax (Linum usitatissimum L.) is a commercially grown oilseed crop, important because of its essential fatty acids and health promoting lignans. Identification and characterization of UDP glycosyltransferase (UGT) genes from flax could provide valuable basic information about this important gene family and help to explain the seed specific glycosylated metabolite accumulation and other processes in plants. Plant genome sequencing projects are useful to discover complexity within this gene family and also pave way for the development of functional genomics approaches. Results Taking advantage of the newly assembled draft genome sequence of flax, we identified 137 UDP glycosyltransferase (UGT) genes from flax using a conserved signature motif. Phylogenetic analysis of these protein sequences clustered them into 14 major groups (A-N). Expression patterns of these genes were investigated using publicly available expressed sequence tag (EST), microarray data and reverse transcription quantitative real time PCR (RT-qPCR). Seventy-three per cent of these genes (100 out of 137) showed expression evidence in 15 tissues examined and indicated varied expression profiles. The RT-qPCR results of 10 selected genes were also coherent with the digital expression analysis. Interestingly, five duplicated UGT genes were identified, which showed differential expression in various tissues. Of the seven intron loss/gain positions detected, two intron positions were conserved among most of the UGTs, although a clear relationship about the evolution of these genes could not be established. Comparison of the flax UGTs with orthologs from four other sequenced dicot genomes indicated that seven UGTs were flax diverged. Conclusions Flax has a large number of UGT genes including few flax diverged ones. Phylogenetic analysis and expression profiles of these genes identified tissue and condition specific repertoire of UGT genes from this crop. This study would facilitate precise selection of candidate genes and their further characterization of substrate specificities and in planta functions. PMID:22568875

Transcriptomic resources for the medicinal legume Mucuna pruriens: de novo transcriptome assembly, annotation, identification and validation of EST-SSR markers.

PubMed

Sathyanarayana, N; Pittala, Ranjith Kumar; Tripathi, Pankaj Kumar; Chopra, Ratan; Singh, Heikham Russiachand; Belamkar, Vikas; Bhardwaj, Pardeep Kumar; Doyle, Jeff J; Egan, Ashley N

2017-05-25

The medicinal legume Mucuna pruriens (L.) DC. has attracted attention worldwide as a source of the anti-Parkinson's drug L-Dopa. It is also a popular green manure cover crop that offers many agronomic benefits including high protein content, nitrogen fixation and soil nutrients. The plant currently lacks genomic resources and there is limited knowledge on gene expression, metabolic pathways, and genetics of secondary metabolite production. Here, we present transcriptomic resources for M. pruriens, including a de novo transcriptome assembly and annotation, as well as differential transcript expression analyses between root, leaf, and pod tissues. We also develop microsatellite markers and analyze genetic diversity and population structure within a set of Indian germplasm accessions. One-hundred ninety-one million two hundred thirty-three thousand two hundred forty-two bp cleaned reads were assembled into 67,561 transcripts with mean length of 626 bp and N50 of 987 bp. Assembled sequences were annotated using BLASTX against public databases with over 80% of transcripts annotated. We identified 7,493 simple sequence repeat (SSR) motifs, including 787 polymorphic repeats between the parents of a mapping population. 134 SSRs from expressed sequenced tags (ESTs) were screened against 23 M. pruriens accessions from India, with 52 EST-SSRs retained after quality control. Population structure analysis using a Bayesian framework implemented in fastSTRUCTURE showed nearly similar groupings as with distance-based (neighbor-joining) and principal component analyses, with most of the accessions clustering per geographical origins. Pair-wise comparison of transcript expression in leaves, roots and pods identified 4,387 differentially expressed transcripts with the highest number occurring between roots and leaves. Differentially expressed transcripts were enriched with transcription factors and transcripts annotated as belonging to secondary metabolite pathways. The M. pruriens transcriptomic resources generated in this study provide foundational resources for gene discovery and development of molecular markers. Polymorphic SSRs identified can be used for genetic diversity, marker-trait analyses, and development of functional markers for crop improvement. The results of differential expression studies can be used to investigate genes involved in L-Dopa synthesis and other key metabolic pathways in M. pruriens.

Comprehensive Profiling of the Androgen Receptor in Liquid Biopsies from Castration-resistant Prostate Cancer Reveals Novel Intra-AR Structural Variation and Splice Variant Expression Patterns.

PubMed

De Laere, Bram; van Dam, Pieter-Jan; Whitington, Tom; Mayrhofer, Markus; Diaz, Emanuela Henao; Van den Eynden, Gert; Vandebroek, Jean; Del-Favero, Jurgen; Van Laere, Steven; Dirix, Luc; Grönberg, Henrik; Lindberg, Johan

2017-08-01

Expression of the androgen receptor splice variant 7 (AR-V7) is associated with poor response to second-line endocrine therapy in castration-resistant prostate cancer (CRPC). However, a large fraction of nonresponding patients are AR-V7-negative. To investigate if a comprehensive liquid biopsy-based AR profile may improve patient stratification in the context of second-line endocrine therapy. Peripheral blood was collected from patients with CRPC (n=30) before initiation of a new line of systemic therapy. We performed profiling of circulating tumour DNA via low-pass whole-genome sequencing and targeted sequencing of the entire AR gene, including introns. Targeted RNA sequencing was performed on enriched circulating tumour cell fractions to assess the expression levels of seven AR splice variants (ARVs). Somatic AR variations, including copy-number alterations, structural variations, and point mutations, were combined with ARV expression patterns and correlated to clinicopathologic parameters. Collectively, any AR perturbation, including ARV, was detected in 25/30 patients. Surprisingly, intra-AR structural variation was present in 15/30 patients, of whom 14 expressed ARVs. The majority of ARV-positive patients expressed multiple ARVs, with AR-V3 the most abundantly expressed. The presence of any ARV was associated with progression-free survival after second-line endocrine treatment (hazard ratio 4.53, 95% confidence interval 1.424-14.41; p=0.0105). Six out of 17 poor responders were AR-V7-negative, but four carried other AR perturbations. Comprehensive AR profiling, which is feasible using liquid biopsies, is necessary to increase our understanding of the mechanisms underpinning resistance to endocrine treatment. Alterations in the androgen receptor are associated with endocrine treatment outcomes. This study demonstrates that it is possible to identify different types of alterations via simple blood draws. Follow-up studies are needed to determine the effect of such alterations on hormonal therapy. Copyright © 2017 European Association of Urology. Published by Elsevier B.V. All rights reserved.

First characterization of three cyclophilin family proteins in the oyster, Crassostrea ariakensis Gould.

PubMed

Xu, Ting; Xie, Jiasong; Yang, Shoubao; Ye, Shigen; Luo, Ming; Wu, Xinzhong

2016-08-01

Cyclophilins (CyPs) are a family of proteins that bind the immunosuppressive agent cyclosporin A (CsA) with high-affinity and belong to one of the three superfamilies of peptidyl-prolyl cis-trans isomerases (PPIase). In this report, three cyclophilin genes (Ca-CyPs), including Ca-CyPA, Ca-CyPB and Ca-PPIL3, were identified from oyster, Crassostrea ariakensis Gould in which Ca-CyPA encodes a protein with 165 amino acid sequences, Ca-CyPB encodes a protein with 217 amino acid sequences and Ca-PPIL3 encodes a protein with 162 amino acid sequences. All of the three Ca-CyPs genes contain a typical CyP-PPIase domain with its signature sequences and Ca-CyPB contains an N-signal peptide sequences. Tissue distribution study revealed that Ca-CyPs were ubiquitously expressed in all examined tissues and the highest levels were observed in hemocytes. RLO incubation upregulated the mRNA expression levels of Ca-CyPs, indicating that three Ca-CyPs might be involved in oyster immune response against RLO infection. Copyright © 2016 Elsevier Ltd. All rights reserved.

Single-cell full-length total RNA sequencing uncovers dynamics of recursive splicing and enhancer RNAs.

PubMed

Hayashi, Tetsutaro; Ozaki, Haruka; Sasagawa, Yohei; Umeda, Mana; Danno, Hiroki; Nikaido, Itoshi

2018-02-12

Total RNA sequencing has been used to reveal poly(A) and non-poly(A) RNA expression, RNA processing and enhancer activity. To date, no method for full-length total RNA sequencing of single cells has been developed despite the potential of this technology for single-cell biology. Here we describe random displacement amplification sequencing (RamDA-seq), the first full-length total RNA-sequencing method for single cells. Compared with other methods, RamDA-seq shows high sensitivity to non-poly(A) RNA and near-complete full-length transcript coverage. Using RamDA-seq with differentiation time course samples of mouse embryonic stem cells, we reveal hundreds of dynamically regulated non-poly(A) transcripts, including histone transcripts and long noncoding RNA Neat1. Moreover, RamDA-seq profiles recursive splicing in >300-kb introns. RamDA-seq also detects enhancer RNAs and their cell type-specific activity in single cells. Taken together, we demonstrate that RamDA-seq could help investigate the dynamics of gene expression, RNA-processing events and transcriptional regulation in single cells.

Applications of nanotechnology, next generation sequencing and microarrays in biomedical research.

PubMed

Elingaramil, Sauli; Li, Xiaolong; He, Nongyue

2013-07-01

Next-generation sequencing technologies, microarrays and advances in bio nanotechnology have had an enormous impact on research within a short time frame. This impact appears certain to increase further as many biomedical institutions are now acquiring these prevailing new technologies. Beyond conventional sampling of genome content, wide-ranging applications are rapidly evolving for next-generation sequencing, microarrays and nanotechnology. To date, these technologies have been applied in a variety of contexts, including whole-genome sequencing, targeted re sequencing and discovery of transcription factor binding sites, noncoding RNA expression profiling and molecular diagnostics. This paper thus discusses current applications of nanotechnology, next-generation sequencing technologies and microarrays in biomedical research and highlights the transforming potential these technologies offer.

A synthetic biology approach to the development of transcriptional regulatory models and custom enhancer design☆,☆☆

PubMed Central

Martinez, Carlos A.; Barr, Kenneth; Kim, Ah-Ram; Reinitz, John

2013-01-01

Synthetic biology offers novel opportunities for elucidating transcriptional regulatory mechanisms and enhancer logic. Complex cis-regulatory sequences—like the ones driving expression of the Drosophila even-skipped gene—have proven difficult to design from existing knowledge, presumably due to the large number of protein-protein interactions needed to drive the correct expression patterns of genes in multicellular organisms. This work discusses two novel computational methods for the custom design of enhancers that employ a sophisticated, empirically validated transcriptional model, optimization algorithms, and synthetic biology. These synthetic elements have both utilitarian and academic value, including improving existing regulatory models as well as evolutionary questions. The first method involves the use of simulated annealing to explore the sequence space for synthetic enhancers whose expression output fit a given search criterion. The second method uses a novel optimization algorithm to find functionally accessible pathways between two enhancer sequences. These paths describe a set of mutations wherein the predicted expression pattern does not significantly vary at any point along the path. Both methods rely on a predictive mathematical framework that maps the enhancer sequence space to functional output. PMID:23732772

The Spatial and Temporal Transcriptomic Landscapes of Ginseng, Panax ginseng C. A. Meyer.

PubMed

Wang, Kangyu; Jiang, Shicui; Sun, Chunyu; Lin, Yanping; Yin, Rui; Wang, Yi; Zhang, Meiping

2015-12-11

Ginseng, including Asian ginseng (Panax ginseng C. A. Meyer) and American ginseng (P. quinquefolius L.), is one of the most important medicinal herbs in Asia and North America, but significantly understudied. This study sequenced and characterized the transcriptomes and expression profiles of genes expressed in 14 tissues and four different aged roots of Asian ginseng. A total of 265.2 million 100-bp clean reads were generated using the high-throughput sequencing platform HiSeq 2000, representing >8.3x of the 3.2-Gb ginseng genome. From the sequences, 248,993 unigenes were assembled for whole plant, 61,912-113,456 unigenes for each tissue and 54,444-65,412 unigenes for different year-old roots. We comprehensively analyzed the unigene sets and gene expression profiles. We found that the number of genes allocated to each functional category is stable across tissues or developmental stages, while the expression profiles of different genes of a gene family or involved in ginsenoside biosynthesis dramatically diversified spatially and temporally. These results provide an overall insight into the spatial and temporal transcriptome dynamics and landscapes of Asian ginseng, and comprehensive resources for advanced research and breeding of ginseng and related species.

CMV-promoter driven codon-optimized expression alters the assembly type and morphology of a reconstituted HERV-K(HML-2).

PubMed

Hohn, Oliver; Hanke, Kirsten; Lausch, Veronika; Zimmermann, Anja; Mostafa, Saeed; Bannert, Norbert

2014-11-11

The HERV-K(HML-2) family contains the most recently integrated and best preserved endogenized proviral sequences in the human genome. All known elements have nevertheless been subjected to mutations or deletions that render expressed particles non-infectious. Moreover, these post-insertional mutations hamper the analysis of the general biological properties of this ancient virus family. The expression of consensus sequences and sequences of elements with reverted post-insertional mutations has therefore been very instrumental in overcoming this limitation. We investigated the particle morphology of a recently reconstituted HERV-K113 element termed oriHERV-K113 using thin-section electron microscopy (EM) and could demonstrate that strong overexpression by substitution of the 5'LTR for a CMV promoter and partial codon optimization altered the virus assembly type and morphology. This included a conversion from the regular C-type to an A-type morphology with a mass of cytoplasmic immature cores tethered to the cell membrane and the membranes of vesicles. Overexpression permitted the release and maturation of virions but reduced the envelope content. A weaker boost of virus expression by Staufen-1 was not sufficient to induce these morphological alterations.

CMV-Promoter Driven Codon-Optimized Expression Alters the Assembly Type and Morphology of a Reconstituted HERV-K(HML-2)

PubMed Central

Hohn, Oliver; Hanke, Kirsten; Lausch, Veronika; Zimmermann, Anja; Mostafa, Saeed; Bannert, Norbert

2014-01-01

The HERV-K(HML-2) family contains the most recently integrated and best preserved endogenized proviral sequences in the human genome. All known elements have nevertheless been subjected to mutations or deletions that render expressed particles non-infectious. Moreover, these post-insertional mutations hamper the analysis of the general biological properties of this ancient virus family. The expression of consensus sequences and sequences of elements with reverted post-insertional mutations has therefore been very instrumental in overcoming this limitation. We investigated the particle morphology of a recently reconstituted HERV-K113 element termed oriHERV-K113 using thin-section electron microscopy (EM) and could demonstrate that strong overexpression by substitution of the 5'LTR for a CMV promoter and partial codon optimization altered the virus assembly type and morphology. This included a conversion from the regular C-type to an A-type morphology with a mass of cytoplasmic immature cores tethered to the cell membrane and the membranes of vesicles. Overexpression permitted the release and maturation of virions but reduced the envelope content. A weaker boost of virus expression by Staufen-1 was not sufficient to induce these morphological alterations. PMID:25393897

HeLa Nucleic Acid Contamination in The Cancer Genome Atlas Leads to the Misidentification of Human Papillomavirus 18

PubMed Central

Cantalupo, Paul G.; Katz, Joshua P.

2015-01-01

ABSTRACT We searched The Cancer Genome Atlas (TCGA) database for viruses by comparing non-human reads present in transcriptome sequencing (RNA-Seq) and whole-exome sequencing (WXS) data to viral sequence databases. Human papillomavirus 18 (HPV18) is an etiologic agent of cervical cancer, and as expected, we found robust expression of HPV18 genes in cervical cancer samples. In agreement with previous studies, we also found HPV18 transcripts in non-cervical cancer samples, including those from the colon, rectum, and normal kidney. However, in each of these cases, HPV18 gene expression was low, and single-nucleotide variants and positions of genomic alignments matched the integrated portion of HPV18 present in HeLa cells. Chimeric reads that match a known virus-cell junction of HPV18 integrated in HeLa cells were also present in some samples. We hypothesize that HPV18 sequences in these non-cervical samples are due to nucleic acid contamination from HeLa cells. This finding highlights the problems that contamination presents in computational virus detection pipelines. IMPORTANCE Viruses associated with cancer can be detected by searching tumor sequence databases. Several studies involving searches of the TCGA database have reported the presence of HPV18, a known cause of cervical cancer, in a small number of additional cancers, including those of the rectum, kidney, and colon. We have determined that the sequences related to HPV18 in non-cervical samples are due to nucleic acid contamination from HeLa cells. To our knowledge, this is the first report of the misidentification of viruses in next-generation sequencing data of tumors due to contamination with a cancer cell line. These results raise awareness of the difficulty of accurately identifying viruses in human sequence databases. PMID:25631090

Comparative analysis of the feline immunoglobulin repertoire.

PubMed

Steiniger, Sebastian C J; Glanville, Jacob; Harris, Douglas W; Wilson, Thomas L; Ippolito, Gregory C; Dunham, Steven A

2017-03-01

Next-Generation Sequencing combined with bioinformatics is a powerful tool for analyzing the large number of DNA sequences present in the expressed antibody repertoire and these data sets can be used to advance a number of research areas including antibody discovery and engineering. The accurate measurement of the immune repertoire sequence composition, diversity and abundance is important for understanding the repertoire response in infections, vaccinations and cancer immunology and could also be useful for elucidating novel molecular targets. In this study 4 individual domestic cats (Felis catus) were subjected to antibody repertoire sequencing with total number of sequences generated 1079863 for VH for IgG, 1050824 VH for IgM, 569518 for VK and 450195 for VL. Our analysis suggests that a similar VDJ expression patterns exists across all cats. Similar to the canine repertoire, the feline repertoire is dominated by a single subgroup, namely VH3. The antibody paratope of felines showed similar amino acid variation when compared to human, mouse and canine counterparts. All animals show a similarly skewed VH CDR-H3 profile and, when compared to canine, human and mouse, distinct differences are observed. Our study represents the first attempt to characterize sequence diversity in the expressed feline antibody repertoire and this demonstrates the utility of using NGS to elucidate entire antibody repertoires from individual animals. These data provide significant insight into understanding the feline immune system function. Copyright © 2017 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.

An insight into the sialotranscriptome of the seed-feeding bug, Oncopeltus fasciatus.

PubMed

Francischetti, Ivo M B; Lopes, Angela H; Dias, Felipe A; Pham, Van M; Ribeiro, José M C

2007-09-01

The salivary transcriptome of the seed-feeding hemipteran, Oncopeltus fasciatus (milkweed bug), is described following assembly of 1025 expressed sequence tags (ESTs) into 305 clusters of related sequences. Inspection of these sequences reveals abundance of low complexity, putative secreted products rich in the amino acids (aa) glycine, serine or threonine, which might function as silk or mucins and assist food canal lubrication and sealing of the feeding site around the mouthparts. Several protease inhibitors were found, including abundant expression of cystatin transcripts that may inhibit cysteine proteases common in seeds that might injure the insect or induce plant apoptosis. Serine proteases and lipases are described that might assist digestion and liquefaction of seed proteins and oils. Finally, several novel putative proteins are described with no known function that might affect plant physiology or act as antimicrobials.

VitisExpDB: a database resource for grape functional genomics.

PubMed

Doddapaneni, Harshavardhan; Lin, Hong; Walker, M Andrew; Yao, Jiqiang; Civerolo, Edwin L

2008-02-28

The family Vitaceae consists of many different grape species that grow in a range of climatic conditions. In the past few years, several studies have generated functional genomic information on different Vitis species and cultivars, including the European grape vine, Vitis vinifera. Our goal is to develop a comprehensive web data source for Vitaceae. VitisExpDB is an online MySQL-PHP driven relational database that houses annotated EST and gene expression data for V. vinifera and non-vinifera grape species and varieties. Currently, the database stores approximately 320,000 EST sequences derived from 8 species/hybrids, their annotation (BLAST top match) details and Gene Ontology based structured vocabulary. Putative homologs for each EST in other species and varieties along with information on their percent nucleotide identities, phylogenetic relationship and common primers can be retrieved. The database also includes information on probe sequence and annotation features of the high density 60-mer gene expression chip consisting of approximately 20,000 non-redundant set of ESTs. Finally, the database includes 14 processed global microarray expression profile sets. Data from 12 of these expression profile sets have been mapped onto metabolic pathways. A user-friendly web interface with multiple search indices and extensively hyperlinked result features that permit efficient data retrieval has been developed. Several online bioinformatics tools that interact with the database along with other sequence analysis tools have been added. In addition, users can submit their ESTs to the database. The developed database provides genomic resource to grape community for functional analysis of genes in the collection and for the grape genome annotation and gene function identification. The VitisExpDB database is available through our website http://cropdisease.ars.usda.gov/vitis_at/main-page.htm.

VitisExpDB: A database resource for grape functional genomics

PubMed Central

Doddapaneni, Harshavardhan; Lin, Hong; Walker, M Andrew; Yao, Jiqiang; Civerolo, Edwin L

2008-01-01

Background The family Vitaceae consists of many different grape species that grow in a range of climatic conditions. In the past few years, several studies have generated functional genomic information on different Vitis species and cultivars, including the European grape vine, Vitis vinifera. Our goal is to develop a comprehensive web data source for Vitaceae. Description VitisExpDB is an online MySQL-PHP driven relational database that houses annotated EST and gene expression data for V. vinifera and non-vinifera grape species and varieties. Currently, the database stores ~320,000 EST sequences derived from 8 species/hybrids, their annotation (BLAST top match) details and Gene Ontology based structured vocabulary. Putative homologs for each EST in other species and varieties along with information on their percent nucleotide identities, phylogenetic relationship and common primers can be retrieved. The database also includes information on probe sequence and annotation features of the high density 60-mer gene expression chip consisting of ~20,000 non-redundant set of ESTs. Finally, the database includes 14 processed global microarray expression profile sets. Data from 12 of these expression profile sets have been mapped onto metabolic pathways. A user-friendly web interface with multiple search indices and extensively hyperlinked result features that permit efficient data retrieval has been developed. Several online bioinformatics tools that interact with the database along with other sequence analysis tools have been added. In addition, users can submit their ESTs to the database. Conclusion The developed database provides genomic resource to grape community for functional analysis of genes in the collection and for the grape genome annotation and gene function identification. The VitisExpDB database is available through our website . PMID:18307813

MytiBase: a knowledgebase of mussel (M. galloprovincialis) transcribed sequences

PubMed Central

Venier, Paola; De Pittà, Cristiano; Bernante, Filippo; Varotto, Laura; De Nardi, Barbara; Bovo, Giuseppe; Roch, Philippe; Novoa, Beatriz; Figueras, Antonio; Pallavicini, Alberto; Lanfranchi, Gerolamo

2009-01-01

Background Although Bivalves are among the most studied marine organisms due to their ecological role, economic importance and use in pollution biomonitoring, very little information is available on the genome sequences of mussels. This study reports the functional analysis of a large-scale Expressed Sequence Tag (EST) sequencing from different tissues of Mytilus galloprovincialis (the Mediterranean mussel) challenged with toxic pollutants, temperature and potentially pathogenic bacteria. Results We have constructed and sequenced seventeen cDNA libraries from different Mediterranean mussel tissues: gills, digestive gland, foot, anterior and posterior adductor muscle, mantle and haemocytes. A total of 24,939 clones were sequenced from these libraries generating 18,788 high-quality ESTs which were assembled into 2,446 overlapping clusters and 4,666 singletons resulting in a total of 7,112 non-redundant sequences. In particular, a high-quality normalized cDNA library (Nor01) was constructed as determined by the high rate of gene discovery (65.6%). Bioinformatic screening of the non-redundant M. galloprovincialis sequences identified 159 microsatellite-containing ESTs. Clusters, consensuses, related similarities and gene ontology searches have been organized in a dedicated, searchable database . Conclusion We defined the first species-specific catalogue of M. galloprovincialis ESTs including 7,112 unique transcribed sequences. Putative microsatellite markers were identified. This annotated catalogue represents a valuable platform for expression studies, marker validation and genetic linkage analysis for investigations in the biology of Mediterranean mussels. PMID:19203376

Bat Accelerated Regions Identify a Bat Forelimb Specific Enhancer in the HoxD Locus

PubMed Central

Mason, Mandy K.; VanderMeer, Julia E.; Zhao, Jingjing; Eckalbar, Walter L.; Logan, Malcolm; Illing, Nicola; Pollard, Katherine S.; Ahituv, Nadav

2016-01-01

The molecular events leading to the development of the bat wing remain largely unknown, and are thought to be caused, in part, by changes in gene expression during limb development. These expression changes could be instigated by variations in gene regulatory enhancers. Here, we used a comparative genomics approach to identify regions that evolved rapidly in the bat ancestor, but are highly conserved in other vertebrates. We discovered 166 bat accelerated regions (BARs) that overlap H3K27ac and p300 ChIP-seq peaks in developing mouse limbs. Using a mouse enhancer assay, we show that five Myotis lucifugus BARs drive gene expression in the developing mouse limb, with the majority showing differential enhancer activity compared to the mouse orthologous BAR sequences. These include BAR116, which is located telomeric to the HoxD cluster and had robust forelimb expression for the M. lucifugus sequence and no activity for the mouse sequence at embryonic day 12.5. Developing limb expression analysis of Hoxd10-Hoxd13 in Miniopterus natalensis bats showed a high-forelimb weak-hindlimb expression for Hoxd10-Hoxd11, similar to the expression trend observed for M. lucifugus BAR116 in mice, suggesting that it could be involved in the regulation of the bat HoxD complex. Combined, our results highlight novel regulatory regions that could be instrumental for the morphological differences leading to the development of the bat wing. PMID:27019019

Analysis of C. elegans VIG-1 expression.

PubMed

Shin, Kyoung-Hwa; Choi, Boram; Park, Yang-Seo; Cho, Nam Jeong

2008-12-31

Double-stranded RNA (dsRNA) induces gene silencing in a sequence-specific manner by a process known as RNA interference (RNAi). The RNA-induced silencing complex (RISC) is a multi-subunit ribonucleoprotein complex that plays a key role in RNAi. VIG (Vasa intronic gene) has been identified as a component of Drosophila RISC; however, the role VIG plays in regulating RNAi is poorly understood. Here, we examined the spatial and temporal expression patterns of VIG-1, the C. elegans ortholog of Drosophila VIG, using a vig-1::gfp fusion construct. This construct contains the 908-bp region immediately upstream of vig-1 gene translation initiation site. Analysis by confocal microscopy demonstrated GFP-VIG-1 expression in a number of tissues including the pharynx, body wall muscle, hypodermis, intestine, reproductive system, and nervous system at the larval and adult stages. Furthermore, western blot analysis showed that VIG-1 is present in each developmental stage examined. To investigate regulatory sequences for vig-1 gene expression, we generated constructs containing deletions in the upstream region. It was determined that the GFP expression pattern of a deletion construct (delta-908 to -597) was generally similar to that of the non-deletion construct. In contrast, removal of a larger segment (delta-908 to -191) resulted in the loss of GFP expression in most cell types. Collectively, these results indicate that the 406-bp upstream region (-596 to -191) contains essential regulatory sequences required for VIG-1 expression.

Digital Gene Expression Analysis Based on De Novo Transcriptome Assembly Reveals New Genes Associated with Floral Organ Differentiation of the Orchid Plant Cymbidium ensifolium

PubMed Central

Yang, Fengxi; Zhu, Genfa

2015-01-01

Cymbidium ensifolium belongs to the genus Cymbidium of the orchid family. Owing to its spectacular flower morphology, C. ensifolium has considerable ecological and cultural value. However, limited genetic data is available for this non-model plant, and the molecular mechanism underlying floral organ identity is still poorly understood. In this study, we characterize the floral transcriptome of C. ensifolium and present, for the first time, extensive sequence and transcript abundance data of individual floral organs. After sequencing, over 10 Gb clean sequence data were generated and assembled into 111,892 unigenes with an average length of 932.03 base pairs, including 1,227 clusters and 110,665 singletons. Assembled sequences were annotated with gene descriptions, gene ontology, clusters of orthologous group terms, the Kyoto Encyclopedia of Genes and Genomes, and the plant transcription factor database. From these annotations, 131 flowering-associated unigenes, 61 CONSTANS-LIKE (COL) unigenes and 90 floral homeotic genes were identified. In addition, four digital gene expression libraries were constructed for the sepal, petal, labellum and gynostemium, and 1,058 genes corresponding to individual floral organ development were identified. Among them, eight MADS-box genes were further investigated by full-length cDNA sequence analysis and expression validation, which revealed two APETALA1/AGL9-like MADS-box genes preferentially expressed in the sepal and petal, two AGAMOUS-like genes particularly restricted to the gynostemium, and four DEF-like genes distinctively expressed in different floral organs. The spatial expression of these genes varied distinctly in different floral mutant corresponding to different floral morphogenesis, which validated the specialized roles of them in floral patterning and further supported the effectiveness of our in silico analysis. This dataset generated in our study provides new insights into the molecular mechanisms underlying floral patterning of Cymbidium and supports a valuable resource for molecular breeding of the orchid plant. PMID:26580566

Mapping-by-sequencing of Ligon-lintless-1 (Li 1 ) reveals a cluster of neighboring genes with correlated expression in developing fibers of Upland cotton (Gossypium hirsutum L.).

PubMed

Thyssen, Gregory N; Fang, David D; Turley, Rickie B; Florane, Christopher; Li, Ping; Naoumkina, Marina

2015-09-01

Mapping-by-sequencing and SNP marker analysis were used to fine map the Ligon-lintless-1 ( Li 1 ) short fiber mutation in tetraploid cotton to a 255-kb region that contains 16 annotated proteins. The Ligon-lintless-1 (Li 1 ) mutant of cotton (Gossypium hirsutum L.) has been studied as a model for cotton fiber development since its identification in 1929; however, the causative mutation has not been identified yet. Here we report the fine genetic mapping of the mutation to a 255-kb region that contains only 16 annotated genes in the reference Gossypium raimondii genome. We took advantage of the incompletely dominant dwarf vegetative phenotype to identify 100 mutants (Li 1 /Li 1 ) and 100 wild-type (li 1 /li 1 ) homozygotes from a mapping population of 2567 F2 plants, which we bulked and deep sequenced. Since only homozygotes were sequenced, we were able to use a high stringency in SNP calling to rapidly narrow down the region harboring the Li 1 locus, and designed subgenome-specific SNP markers to test the population. We characterized the expression of all sixteen genes in the region by RNA sequencing of elongating fibers and by RT-qPCR at seven time points spanning fiber development. One of the most highly expressed genes found in this interval in wild-type fiber cells is 40-fold under-expressed at the day of anthesis (DOA) in the mutant fiber cells.  This gene is a major facilitator superfamily protein, part of the large family of proteins that includes auxin and sugar transporters. Interestingly, nearly all genes in this region were most highly expressed at DOA and showed a high degree of co-expression. Further characterization is required to determine if transport of hormones or carbohydrates is involved in both the dwarf and lintless phenotypes of Li 1 plants.

Simultaneous Characterization of Somatic Events and HPV-18 Integration in a Metastatic Cervical Carcinoma Patient Using DNA and RNA Sequencing

PubMed Central

Liang, Winnie S.; Aldrich, Jessica; Nasser, Sara; Kurdoglu, Ahmet; Phillips, Lori; Reiman, Rebecca; McDonald, Jacquelyn; Izatt, Tyler; Christoforides, Alexis; Baker, Angela; Craig, Christine; Egan, Jan B.; Chase, Dana M.; Farley, John H.; Bryce, Alan H.; Stewart, A. Keith; Borad, Mitesh J.; Carpten, John D.; Craig, David W.; Monk, Bradley J.

2014-01-01

Objective Integration of carcinogenic human papillomaviruses (HPVs) into the host genome is a significant tumorigenic factor in specific cancers including cervical carcinoma. Although major strides have been made with respect to HPV diagnosis and prevention, identification and development of efficacious treatments for cervical cancer patients remains a goal and thus requires additional detailed characterization of both somatic events and HPV integration. Given this need, the goal of this study was to use the next generation sequencing to simultaneously evaluate somatic alterations and expression changes in a patient’s cervical squamous carcinoma lesion metastatic to the lung and to detect and analyze HPV infection in the same sample. Materials and Methods We performed tumor and normal exome, tumor and normal shallow whole-genome sequencing, and RNA sequencing of the patient’s lung metastasis. Results We generated over 1.2 billion mapped reads and identified 130 somatic point mutations and indels, 21 genic translocations, 16 coding regions demonstrating copy number changes, and over 36 genes demonstrating altered expression in the tumor (corrected P < 0.05). Sequencing also revealed the HPV type 18 (HPV-18) integration in the metastasis. Using both DNA and RNA reads, we pinpointed 3 major events indicating HPV-18 integration into an intronic region of chromosome 6p25.1 in the patient’s tumor and validated these events with Sanger sequencing. This integration site has not been reported for HPV-18. Conclusions We demonstrate that DNA and RNA sequencing can be used to concurrently characterize somatic alterations and expression changes in a biopsy and delineate HPV integration at base resolution in cervical cancer. Further sequencing will allow us to better understand the molecular basis of cervical cancer pathogenesis. PMID:24418928

VIZARD: analysis of Affymetrix Arabidopsis GeneChip data

NASA Technical Reports Server (NTRS)

Moseyko, Nick; Feldman, Lewis J.

2002-01-01

SUMMARY: The Affymetrix GeneChip Arabidopsis genome array has proved to be a very powerful tool for the analysis of gene expression in Arabidopsis thaliana, the most commonly studied plant model organism. VIZARD is a Java program created at the University of California, Berkeley, to facilitate analysis of Arabidopsis GeneChip data. It includes several integrated tools for filtering, sorting, clustering and visualization of gene expression data as well as tools for the discovery of regulatory motifs in upstream sequences. VIZARD also includes annotation and upstream sequence databases for the majority of genes represented on the Affymetrix Arabidopsis GeneChip array. AVAILABILITY: VIZARD is available free of charge for educational, research, and not-for-profit purposes, and can be downloaded at http://www.anm.f2s.com/research/vizard/ CONTACT: moseyko@uclink4.berkeley.edu.

Molecular cloning and expression of the hyu genes from Microbacterium liquefaciens AJ 3912, responsible for the conversion of 5-substituted hydantoins to alpha-amino acids, in Escherichia coli.

PubMed

Suzuki, Shun'ichi; Takenaka, Yasuhiro; Onishi, Norimasa; Yokozeki, Kenzo

2005-08-01

A DNA fragment from Microbacterium liquefaciens AJ 3912, containing the genes responsible for the conversion of 5-substituted-hydantoins to alpha-amino acids, was cloned in Escherichia coli and sequenced. Seven open reading frames (hyuP, hyuA, hyuH, hyuC, ORF1, ORF2, and ORF3) were identified on the 7.5 kb fragment. The deduced amino acid sequence encoded by the hyuA gene included the N-terminal amino acid sequence of the hydantoin racemase from M. liquefaciens AJ 3912. The hyuA, hyuH, and hyuC genes were heterologously expressed in E. coli; their presence corresponded with the detection of hydantoin racemase, hydantoinase, and N-carbamoyl alpha-amino acid amido hydrolase enzymatic activities respectively. The deduced amino acid sequences of hyuP were similar to those of the allantoin (5-ureido-hydantoin) permease from Saccharomyces cerevisiae, suggesting that hyuP protein might function as a hydantoin transporter.

A conserved post-transcriptional BMP2 switch in lung cells.

PubMed

Jiang, Shan; Fritz, David T; Rogers, Melissa B

2010-05-15

An ultra-conserved sequence in the bone morphogenetic protein 2 (BMP2) 3' untranslated region (UTR) markedly represses BMP2 expression in non-transformed lung cells. In contrast, the ultra-conserved sequence stimulates BMP2 expression in transformed lung cells. The ultra-conserved sequence functions as a post-transcriptional cis-regulatory switch. A common single-nucleotide polymorphism (SNP, rs15705, +A1123C), which has been shown to influence human morphology, disrupts a conserved element within the ultra-conserved sequence and altered reporter gene activity in non-transformed lung cells. This polymorphism changed the affinity of the BMP2 RNA for several proteins including nucleolin, which has an increased affinity for the C allele. Elevated BMP2 synthesis is associated with increased malignancy in mouse models of lung cancer and poor lung cancer patient prognosis. Understanding the cis- and trans-regulatory factors that control BMP2 synthesis is relevant to the initiation or progression of pathologies associated with abnormal BMP2 levels. (c) 2010 Wiley-Liss, Inc.

Recombinant soluble adenovirus receptor

DOEpatents

Freimuth, Paul I.

2002-01-01

Disclosed are isolated polypeptides from human CAR (coxsackievirus and adenovirus receptor) protein which bind adenovirus. Specifically disclosed are amino acid sequences which corresponds to adenovirus binding domain D1 and the entire extracellular domain of human CAR protein comprising D1 and D2. In other aspects, the disclosure relates to nucleic acid sequences encoding these domains as well as expression vectors which encode the domains and bacterial cells containing such vectors. Also disclosed is an isolated fusion protein comprised of the D1 polypeptide sequence fused to a polypeptide sequence which facilitates folding of D1 into a functional, soluble domain when expressed in bacteria. The functional D1 domain finds application for example in a therapeutic method for treating a patient infected with a virus which binds to D1, and also in a method for identifying an antiviral compound which interferes with viral attachment. Also included is a method for specifically targeting a cell for infection by a virus which binds to D1.

Plant nitrogen regulatory P-PII polypeptides

DOEpatents

Coruzzi, Gloria M.; Lam, Hon-Ming; Hsieh, Ming-Hsiun

2004-11-23

The present invention generally relates to plant nitrogen regulatory PII gene (hereinafter P-PII gene), a gene involved in regulating plant nitrogen metabolism. The invention provides P-PII nucleotide sequences, expression constructs comprising said nucleotide sequences, and host cells and plants having said constructs and, optionally expressing the P-PII gene from said constructs. The invention also provides substantially pure P-PII proteins. The P-PII nucleotide sequences and constructs of the invention may be used to engineer organisms to overexpress wild-type or mutant P-PII regulatory protein. Engineered plants that overexpress or underexpress P-PII regulatory protein may have increased nitrogen assimilation capacity. Engineered organisms may be used to produce P-PII proteins which, in turn, can be used for a variety of purposes including in vitro screening of herbicides. P-PII nucleotide sequences have additional uses as probes for isolating additional genomic clones having the promoters of P-PII gene. P-PII promoters are light- and/or sucrose-inducible and may be advantageously used in genetic engineering of plants.

Synaptotagmin 1 causes phosphatidyl inositol lipid-dependent actin remodeling in cultured non-neuronal and neuronal cells

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

Johnsson, Anna-Karin; Karlsson, Roger, E-mail: roger.karlsson@wgi.su.se

2012-01-15

Here we demonstrate that a dramatic actin polymerizing activity caused by ectopic expression of the synaptic vesicle protein synaptotagmin 1 that results in extensive filopodia formation is due to the presence of a lysine rich sequence motif immediately at the cytoplasmic side of the transmembrane domain of the protein. This polybasic sequence interacts with anionic phospholipids in vitro, and, consequently, the actin remodeling caused by this sequence is interfered with by expression of a phosphatidyl inositol (4,5)-bisphosphate (PIP2)-targeted phosphatase, suggesting that it intervenes with the function of PIP2-binding actin control proteins. The activity drastically alters the behavior of a rangemore » of cultured cells including the neuroblastoma cell line SH-SY5Y and primary cortical mouse neurons, and, since the sequence is conserved also in synaptotagmin 2, it may reflect an important fine-tuning role for these two proteins during synaptic vesicle fusion and neurotransmitter release.« less

Amino acid sequence of a trypsin inhibitor from a Spirometra (Spirometra erinaceieuropaei).

PubMed

Sanda, A; Uchida, A; Itagaki, T; Kobayashi, H; Inokuchi, N; Koyama, T; Iwama, M; Ohgi, K; Irie, M

2001-12-01

A trypsin inhibitor that is highly homologous with bovine pancreatic trypsin inhibitor (BPTI) was co-purified along with RNase from Spirometra (Spirometra erinaceieuropaei). The amino acid sequence of this inhibitor (SETI) and the nucleotide sequence of the cDNA encoding this protein were determined by protein chemistry and gene technology. SETI contains 68 amino acid residues and has a molecular mass of 7,798 Da. SETI has 31 amino acid residues that are identical with BPTI's sequence, including 6 half-cystine and 5 aromatic amino acid residues. The active site Lys residue in BPTI is replaced by an Arg residue in SETI. SETI is an effective inhibitor of trypsin and moderately inhibits a-chymotrypsin, but less inhibits elastase or subtilisin. SETI was expressed by E. coli containing a PelB vector carrying the SETI encoding cDNA; an expression yield of 0.68 mg/l was obtained. The phylogenetic relationship of SETI and the other BPTI-like trypsin inhibitors was analyzed using most likelihood inference methods.

Sox2 regulatory region 2 sequence works as a DNA nuclear targeting sequence enhancing the efficiency of an exogenous gene expression in ES cells

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

Funabashi, Hisakage; Takatsu, Makoto; Saito, Mikako

2010-10-01

Research highlights: {yields} SV40-DTS worked as a DTS in ES cells as well as other types of cells. {yields} Sox2 regulatory region 2 worked as a DTS in ES cells and thus was termed as SRR2-DTS. {yields} SRR2-DTS was suggested as an ES cell-specific DTS. -- Abstract: In this report, the effects of two DNA nuclear targeting sequence (DTS) candidates on the gene expression efficiency in ES cells were investigated. Reporter plasmids containing the simian virus 40 (SV40) promoter/enhancer sequence (SV40-DTS), a DTS for various types of cells but not being reported yet for ES cells, and the 81 basemore » pairs of Sox2 regulatory region 2 (SRR2) where two transcriptional factors in ES cells, Oct3/4 and Sox2, are bound (SRR2-DTS), were introduced into cytoplasm in living cells by femtoinjection. The gene expression efficiencies of each plasmid in mouse insulinoma cell line MIN6 cells and mouse ES cells were then evaluated. Plasmids including SV40-DTS and SRR2-DTS exhibited higher gene expression efficiency comparing to plasmids without these DTSs, and thus it was concluded that both sequences work as a DTS in ES cells. In addition, it was suggested that SRR2-DTS works as an ES cell-specific DTS. To the best of our knowledge, this is the first report to confirm the function of DTSs in ES cells.« less

A powerful graphical pulse sequence programming tool for magnetic resonance imaging.

PubMed

Jie, Shen; Ying, Liu; Jianqi, Li; Gengying, Li

2005-12-01

A powerful graphical pulse sequence programming tool has been designed for creating magnetic resonance imaging (MRI) applications. It allows rapid development of pulse sequences in graphical mode (allowing for the visualization of sequences), and consists of three modules which include a graphical sequence editor, a parameter management module and a sequence compiler. Its key features are ease to use, flexibility and hardware independence. When graphic elements are combined with a certain text expressions, the graphical pulse sequence programming is as flexible as text-based programming tool. In addition, a hardware-independent design is implemented by using the strategy of two step compilations. To demonstrate the flexibility and the capability of this graphical sequence programming tool, a multi-slice fast spin echo experiment is performed on our home-made 0.3 T permanent magnet MRI system.

Genome-wide identification of wheat (Triticum aestivum) expansins and expansin expression analysis in cold-tolerant and cold-sensitive wheat cultivars

PubMed Central

Zhang, Jun-Feng; Xu, Yong-Qing; Dong, Jia-Min; Peng, Li-Na; Feng, Xu; Wang, Xu; Li, Fei; Miao, Yu; Yao, Shu-Kuan; Zhao, Qiao-Qin; Feng, Shan-Shan; Hu, Bao-Zhong

2018-01-01

Plant expansins are proteins involved in cell wall loosening, plant growth, and development, as well as in response to plant diseases and other stresses. In this study, we identified 128 expansin coding sequences from the wheat (Triticum aestivum) genome. These sequences belong to 45 homoeologous copies of TaEXPs, including 26 TaEXPAs, 15 TaEXPBs and four TaEXLAs. No TaEXLB was identified. Gene expression and sub-expression profiles revealed that most of the TaEXPs were expressed either only in root tissues or in multiple organs. Real-time qPCR analysis showed that many TaEXPs were differentially expressed in four different tissues of the two wheat cultivars—the cold-sensitive ‘Chinese Spring (CS)’ and the cold-tolerant ‘Dongnongdongmai 1 (D1)’ cultivars. Our results suggest that the differential expression of TaEXPs could be related to low-temperature tolerance or sensitivity of different wheat cultivars. Our study expands our knowledge on wheat expansins and sheds new light on the functions of expansins in plant development and stress response. PMID:29596529

A prokaryotic viral sequence is expressed and conserved in mammalian brain.

PubMed

Yeh, Yang-Hui; Gunasekharan, Vignesh; Manuelidis, Laura

2017-07-03

A natural and permanent transfer of prokaryotic viral sequences to mammals has not been reported by others. Circular "SPHINX" DNAs <5 kb were previously isolated from nuclease-protected cytoplasmic particles in rodent neuronal cell lines and brain. Two of these DNAs were sequenced after Φ29 polymerase amplification, and they revealed significant but imperfect homology to segments of commensal Acinetobacter phage viruses. These findings were surprising because the brain is isolated from environmental microorganisms. The 1.76-kb DNA sequence (SPHINX 1.8), with an iteron before its ORF, was evaluated here for its expression in neural cells and brain. A rabbit affinity purified antibody generated against a peptide without homology to mammalian sequences labeled a nonglycosylated ∼41-kDa protein (spx1) on Western blots, and the signal was efficiently blocked by the competing peptide. Spx1 was resistant to limited proteinase K digestion, but was unrelated to the expression of host prion protein or its pathologic amyloid form. Remarkably, spx1 concentrated in selected brain synapses, such as those on anterior motor horn neurons that integrate many complex neural inputs. SPHINX 1.8 appears to be involved in tissue-specific differentiation, including essential functions that preserve its propagation during mammalian evolution, possibly via maternal inheritance. The data here indicate that mammals can share and exchange a larger world of prokaryotic viruses than previously envisioned.

Effects of Self-Graphing on Written Expression of Fourth Grade Students with High-Incidence Disabilities

ERIC Educational Resources Information Center

Stotz, Kate E.; Itoi, Madoka; Konrad, Moira; Alber-Morgan, Sheila R.

2008-01-01

The purpose of this study was to determine the effects of self-graphing on the writing of 3 fourth grade students with high-incidence disabilities. Measures of written expression included total number of words written and number of correct word sequences. During intervention, students self-graphed their total number of words written in response to…

A Brassica oleracea gene expressed in a variety-specific manner may encode a novel plant transmembrane receptor.

PubMed

Palmer, J E; Dikeman, D A; Fujinuma, T; Kim, B; Jones, J I; Denda, M; Martínez-Zapater, J M; Cruz-Alvarez, M

2001-04-01

The species Brassica oleracea includes several agricultural varieties characterized by the proliferation of different types of meristems. Using a combination of subtractive hybridization and PCR (polymerase chain reaction) techniques we have identified several genes which are expressed in the reproductive meristems of the cauliflower curd (B. oleracea var. botrytis) but not in the vegetative meristems of Brussels sprouts (B. oleracea var. gemmifera) axillary buds. One of the cloned genes, termed CCE1 (CAULIFLOWER CURD EXPRESSION 1) shows specific expression in the botrytis variety. Preferential expression takes place in this variety in the meristems of the curd and in the stem throughout the vegetative and reproductive stages of plant growth. CCE1 transcripts are not detected in any of the organs of other B. oleracea varieties analyzed. Based on the nucleotide sequence of a cDNA encompassing the complete coding region, we predict that this gene encodes a transmembrane protein, with three transmembrane domains. The deduced amino acid sequence includes motifs conserved in G-protein-coupled receptors (GPCRs) from yeast and animal species. Our results suggest that the cloned gene encodes a protein belonging to a new, so far unidentified, family of transmembrane receptors in plants. The expression pattern of the gene suggests that the receptor may be involved in the control of meristem development/arrest that takes place in cauliflower.

Coordinate cytokine regulatory sequences

DOEpatents

Frazer, Kelly A.; Rubin, Edward M.; Loots, Gabriela G.

2005-05-10

The present invention provides CNS sequences that regulate the cytokine gene expression, expression cassettes and vectors comprising or lacking the CNS sequences, host cells and non-human transgenic animals comprising the CNS sequences or lacking the CNS sequences. The present invention also provides methods for identifying compounds that modulate the functions of CNS sequences as well as methods for diagnosing defects in the CNS sequences of patients.

An upstream sequence modulates phenazine production at the level of transcription and translation in the biological control strain Pseudomonas chlororaphis 30-84

PubMed Central

Wang, Dongping; Ries, Tessa R.; Pierson, Leland S.; Pierson, Elizabeth A.

2018-01-01

Phenazines are bacterial secondary metabolites and play important roles in the antagonistic activity of the biological control strain P. chlororaphis 30–84 against take-all disease of wheat. The expression of the P. chlororaphis 30–84 phenazine biosynthetic operon (phzXYFABCD) is dependent on the PhzR/PhzI quorum sensing system located immediately upstream of the biosynthetic operon as well as other regulatory systems including Gac/Rsm. Bioinformatic analysis of the sequence between the divergently oriented phzR and phzX promoters identified features within the 5’-untranslated region (5’-UTR) of phzX that are conserved only among 2OHPCA producing Pseudomonas. The conserved sequence features are potentially capable of producing secondary structures that negatively modulate one or both promoters. Transcriptional and translational fusion assays revealed that deletion of 90-bp of sequence at the 5’-UTR of phzX led to up to 4-fold greater expression of the reporters with the deletion compared to the controls, which indicated this sequence negatively modulates phenazine gene expression both transcriptionally and translationally. This 90-bp sequence was deleted from the P. chlororaphis 30–84 chromosome, resulting in 30-84Enh, which produces significantly more phenazine than the wild-type while retaining quorum sensing control. The transcriptional expression of phzR/phzI and amount of AHL signal produced by 30-84Enh also were significantly greater than for the wild-type, suggesting this 90-bp sequence also negatively affects expression of the quorum sensing genes. In addition, deletion of the 90-bp partially relieved RsmE-mediated translational repression, indicating a role for Gac/RsmE interaction. Compared to the wild-type, enhanced phenazine production by 30-84Enh resulted in improvement in fungal inhibition, biofilm formation, extracellular DNA release and suppression of take-all disease of wheat in soil without negative consequences on growth or rhizosphere persistence. This work provides greater insight into the regulation of phenazine biosynthesis with potential applications for improved biological control. PMID:29451920

IDENTIFICATION AND EXPRESSION OF MACROPHAGE MIGRATION INHIBITORY FACTOR IN SARCOPTES SCABIEI

PubMed Central

COTE’, N.M.; JAWORSKI, D.C.; WASALA, N.B.; MORGAN, M.S.; ARLIAN, L. G.

2013-01-01

Macrophage migration inhibitory factor (MIF) is a pleiotropic proinflammatory cytokine produced by many mammalian tissues including skin. It is also found in many invertebrate parasites of mammals including ticks and may function to aid the parasite to evade the innate and adaptive immune responses in the host. In this study, the cDNA for a MIF gene was sequenced from Sarcoptes scabiei, the scabies mite, using RT-PCR and RACE molecular techniques. The resulting nucleotide sequence had a length of 405 base pairs and the putative amino acid sequences for the mite and tick (Dermacentor variabilis) proteins were identical. The initial steps for the project resulted in the production of expressed scabies mite cDNAs. A real time (qPCR) assay was performed with MIF from scabies mites and various tick species. Results show that mRNA encoding MIF homologues was three times more abundant in the mite samples when compared to RNA prepared from D. variabilis salivary glands and 1.3 times more abundant when compared with RNA prepared from D. variabilis midgut. PMID:23831036

Molecular cloning, sequence characterization and recombinant expression of Nanog gene in goat fibroblast cells using lentiviral based expression system.

PubMed

Singhal, Dinesh K; Singhal, Raxita; Malik, Hruda N; Kumar, Surender; Kumar, Sudarshan; Mohanty, Ashok K; Kaushik, Jai K; Malakar, Dhruba

2014-01-01

Nanog is a homeodomain containing protein which plays important roles in regulation of signaling pathways for maintenance and induction of pluripotency in stem cells. Because of its unique expression in stem cells it is also regarded as pluripotency marker. In this study goat Nanog (gNanog) gene has been amplified, cloned and characterized at sequence level with successful over-expression in CHO-K1 cell line using a lentiviral based system. gNanog ORF is 903 bp long which codes for Nanog protein of size 300 amino acids (aas). Complete nucleotide sequence shows some evolutionary mutation in goat in comparision to other species. Protein sequence of goat is highly similar to other species. Overall, gNanog nucleotide sequence and predicted protein sequence showed high similarity and minimum divergence with cattle (96 % identity/4 % divergence) and buffalo (94/5 %) while low similarity and high divergence with pig (84/15 %), human (81/23 %) and mouse (69/40 %) indicating evolutionary closeness of gNanog to cattle and buffalo. gNanog lentiviral expression construct was prepared for over-expression of Nanog gene in adult goat fibroblast cells. Lentiviral expression construct of Nanog enabled continuous protein expression for induction and maintenance of pluripotency. Western blotting revealed the expression of Nanog gene at protein level which supported that the lentiviral expression system is highly promising for Nanog protein expression in differentiated goat cell.

Generation and analysis of expression sequence tags from haustoria of the wheat stripe rust fungus Puccinia striiformis f. sp. Tritici

PubMed Central

2009-01-01

Background Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is one of the most destructive diseases of wheat (Triticum aestivum L.) worldwide. In spite of its agricultural importance, the genomics and genetics of the pathogen are poorly characterized. Pst transcripts from urediniospores and germinated urediniospores have been examined previously, but little is known about genes expressed during host infection. Some genes involved in virulence in other rust fungi have been found to be specifically expressed in haustoria. Therefore, the objective of this study was to generate a cDNA library to characterize genes expressed in haustoria of Pst. Results A total of 5,126 EST sequences of high quality were generated from haustoria of Pst, from which 287 contigs and 847 singletons were derived. Approximately 10% and 26% of the 1,134 unique sequences were homologous to proteins with known functions and hypothetical proteins, respectively. The remaining 64% of the unique sequences had no significant similarities in GenBank. Fifteen genes were predicted to be proteins secreted from Pst haustoria. Analysis of ten genes, including six secreted protein genes, using quantitative RT-PCR revealed changes in transcript levels in different developmental and infection stages of the pathogen. Conclusions The haustorial cDNA library was useful in identifying genes of the stripe rust fungus expressed during the infection process. From the library, we identified 15 genes encoding putative secreted proteins and six genes induced during the infection process. These genes are candidates for further studies to determine their functions in wheat-Pst interactions. PMID:20028560

Expressed sequence tags from heat-shocked seagrass Zostera noltii (Hornemann) from its southern distribution range.

PubMed

Massa, Sónia I; Pearson, Gareth A; Aires, Tânia; Kube, Michael; Olsen, Jeanine L; Reinhardt, Richard; Serrão, Ester A; Arnaud-Haond, Sophie

2011-09-01

Predicted global climate change threatens the distributional ranges of species worldwide. We identified genes expressed in the intertidal seagrass Zostera noltii during recovery from a simulated low tide heat-shock exposure. Five Expressed Sequence Tag (EST) libraries were compared, corresponding to four recovery times following sub-lethal temperature stress, and a non-stressed control. We sequenced and analyzed 7009 sequence reads from 30min, 2h, 4h and 24h after the beginning of the heat-shock (AHS), and 1585 from the control library, for a total of 8594 sequence reads. Among 51 Tentative UniGenes (TUGs) exhibiting significantly different expression between libraries, 19 (37.3%) were identified as 'molecular chaperones' and were over-expressed following heat-shock, while 12 (23.5%) were 'photosynthesis TUGs' generally under-expressed in heat-shocked plants. A time course analysis of expression showed a rapid increase in expression of the molecular chaperone class, most of which were heat-shock proteins; which increased from 2 sequence reads in the control library to almost 230 in the 30min AHS library, followed by a slow decrease during further recovery. In contrast, 'photosynthesis TUGs' were under-expressed 30min AHS compared with the control library, and declined progressively with recovery time in the stress libraries, with a total of 29 sequence reads 24h AHS, compared with 125 in the control. A total of 4734 TUGs were screened for EST-Single Sequence Repeats (EST-SSRs) and 86 microsatellites were identified. Copyright © 2011 Elsevier B.V. All rights reserved.

The MPI Emotional Body Expressions Database for Narrative Scenarios

PubMed Central

Volkova, Ekaterina; de la Rosa, Stephan; Bülthoff, Heinrich H.; Mohler, Betty

2014-01-01

Emotion expression in human-human interaction takes place via various types of information, including body motion. Research on the perceptual-cognitive mechanisms underlying the processing of natural emotional body language can benefit greatly from datasets of natural emotional body expressions that facilitate stimulus manipulation and analysis. The existing databases have so far focused on few emotion categories which display predominantly prototypical, exaggerated emotion expressions. Moreover, many of these databases consist of video recordings which limit the ability to manipulate and analyse the physical properties of these stimuli. We present a new database consisting of a large set (over 1400) of natural emotional body expressions typical of monologues. To achieve close-to-natural emotional body expressions, amateur actors were narrating coherent stories while their body movements were recorded with motion capture technology. The resulting 3-dimensional motion data recorded at a high frame rate (120 frames per second) provides fine-grained information about body movements and allows the manipulation of movement on a body joint basis. For each expression it gives the positions and orientations in space of 23 body joints for every frame. We report the results of physical motion properties analysis and of an emotion categorisation study. The reactions of observers from the emotion categorisation study are included in the database. Moreover, we recorded the intended emotion expression for each motion sequence from the actor to allow for investigations regarding the link between intended and perceived emotions. The motion sequences along with the accompanying information are made available in a searchable MPI Emotional Body Expression Database. We hope that this database will enable researchers to study expression and perception of naturally occurring emotional body expressions in greater depth. PMID:25461382

Methods and compositions for regulating gene expression in plant cells

NASA Technical Reports Server (NTRS)

Dai, Shunhong (Inventor); Beachy, Roger N. (Inventor); Luis, Maria Isabel Ordiz (Inventor)

2010-01-01

Novel chimeric plant promoter sequences are provided, together with plant gene expression cassettes comprising such sequences. In certain preferred embodiments, the chimeric plant promoters comprise the BoxII cis element and/or derivatives thereof. In addition, novel transcription factors are provided, together with nucleic acid sequences encoding such transcription factors and plant gene expression cassettes comprising such nucleic acid sequences. In certain preferred embodiments, the novel transcription factors comprise the acidic domain, or fragments thereof, of the RF2a transcription factor. Methods for using the chimeric plant promoter sequences and novel transcription factors in regulating the expression of at least one gene of interest are provided, together with transgenic plants comprising such chimeric plant promoter sequences and novel transcription factors.

RNA sequencing reveals sexually dimorphic gene expression before gonadal differentiation in chicken and allows comprehensive annotation of the W-chromosome

PubMed Central

2013-01-01

Background Birds have a ZZ male: ZW female sex chromosome system and while the Z-linked DMRT1 gene is necessary for testis development, the exact mechanism of sex determination in birds remains unsolved. This is partly due to the poor annotation of the W chromosome, which is speculated to carry a female determinant. Few genes have been mapped to the W and little is known of their expression. Results We used RNA-seq to produce a comprehensive profile of gene expression in chicken blastoderms and embryonic gonads prior to sexual differentiation. We found robust sexually dimorphic gene expression in both tissues pre-dating gonadogenesis, including sex-linked and autosomal genes. This supports the hypothesis that sexual differentiation at the molecular level is at least partly cell autonomous in birds. Different sets of genes were sexually dimorphic in the two tissues, indicating that molecular sexual differentiation is tissue specific. Further analyses allowed the assembly of full-length transcripts for 26 W chromosome genes, providing a view of the W transcriptome in embryonic tissues. This is the first extensive analysis of W-linked genes and their expression profiles in early avian embryos. Conclusion Sexual differentiation at the molecular level is established in chicken early in embryogenesis, before gonadal sex differentiation. We find that the W chromosome is more transcriptionally active than previously thought, expand the number of known genes to 26 and present complete coding sequences for these W genes. This includes two novel W-linked sequences and three small RNAs reassigned to the W from the Un_Random chromosome. PMID:23531366

Genome and transcriptome sequencing in prospective metastatic triple-negative breast cancer uncovers therapeutic vulnerabilities.

PubMed

Craig, David W; O'Shaughnessy, Joyce A; Kiefer, Jeffrey A; Aldrich, Jessica; Sinari, Shripad; Moses, Tracy M; Wong, Shukmei; Dinh, Jennifer; Christoforides, Alexis; Blum, Joanne L; Aitelli, Cristi L; Osborne, Cynthia R; Izatt, Tyler; Kurdoglu, Ahmet; Baker, Angela; Koeman, Julie; Barbacioru, Catalin; Sakarya, Onur; De La Vega, Francisco M; Siddiqui, Asim; Hoang, Linh; Billings, Paul R; Salhia, Bodour; Tolcher, Anthony W; Trent, Jeffrey M; Mousses, Spyro; Von Hoff, Daniel; Carpten, John D

2013-01-01

Triple-negative breast cancer (TNBC) is characterized by the absence of expression of estrogen receptor, progesterone receptor, and HER-2. Thirty percent of patients recur after first-line treatment, and metastatic TNBC (mTNBC) has a poor prognosis with median survival of one year. Here, we present initial analyses of whole genome and transcriptome sequencing data from 14 prospective mTNBC. We have cataloged the collection of somatic genomic alterations in these advanced tumors, particularly those that may inform targeted therapies. Genes mutated in multiple tumors included TP53, LRP1B, HERC1, CDH5, RB1, and NF1. Notable genes involved in focal structural events were CTNNA1, PTEN, FBXW7, BRCA2, WT1, FGFR1, KRAS, HRAS, ARAF, BRAF, and PGCP. Homozygous deletion of CTNNA1 was detected in 2 of 6 African Americans. RNA sequencing revealed consistent overexpression of the FOXM1 gene when tumor gene expression was compared with nonmalignant breast samples. Using an outlier analysis of gene expression comparing one cancer with all the others, we detected expression patterns unique to each patient's tumor. Integrative DNA/RNA analysis provided evidence for deregulation of mutated genes, including the monoallelic expression of TP53 mutations. Finally, molecular alterations in several cancers supported targeted therapeutic intervention on clinical trials with known inhibitors, particularly for alterations in the RAS/RAF/MEK/ERK and PI3K/AKT/mTOR pathways. In conclusion, whole genome and transcriptome profiling of mTNBC have provided insights into somatic events occurring in this difficult to treat cancer. These genomic data have guided patients to investigational treatment trials and provide hypotheses for future trials in this irremediable cancer.

High-Throughput Sequencing Reveals Differential Expression of miRNAs in Intestine from Sea Cucumber during Aestivation

PubMed Central

Chen, Muyan; Zhang, Xiumei; Liu, Jianning; Storey, Kenneth B.

2013-01-01

The regulatory role of miRNA in gene expression is an emerging hot new topic in the control of hypometabolism. Sea cucumber aestivation is a complicated physiological process that includes obvious hypometabolism as evidenced by a decrease in the rates of oxygen consumption and ammonia nitrogen excretion, as well as a serious degeneration of the intestine into a very tiny filament. To determine whether miRNAs play regulatory roles in this process, the present study analyzed profiles of miRNA expression in the intestine of the sea cucumber (Apostichopus japonicus), using Solexa deep sequencing technology. We identified 308 sea cucumber miRNAs, including 18 novel miRNAs specific to sea cucumber. Animals sampled during deep aestivation (DA) after at least 15 days of continuous torpor, were compared with animals from a non-aestivation (NA) state (animals that had passed through aestivation and returned to the active state). We identified 42 differentially expressed miRNAs [RPM (reads per million) >10, |FC| (|fold change|) ≥1, FDR (false discovery rate) <0.01] during aestivation, which were validated by two other miRNA profiling methods: miRNA microarray and real-time PCR. Among the most prominent miRNA species, miR-200-3p, miR-2004, miR-2010, miR-22, miR-252a, miR-252a-3p and miR-92 were significantly over-expressed during deep aestivation compared with non-aestivation animals. Preliminary analyses of their putative target genes and GO analysis suggest that these miRNAs could play important roles in global transcriptional depression and cell differentiation during aestivation. High-throughput sequencing data and microarray data have been submitted to GEO database. PMID:24143179

ICAM-1-related long non-coding RNA: promoter analysis and expression in human retinal endothelial cells.

PubMed

Lumsden, Amanda L; Ma, Yuefang; Ashander, Liam M; Stempel, Andrew J; Keating, Damien J; Smith, Justine R; Appukuttan, Binoy

2018-05-09

Regulation of intercellular adhesion molecule (ICAM)-1 in retinal endothelial cells is a promising druggable target for retinal vascular diseases. The ICAM-1-related (ICR) long non-coding RNA stabilizes ICAM-1 transcript, increasing protein expression. However, studies of ICR involvement in disease have been limited as the promoter is uncharacterized. To address this issue, we undertook a comprehensive in silico analysis of the human ICR gene promoter region. We used genomic evolutionary rate profiling to identify a 115 base pair (bp) sequence within 500 bp upstream of the transcription start site of the annotated human ICR gene that was conserved across 25 eutherian genomes. A second constrained sequence upstream of the orthologous mouse gene (68 bp; conserved across 27 Eutherian genomes including human) was also discovered. Searching these elements identified 33 matrices predictive of binding sites for transcription factors known to be responsive to a broad range of pathological stimuli, including hypoxia, and metabolic and inflammatory proteins. Five phenotype-associated single nucleotide polymorphisms (SNPs) in the immediate vicinity of these elements included four SNPs (i.e. rs2569693, rs281439, rs281440 and rs11575074) predicted to impact binding motifs of transcription factors, and thus the expression of ICR and ICAM-1 genes, with potential to influence disease susceptibility. We verified that human retinal endothelial cells expressed ICR, and observed induction of expression by tumor necrosis factor-α.

Co-expression of the Thermotoga neapolitana aglB gene with an upstream 3'-coding fragment of the malG gene improves enzymatic characteristics of recombinant AglB cyclomaltodextrinase.

PubMed

Lunina, Natalia A; Agafonova, Elena V; Chekanovskaya, Lyudmila A; Dvortsov, Igor A; Berezina, Oksana V; Shedova, Ekaterina N; Kostrov, Sergey V; Velikodvorskaya, Galina A

2007-07-01

A cluster of Thermotoga neapolitana genes participating in starch degradation includes the malG gene of sugar transport protein and the aglB gene of cyclomaltodextrinase. The start and stop codons of these genes share a common overlapping sequence, aTGAtg. Here, we compared properties of expression products of three different constructs with aglB from T. neapolitana. The first expression vector contained the aglB gene linked to an upstream 90-bp 3'-terminal region of the malG gene with the stop codon overlapping with the start codon of aglB. The second construct included the isolated coding sequence of aglB with two tandem potential start codons. The expression product of this construct in Escherichia coli had two tandem Met residues at its N terminus and was characterized by low thermostability and high tendency to aggregate. In contrast, co-expression of aglB and the 3'-terminal region of malG (the first construct) resulted in AglB with only one N-terminal Met residue and a much higher specific activity of cyclomaltodextrinase. Moreover, the enzyme expressed by such a construct was more thermostable and less prone to aggregation. The third construct was the same as the second one except that it contained only one ATG start codon. The product of its expression had kinetic and other properties similar to those of the enzyme with only one N-terminal Met residue.

Sequences 5' to translation start regulate expression of petunia rbcS genes.

PubMed Central

Dean, C; Favreau, M; Bedbrook, J; Dunsmuir, P

1989-01-01

The promoter sequences that contribute to quantitative differences in expression of the petunia genes (rbcS) encoding the small subunit of ribulose bisphosphate carboxylase have been characterized. The promoter regions of the two most abundantly expressed petunia rbcS genes, SSU301 and SSU611, show sequence similarity not present in other rbcS genes. We investigated the significance of these and other sequences by adding specific regions from the SSU301 promoter (the most strongly expressed gene) to equivalent regions in the SSU911 promoter (the least strongly expressed gene) and assaying the expression of the fusions in transgenic tobacco plants. In this way, we characterized an SSU301 promoter region (either from -285 to -178 or -291 to -204) which, when added to SSU911, in either orientation, increased SSU911 expression 25-fold. This increase was equivalent to that caused by addition of the entire SSU301 5'-flanking region. Replacement of SSU911 promoter sequences between -198 and the start codon with sequences from the equivalent region of SSU301 did not increase SSU911 expression significantly. The -291 to -204 SSU301 promoter fragment contributes significantly to quantitative differences in expression between the petunia rbcS genes. PMID:2535543

miRNEST database: an integrative approach in microRNA search and annotation

PubMed Central

Szcześniak, Michał Wojciech; Deorowicz, Sebastian; Gapski, Jakub; Kaczyński, Łukasz; Makałowska, Izabela

2012-01-01

Despite accumulating data on animal and plant microRNAs and their functions, existing public miRNA resources usually collect miRNAs from a very limited number of species. A lot of microRNAs, including those from model organisms, remain undiscovered. As a result there is a continuous need to search for new microRNAs. We present miRNEST (http://mirnest.amu.edu.pl), a comprehensive database of animal, plant and virus microRNAs. The core part of the database is built from our miRNA predictions conducted on Expressed Sequence Tags of 225 animal and 202 plant species. The miRNA search was performed based on sequence similarity and as many as 10 004 miRNA candidates in 221 animal and 199 plant species were discovered. Out of them only 299 have already been deposited in miRBase. Additionally, miRNEST has been integrated with external miRNA data from literature and 13 databases, which includes miRNA sequences, small RNA sequencing data, expression, polymorphisms and targets data as well as links to external miRNA resources, whenever applicable. All this makes miRNEST a considerable miRNA resource in a sense of number of species (544) that integrates a scattered miRNA data into a uniform format with a user-friendly web interface. PMID:22135287

The Eimeria Transcript DB: an integrated resource for annotated transcripts of protozoan parasites of the genus Eimeria

PubMed Central

Rangel, Luiz Thibério; Novaes, Jeniffer; Durham, Alan M.; Madeira, Alda Maria B. N.; Gruber, Arthur

2013-01-01

Parasites of the genus Eimeria infect a wide range of vertebrate hosts, including chickens. We have recently reported a comparative analysis of the transcriptomes of Eimeria acervulina, Eimeria maxima and Eimeria tenella, integrating ORESTES data produced by our group and publicly available Expressed Sequence Tags (ESTs). All cDNA reads have been assembled, and the reconstructed transcripts have been submitted to a comprehensive functional annotation pipeline. Additional studies included orthology assignment across apicomplexan parasites and clustering analyses of gene expression profiles among different developmental stages of the parasites. To make all this body of information publicly available, we constructed the Eimeria Transcript Database (EimeriaTDB), a web repository that provides access to sequence data, annotation and comparative analyses. Here, we describe the web interface, available sequence data sets and query tools implemented on the site. The main goal of this work is to offer a public repository of sequence and functional annotation data of reconstructed transcripts of parasites of the genus Eimeria. We believe that EimeriaTDB will represent a valuable and complementary resource for the Eimeria scientific community and for those researchers interested in comparative genomics of apicomplexan parasites. Database URL: http://www.coccidia.icb.usp.br/eimeriatdb/ PMID:23411718

Gene expression profiling via LongSAGE in a non-model plant species: a case study in seeds of Brassica napus

PubMed Central

Obermeier, Christian; Hosseini, Bashir; Friedt, Wolfgang; Snowdon, Rod

2009-01-01

Background Serial analysis of gene expression (LongSAGE) was applied for gene expression profiling in seeds of oilseed rape (Brassica napus ssp. napus). The usefulness of this technique for detailed expression profiling in a non-model organism was demonstrated for the highly complex, neither fully sequenced nor annotated genome of B. napus by applying a tag-to-gene matching strategy based on Brassica ESTs and the annotated proteome of the closely related model crucifer A. thaliana. Results Transcripts from 3,094 genes were detected at two time-points of seed development, 23 days and 35 days after pollination (DAP). Differential expression showed a shift from gene expression involved in diverse developmental processes including cell proliferation and seed coat formation at 23 DAP to more focussed metabolic processes including storage protein accumulation and lipid deposition at 35 DAP. The most abundant transcripts at 23 DAP were coding for diverse protease inhibitor proteins and proteases, including cysteine proteases involved in seed coat formation and a number of lipid transfer proteins involved in embryo pattern formation. At 35 DAP, transcripts encoding napin, cruciferin and oleosin storage proteins were most abundant. Over both time-points, 18.6% of the detected genes were matched by Brassica ESTs identified by LongSAGE tags in antisense orientation. This suggests a strong involvement of antisense transcript expression in regulatory processes during B. napus seed development. Conclusion This study underlines the potential of transcript tagging approaches for gene expression profiling in Brassica crop species via EST matching to annotated A. thaliana genes. Limits of tag detection for low-abundance transcripts can today be overcome by ultra-high throughput sequencing approaches, so that tag-based gene expression profiling may soon become the method of choice for global expression profiling in non-model species. PMID:19575793

An Epstein-Barr virus immediate-early gene product trans-activates gene expression from the human immunodeficiency virus long terminal repeat.

PubMed

Kenney, S; Kamine, J; Markovitz, D; Fenrick, R; Pagano, J

1988-03-01

Acquired immunodeficiency syndrome patients are frequently coinfected with Epstein-Barr virus (EBV). In this report, we demonstrate that an EBV immediate-early gene product, BamHI MLF1, stimulates expression of the bacterial chloramphenicol acetyltransferase (CAT) gene linked to the human immunodeficiency virus (HIV) promoter. The HIV promoter sequences necessary for trans-activation by EBV do not include the tat-responsive sequences. In addition, in contrast to the other herpesvirus trans-activators previously studied, the EBV BamHI MLF1 gene product appears to function in part by a posttranscriptional mechanism, since it increases pHIV-CAT protein activity more than it increases HIV-CAT mRNA. This ability of an EBV gene product to activate HIV gene expression may have biologic consequences in persons coinfected with both viruses.

Transcriptome Sequencing Revealed Significant Alteration of Cortical Promoter Usage and Splicing in Schizophrenia

PubMed Central

Wu, Jing Qin; Wang, Xi; Beveridge, Natalie J.; Tooney, Paul A.; Scott, Rodney J.; Carr, Vaughan J.; Cairns, Murray J.

2012-01-01

Background While hybridization based analysis of the cortical transcriptome has provided important insight into the neuropathology of schizophrenia, it represents a restricted view of disease-associated gene activity based on predetermined probes. By contrast, sequencing technology can provide un-biased analysis of transcription at nucleotide resolution. Here we use this approach to investigate schizophrenia-associated cortical gene expression. Methodology/Principal Findings The data was generated from 76 bp reads of RNA-Seq, aligned to the reference genome and assembled into transcripts for quantification of exons, splice variants and alternative promoters in postmortem superior temporal gyrus (STG/BA22) from 9 male subjects with schizophrenia and 9 matched non-psychiatric controls. Differentially expressed genes were then subjected to further sequence and functional group analysis. The output, amounting to more than 38 Gb of sequence, revealed significant alteration of gene expression including many previously shown to be associated with schizophrenia. Gene ontology enrichment analysis followed by functional map construction identified three functional clusters highly relevant to schizophrenia including neurotransmission related functions, synaptic vesicle trafficking, and neural development. Significantly, more than 2000 genes displayed schizophrenia-associated alternative promoter usage and more than 1000 genes showed differential splicing (FDR<0.05). Both types of transcriptional isoforms were exemplified by reads aligned to the neurodevelopmentally significant doublecortin-like kinase 1 (DCLK1) gene. Conclusions This study provided the first deep and un-biased analysis of schizophrenia-associated transcriptional diversity within the STG, and revealed variants with important implications for the complex pathophysiology of schizophrenia. PMID:22558445

Integrating Transcriptome and Genome Re-Sequencing Data to Identify Key Genes and Mutations Affecting Chicken Eggshell Qualities

PubMed Central

Liu, Long; Zheng, Chuan Wei; Wang, De He; Hou, Zhuo Cheng; Ning, Zhong Hua

2015-01-01

Eggshell damages lead to economic losses in the egg production industry and are a threat to human health. We examined 49-wk-old Rhode Island White hens (Gallus gallus) that laid eggs having shells with significantly different strengths and thicknesses. We used HiSeq 2000 (Illumina) sequencing to characterize the chicken transcriptome and whole genome to identify the key genes and genetic mutations associated with eggshell calcification. We identified a total of 14,234 genes expressed in the chicken uterus, representing 89% of all annotated chicken genes. A total of 889 differentially expressed genes were identified by comparing low eggshell strength (LES) and normal eggshell strength (NES) genomes. The DEGs are enriched in calcification-related processes, including calcium ion transport and calcium signaling pathways as reveled by gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis. Some important matrix proteins, such as OC-116, LTF and SPP1, were also expressed differentially between two groups. A total of 3,671,919 single-nucleotide polymorphisms (SNPs) and 508,035 Indels were detected in protein coding genes by whole-genome re-sequencing, including 1775 non-synonymous variations and 19 frame-shift Indels in DEGs. SNPs and Indels found in this study could be further investigated for eggshell traits. This is the first report to integrate the transcriptome and genome re-sequencing to target the genetic variations which decreased the eggshell qualities. These findings further advance our understanding of eggshell calcification in the chicken uterus. PMID:25974068

Characterization of defensin gene from abalone Haliotis discus hannai and its deduced protein

NASA Astrophysics Data System (ADS)

Hong, Xuguang; Sun, Xiuqin; Zheng, Minggang; Qu, Lingyun; Zan, Jindong; Zhang, Jinxing

2008-11-01

Defensin is one of preserved ancient host defensive materials formed in biological evolution. As a regulator and effector molecule, it is very important in animals’ acquired immune system. This paper reports the defensin gene from the mixed liver and kidney cDNA library of abalone Haliotis discus hannai Ino. Sequence analysis shows that the gene sequence of full-length cDNA encodes 42 mature peptides (including six Cys), molecular weight of 4 323 Da, and pI of 8.02. Amino acid sequence homology analysis shows that the peptides are highly similar (70% in common) to other insects defensin. Because of a typical insect-defensin structural character of mature peptide in the secondary structure, the polypeptide named Haliotis discus defensin (hd-def), a novel of antimicrobial peptides, belongs to insects defensin subfamily. The RT-PCR result of Haliotis discus defensin shows that the gene can be expressed only in the hepatopancreas by Gram-negative and positive bacteria stimulation, which is ascribed to inducible expression. Therefore, it is revealed that the Haliotis discus defensin gene expression was related to the antibacterial infection of Haliotis discus hannai Ino.

Diversity amongst trigeminal neurons revealed by high throughput single cell sequencing

PubMed Central

Nguyen, Minh Q.; Wu, Youmei; Bonilla, Lauren S.; von Buchholtz, Lars J.

2017-01-01

The trigeminal ganglion contains somatosensory neurons that detect a range of thermal, mechanical and chemical cues and innervate unique sensory compartments in the head and neck including the eyes, nose, mouth, meninges and vibrissae. We used single-cell sequencing and in situ hybridization to examine the cellular diversity of the trigeminal ganglion in mice, defining thirteen clusters of neurons. We show that clusters are well conserved in dorsal root ganglia suggesting they represent distinct functional classes of somatosensory neurons and not specialization associated with their sensory targets. Notably, functionally important genes (e.g. the mechanosensory channel Piezo2 and the capsaicin gated ion channel Trpv1) segregate into multiple clusters and often are expressed in subsets of cells within a cluster. Therefore, the 13 genetically-defined classes are likely to be physiologically heterogeneous rather than highly parallel (i.e., redundant) lines of sensory input. Our analysis harnesses the power of single-cell sequencing to provide a unique platform for in silico expression profiling that complements other approaches linking gene-expression with function and exposes unexpected diversity in the somatosensory system. PMID:28957441

Non-coding variants contribute to the clinical heterogeneity of TTR amyloidosis.

PubMed

Iorio, Andrea; De Lillo, Antonella; De Angelis, Flavio; Di Girolamo, Marco; Luigetti, Marco; Sabatelli, Mario; Pradotto, Luca; Mauro, Alessandro; Mazzeo, Anna; Stancanelli, Claudia; Perfetto, Federico; Frusconi, Sabrina; My, Filomena; Manfellotto, Dario; Fuciarelli, Maria; Polimanti, Renato

2017-09-01

Coding mutations in TTR gene cause a rare hereditary form of systemic amyloidosis, which has a complex genotype-phenotype correlation. We investigated the role of non-coding variants in regulating TTR gene expression and consequently amyloidosis symptoms. We evaluated the genotype-phenotype correlation considering the clinical information of 129 Italian patients with TTR amyloidosis. Then, we conducted a re-sequencing of TTR gene to investigate how non-coding variants affect TTR expression and, consequently, phenotypic presentation in carriers of amyloidogenic mutations. Polygenic scores for genetically determined TTR expression were constructed using data from our re-sequencing analysis and the GTEx (Genotype-Tissue Expression) project. We confirmed a strong phenotypic heterogeneity across coding mutations causing TTR amyloidosis. Considering the effects of non-coding variants on TTR expression, we identified three patient clusters with specific expression patterns associated with certain phenotypic presentations, including late onset, autonomic neurological involvement, and gastrointestinal symptoms. This study provides novel data regarding the role of non-coding variation and the gene expression profiles in patients affected by TTR amyloidosis, also putting forth an approach that could be used to investigate the mechanisms at the basis of the genotype-phenotype correlation of the disease.

Identification and characterization of novel and differentially expressed microRNAs in peripheral blood from healthy and mastitis Holstein cattle by deep sequencing.

PubMed

Li, Zhixiong; Wang, Hongliang; Chen, Ling; Wang, Lijun; Liu, Xiaolin; Ru, Caixia; Song, Ailong

2014-02-01

MicroRNA (miRNA) mediates post-transcriptional gene regulation and plays an important role in regulating the development of immune cells and in modulating innate and adaptive immune responses in mammals, including cattle. In the present study, we identified novel and differentially expressed miRNAs in peripheral blood from healthy and mastitis Holstein cattle by Solexa sequencing and bioinformatics. In total, 608 precursor hairpins (pre-miRNAs) encoding for 753 mature miRNAs were detected. Statistically, 173 unique miRNAs (of 753, 22.98%) were identified that had significant differential expression between healthy and mastitis Holstein cattle (P < 0.001). Most differentially expressed miRNAs (118 of 173, 68.21%) belonged to the chemokine signaling pathway involved in the immune responses. This study expands the number of miRNAs known to be expressed in cattle. The patterns of miRNAs expression differed significantly between the peripheral blood from healthy and mastitis Holstein cattle, which provide important information on mastitis in miRNAs expression. Diverse miRNAs may play an important role in the treatment of mastitis in Holstein cattle. © 2013 Stichting International Foundation for Animal Genetics.

Regulation of miRNA Processing and miRNA Mediated Gene Repression in Cancer

PubMed Central

Bajan, Sarah; Hutvagner, Gyorgy

2014-01-01

The majority of human protein-coding genes are predicted to be targets of miRNA-mediated post-transcriptional regulation. The widespread influence of miRNAs is illustrated by their essential roles in all biological processes. Regulated miRNA expression is essential for maintaining cellular differentiation; therefore alterations in miRNA expression patterns are associated with several diseases, including various cancers. High-throughput sequencing technologies revealed low level expressing miRNA isoforms, termed isomiRs. IsomiRs may differ in sequence, length, target preference and expression patterns from their parental miRNA and can arise from differences in miRNA biosynthesis, RNA editing, or SNPs inherent to the miRNA gene. The association between isomiR expression and disease progression is largely unknown. Misregulated miRNA expression is thought to contribute to the formation and/or progression of cancer. However, due to the diversity of targeted transcripts, miRNAs can function as both tumor-suppressor genes and oncogenes as defined by cellular context. Despite this, miRNA profiling studies concluded that the differential expression of particular miRNAs in diseased tissue could aid the diagnosis and treatment of some cancers. PMID:25069508

Differential Expression of HERV-K (HML-2) Proviruses in Cells and Virions of the Teratocarcinoma Cell Line Tera-1

PubMed Central

Bhardwaj, Neeru; Montesion, Meagan; Roy, Farrah; Coffin, John M.

2015-01-01

Human endogenous retrovirus (HERV-K (HML-2)) proviruses are among the few endogenous retroviral elements in the human genome that retain coding sequence. HML-2 expression has been widely associated with human disease states, including different types of cancers as well as with HIV-1 infection. Understanding of the potential impact of this expression requires that it be annotated at the proviral level. Here, we utilized the high throughput capabilities of next-generation sequencing to profile HML-2 expression at the level of individual proviruses and secreted virions in the teratocarcinoma cell line Tera-1. We identified well-defined expression patterns, with transcripts emanating primarily from two proviruses located on chromosome 22, only one of which was efficiently packaged. Interestingly, there was a preference for transcripts of recently integrated proviruses, over those from other highly expressed but older elements, to be packaged into virions. We also assessed the promoter competence of the 5’ long terminal repeats (LTRs) of expressed proviruses via a luciferase assay following transfection of Tera-1 cells. Consistent with the RNASeq results, we found that the activity of most LTRs corresponded to their transcript levels. PMID:25746218

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

Jenny, Matthew J.; Department of Biological Sciences, University of Alabama, Tuscaloosa, AL 35487; Aluru, Neelakanteswar

Although many drugs and environmental chemicals are teratogenic, the mechanisms by which most toxicants disrupt embryonic development are not well understood. MicroRNAs, single-stranded RNA molecules of ∼ 22 nt that regulate protein expression by inhibiting mRNA translation and promoting mRNA sequestration or degradation, are important regulators of a variety of cellular processes including embryonic development and cellular differentiation. Recent studies have demonstrated that exposure to xenobiotics can alter microRNA expression and contribute to the mechanisms by which environmental chemicals disrupt embryonic development. In this study we tested the hypothesis that developmental exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a well-known teratogen, alters microRNAmore » expression during zebrafish development. We exposed zebrafish embryos to DMSO (0.1%) or TCDD (5 nM) for 1 h at 30 hours post fertilization (hpf) and measured microRNA expression using several methods at 36 and 60 hpf. TCDD caused strong induction of CYP1A at 36 hpf (62-fold) and 60 hpf (135-fold) as determined by real-time RT-PCR, verifying the effectiveness of the exposure. MicroRNA expression profiles were determined using microarrays (Agilent and Exiqon), next-generation sequencing (SOLiD), and real-time RT-PCR. The two microarray platforms yielded results that were similar but not identical; both showed significant changes in expression of miR-451, 23a, 23b, 24 and 27e at 60 hpf. Multiple analyses were performed on the SOLiD sequences yielding a total of 16 microRNAs as differentially expressed by TCDD in zebrafish embryos. However, miR-27e was the only microRNA to be identified as differentially expressed by all three methods (both microarrays, SOLiD sequencing, and real-time RT-PCR). These results suggest that TCDD exposure causes modest changes in expression of microRNAs, including some (miR-451, 23a, 23b, 24 and 27e) that are critical for hematopoiesis and cardiovascular development. -- Highlights: ► Zebrafish embryos were exposed to TCDD at two different developmental timepoints. ► Compared different methods in detecting global changes in microRNA expression. ► TCDD caused significant changes in microRNA expression in zebrafish embryos. ► Differentially expressed microRNAs have roles related to TCDD-induced phenotypes.« less

Identifying Novel Helix–Loop–Helix Genes in Caenorhabditis elegans through a Classroom Demonstration of Functional Genomics

PubMed Central

Griffin, Vernetta; McMiller, Tracee; Jones, Erika; Johnson, Casonya M.

2003-01-01

A 14-week, undergraduate-level Genetics and Population Biology course at Morgan State University was modified to include a demonstration of functional genomics in the research laboratory. Students performed a rudimentary sequence analysis of the Caenorhabditis elegans genome and further characterized three sequences that were predicted to encode helix–loop–helix proteins. Students then used reverse transcription–polymerase chain reaction to determine which of the three genes is normally expressed in C. elegans. At the end of this laboratory activity, students were 1) to demonstrate a rudimentary knowledge of bioinformatics, including the ability to differentiate between “having” a gene and “expressing” a gene, and 2) to understand basic approaches to functional genomics, including one specific technique for assaying for gene expression. It was also anticipated that students would increase their skills at effectively communicating their research activities through written and/or oral presentation. This article describes the laboratory activity and the assessment of the effectiveness of the activity. PMID:12822036

Characterization of microRNAs from goat (Capra hircus) by Solexa deep-sequencing technology.

PubMed

Ling, Y H; Ding, J P; Zhang, X D; Wang, L J; Zhang, Y H; Li, Y S; Zhang, Z J; Zhang, X R

2013-06-13

MicroRNAs (miRNAs) are an important class of small noncoding RNAs that are highly conserved in plants and animals. Many miRNAs are known to mediate a myriad of cell processes, including proliferation and differentiation, via the regulation of some transcription and signaling factors, which are closely related to muscle development and disease. In this study, small RNA cDNA libraries of Boer goats were constructed. In addition, we obtained the goat muscle miRNAs by using Solexa deep-sequencing technology and analyzed these miRNA characteristics by combining it with the bioinformatics technology. Based on Solexa sequencing and bioinformatics analysis, 562 species-conserved and 5 goat genome-specific miRNAs were identified, 322 of which exceeded 100 in the expression levels. The results of real-time quantitative polymerase chain reaction from 8 randomly selected miRNAs showed that the 8 miRNAs were expressed in goat muscle, and the expression patterns were consistent with the Solexa sequencing results. The identification and characterization of miRNAs in goat muscle provide important information on the role of miRNA regulation in muscle growth and development. These data will help to facilitate studies on the regulatory roles played by miRNAs during goat growth and development.

Antisense Transcription Is Pervasive but Rarely Conserved in Enteric Bacteria

PubMed Central

Raghavan, Rahul; Sloan, Daniel B.; Ochman, Howard

2012-01-01

ABSTRACT Noncoding RNAs, including antisense RNAs (asRNAs) that originate from the complementary strand of protein-coding genes, are involved in the regulation of gene expression in all domains of life. Recent application of deep-sequencing technologies has revealed that the transcription of asRNAs occurs genome-wide in bacteria. Although the role of the vast majority of asRNAs remains unknown, it is often assumed that their presence implies important regulatory functions, similar to those of other noncoding RNAs. Alternatively, many antisense transcripts may be produced by chance transcription events from promoter-like sequences that result from the degenerate nature of bacterial transcription factor binding sites. To investigate the biological relevance of antisense transcripts, we compared genome-wide patterns of asRNA expression in closely related enteric bacteria, Escherichia coli and Salmonella enterica serovar Typhimurium, by performing strand-specific transcriptome sequencing. Although antisense transcripts are abundant in both species, less than 3% of asRNAs are expressed at high levels in both species, and only about 14% appear to be conserved among species. And unlike the promoters of protein-coding genes, asRNA promoters show no evidence of sequence conservation between, or even within, species. Our findings suggest that many or even most bacterial asRNAs are nonadaptive by-products of the cell’s transcription machinery. PMID:22872780

Transcriptome sequencing and annotation of the halophytic microalga Dunaliella salina * #

PubMed Central

Hong, Ling; Liu, Jun-li; Midoun, Samira Z.; Miller, Philip C.

2017-01-01

The unicellular green alga Dunaliella salina is well adapted to salt stress and contains compounds (including β-carotene and vitamins) with potential commercial value. A large transcriptome database of D. salina during the adjustment, exponential and stationary growth phases was generated using a high throughput sequencing platform. We characterized the metabolic processes in D. salina with a focus on valuable metabolites, with the aim of manipulating D. salina to achieve greater economic value in large-scale production through a bioengineering strategy. Gene expression profiles under salt stress verified using quantitative polymerase chain reaction (qPCR) implied that salt can regulate the expression of key genes. This study generated a substantial fraction of D. salina transcriptional sequences for the entire growth cycle, providing a basis for the discovery of novel genes. This first full-scale transcriptome study of D. salina establishes a foundation for further comparative genomic studies. PMID:28990374

Identification of an expressed gene in Dipylidium caninum.

PubMed

Miranda, Rodrigo R C; Costa-Júnior, Livio M; Campos, Artur K; Santos, Hudson A; Rabelo, Elida M L

2004-10-01

Recombinant DNA studies have been focused on developing vaccines to different cestodes. But few studies involving Dipylidium caninum molecular biology and genes have been done. Only partial sequences of mitochondrial DNA and ribosomal RNA gene are available in databases. Any molecular work with this parasite, including epidemiology, study of drug-resistant strains, and vaccine development, is hampered by the lack of knowledge of its genome. Thus, the knowledge of specific genes of different developmental stages of D. caninum is crucial to locate potential targets to be used as candidates to develop a vaccine and/or new drugs against this parasite. Here we report, for the first time, the sequencing of a fragment of a D. caninum expressed gene.

Evaluation of anonymous and expressed sequence tag derived polymorphic microsatellite markers in the tobacco budworm Heliothis virescens (Lepidoptera: noctuidae)

USDA-ARS?s Scientific Manuscript database

Polymorphic genetic markers were identified and characterized using a partial genomic library of Heliothis virescens enriched for simple sequence repeats (SSR) and nucleotide sequences of expressed sequence tags (EST). Nucleotide sequences of 192 clones from the partial genomic library yielded 147 u...

An Automated Pipeline for Engineering Many-Enzyme Pathways: Computational Sequence Design, Pathway Expression-Flux Mapping, and Scalable Pathway Optimization.

PubMed

Halper, Sean M; Cetnar, Daniel P; Salis, Howard M

2018-01-01

Engineering many-enzyme metabolic pathways suffers from the design curse of dimensionality. There are an astronomical number of synonymous DNA sequence choices, though relatively few will express an evolutionary robust, maximally productive pathway without metabolic bottlenecks. To solve this challenge, we have developed an integrated, automated computational-experimental pipeline that identifies a pathway's optimal DNA sequence without high-throughput screening or many cycles of design-build-test. The first step applies our Operon Calculator algorithm to design a host-specific evolutionary robust bacterial operon sequence with maximally tunable enzyme expression levels. The second step applies our RBS Library Calculator algorithm to systematically vary enzyme expression levels with the smallest-sized library. After characterizing a small number of constructed pathway variants, measurements are supplied to our Pathway Map Calculator algorithm, which then parameterizes a kinetic metabolic model that ultimately predicts the pathway's optimal enzyme expression levels and DNA sequences. Altogether, our algorithms provide the ability to efficiently map the pathway's sequence-expression-activity space and predict DNA sequences with desired metabolic fluxes. Here, we provide a step-by-step guide to applying the Pathway Optimization Pipeline on a desired multi-enzyme pathway in a bacterial host.

Control of total GFP expression by alterations to the 3′ region nucleotide sequence

PubMed Central

2013-01-01

Background Previously, we distinguished the Escherichia coli type II cytoplasmic membrane translocation pathways of Tat, Yid, and Sec for unfolded and folded soluble target proteins. The translocation of folded protein to the periplasm for soluble expression via the Tat pathway was controlled by an N-terminal hydrophilic leader sequence. In this study, we investigated the effect of the hydrophilic C-terminal end and its nucleotide sequence on total and soluble protein expression. Results The native hydrophilic C-terminal end of GFP was obtained by deleting the C-terminal peptide LeuGlu-6×His, derived from pET22b(+). The corresponding clones induced total and soluble GFP expression that was either slightly increased or dramatically reduced, apparently through reconstruction of the nucleotide sequence around the stop codon in the 3′ region. In the expression-induced clones, the hydrophilic C-terminus showed increased Tat pathway specificity for soluble expression. However, in the expression-reduced clone, after analyzing the role of the 5′ poly(A) coding sequence with a substituted synonymous codon, we proved that the longer 5′ poly(A) coding sequence interacted with the reconstructed 3′ region nucleotide sequence to create a new mRNA tertiary structure between the 5′ and 3′ regions, which resulted in reduced total GFP expression. Further, to recover the reduced expression by changing the 3′ nucleotide sequence, after replacing selected C-terminal 5′ codons and the stop codon in the ORF with synonymous codons, total GFP expression in most of the clones was recovered to the undeleted control level. The insertion of trinucleotides after the stop codon in the 3′-UTR recovered or reduced total GFP expression. RT-PCR revealed that the level of total protein expression was controlled by changes in translational or transcriptional regulation, which were induced or reduced by the substitution or insertion of 3′ region nucleotides. Conclusions We found that the hydrophilic C-terminal end of GFP increased Tat pathway specificity and that the 3′ nucleotide sequence played an important role in total protein expression through translational and transcriptional regulation. These findings may be useful for efficiently producing recombinant proteins as well as for potentially controlling the expression level of specific genes in the body for therapeutic purposes. PMID:23834827

Differential expression of copper-zinc superoxide dismutase gene of Polygonum sibiricum leaves, stems and underground stems, subjected to high-salt stress.

PubMed

Qu, Chun-Pu; Xu, Zhi-Ru; Liu, Guan-Jun; Liu, Chun; Li, Yang; Wei, Zhi-Gang; Liu, Gui-Feng

2010-01-01

In aerobic organisms, protection against oxidative damage involves the combined action of highly specialized antioxidant enzymes, such as copper-zinc superoxide dismutase. In this work, a cDNA clone which encodes a copper-zinc superoxide dismutase gene, named PS-CuZnSOD, has been identified from P. sibiricum Laxm. by the rapid amplification of cDNA ends method (RACE). Analysis of the nucleotide sequence reveals that the PS-CuZnSOD gene cDNA clone consists of 669 bp, containing 87 bp in the 5' untranslated region; 459 bp in the open reading frame (ORF) encoding 152 amino acids; and 123 bp in 3' untranslated region. The gene accession nucleotide sequence number in GenBank is GQ472846. Sequence analysis indicates that the protein, like most plant superoxide dismutases (SOD), includes two conserved ecCuZnSOD signatures that are from the amino acids 43 to 51, and from the amino acids 137 to 148, and it has a signal peptide extension in the front of the N-terminus (1-16 aa). Expression analysis by real-time quantitative PCR reveals that the PS-CuZnSOD gene is expressed in leaves, stems and underground stems. PS-CuZnSOD gene expression can be induced by 3% NaHCO(3). The different mRNA levels' expression of PS-CuZnSOD show the gene's different expression modes in leaves, stems and underground stems under the salinity-alkalinity stress.

Global analysis of gene expression profiles in developing physic nut (Jatropha curcas L.) seeds.

PubMed

Jiang, Huawu; Wu, Pingzhi; Zhang, Sheng; Song, Chi; Chen, Yaping; Li, Meiru; Jia, Yongxia; Fang, Xiaohua; Chen, Fan; Wu, Guojiang

2012-01-01

Physic nut (Jatropha curcas L.) is an oilseed plant species with high potential utility as a biofuel. Furthermore, following recent sequencing of its genome and the availability of expressed sequence tag (EST) libraries, it is a valuable model plant for studying carbon assimilation in endosperms of oilseed plants. There have been several transcriptomic analyses of developing physic nut seeds using ESTs, but they have provided limited information on the accumulation of stored resources in the seeds. We applied next-generation Illumina sequencing technology to analyze global gene expression profiles of developing physic nut seeds 14, 19, 25, 29, 35, 41, and 45 days after pollination (DAP). The acquired profiles reveal the key genes, and their expression timeframes, involved in major metabolic processes including: carbon flow, starch metabolism, and synthesis of storage lipids and proteins in the developing seeds. The main period of storage reserves synthesis in the seeds appears to be 29-41 DAP, and the fatty acid composition of the developing seeds is consistent with relative expression levels of different isoforms of acyl-ACP thioesterase and fatty acid desaturase genes. Several transcription factor genes whose expression coincides with storage reserve deposition correspond to those known to regulate the process in Arabidopsis. The results will facilitate searches for genes that influence de novo lipid synthesis, accumulation and their regulatory networks in developing physic nut seeds, and other oil seeds. Thus, they will be helpful in attempts to modify these plants for efficient biofuel production.

Prunus transcription factors: breeding perspectives

PubMed Central

Bianchi, Valmor J.; Rubio, Manuel; Trainotti, Livio; Verde, Ignazio; Bonghi, Claudio; Martínez-Gómez, Pedro

2015-01-01

Many plant processes depend on differential gene expression, which is generally controlled by complex proteins called transcription factors (TFs). In peach, 1533 TFs have been identified, accounting for about 5.5% of the 27,852 protein-coding genes. These TFs are the reference for the rest of the Prunus species. TF studies in Prunus have been performed on the gene expression analysis of different agronomic traits, including control of the flowering process, fruit quality, and biotic and abiotic stress resistance. These studies, using quantitative RT-PCR, have mainly been performed in peach, and to a lesser extent in other species, including almond, apricot, black cherry, Fuji cherry, Japanese apricot, plum, and sour and sweet cherry. Other tools have also been used in TF studies, including cDNA-AFLP, LC-ESI-MS, RNA, and DNA blotting or mapping. More recently, new tools assayed include microarray and high-throughput DNA sequencing (DNA-Seq) and RNA sequencing (RNA-Seq). New functional genomics opportunities include genome resequencing and the well-known synteny among Prunus genomes and transcriptomes. These new functional studies should be applied in breeding programs in the development of molecular markers. With the genome sequences available, some strategies that have been used in model systems (such as SNP genotyping assays and genotyping-by-sequencing) may be applicable in the functional analysis of Prunus TFs as well. In addition, the knowledge of the gene functions and position in the peach reference genome of the TFs represents an additional advantage. These facts could greatly facilitate the isolation of genes via QTL (quantitative trait loci) map-based cloning in the different Prunus species, following the association of these TFs with the identified QTLs using the peach reference genome. PMID:26124770

Expressed sequence tag analysis of functional genes associated with adventitious rooting in Liriodendron hybrids.

PubMed

Zhong, Y D; Sun, X Y; Liu, E Y; Li, Y Q; Gao, Z; Yu, F X

2016-06-24

Liriodendron hybrids (Liriodendron chinense x L. tulipifera) are important landscaping and afforestation hardwood trees. To date, little genomic research on adventitious rooting has been reported in these hybrids, as well as in the genus Liriodendron. In the present study, we used adventitious roots to construct the first cDNA library for Liriodendron hybrids. A total of 5176 expressed sequence tags (ESTs) were generated and clustered into 2921 unigenes. Among these unigenes, 2547 had significant homology to the non-redundant protein database representing a wide variety of putative functions. Homologs of these genes regulated many aspects of adventitious rooting, including those for auxin signal transduction and root hair development. Results of quantitative real-time polymerase chain reaction showed that AUX1, IRE, and FB1 were highly expressed in adventitious roots and the expression of AUX1, ARF1, NAC1, RHD1, and IRE increased during the development of adventitious roots. Additionally, 181 simple sequence repeats were identified from 166 ESTs and more than 91.16% of these were dinucleotide and trinucleotide repeats. To the best of our knowledge, the present study reports the identification of the genes associated with adventitious rooting in the genus Liriodendron for the first time and provides a valuable resource for future genomic studies. Expression analysis of selected genes could allow us to identify regulatory genes that may be essential for adventitious rooting.

Site-targeted mutagenesis for stabilization of recombinant monoclonal antibody expressed in tobacco (Nicotiana tabacum) plants

PubMed Central

Hehle, Verena K.; Paul, Matthew J.; Roberts, Victoria A.; van Dolleweerd, Craig J.; Ma, Julian K.-C.

2016-01-01

This study examined the degradation pattern of a murine IgG1κ monoclonal antibody expressed in and extracted from transformed Nicotiana tabacum. Gel electrophoresis of leaf extracts revealed a consistent pattern of recombinant immunoglobulin bands, including intact and full-length antibody, as well as smaller antibody fragments. N-terminal sequencing revealed these smaller fragments to be proteolytic cleavage products and identified a limited number of protease-sensitive sites in the antibody light and heavy chain sequences. No strictly conserved target sequence was evident, although the peptide bonds that were susceptible to proteolysis were predominantly and consistently located within or near to the interdomain or solvent-exposed regions in the antibody structure. Amino acids surrounding identified cleavage sites were mutated in an attempt to increase resistance. Different Guy’s 13 antibody heavy and light chain mutant combinations were expressed transiently in N. tabacum and demonstrated intensity shifts in the fragmentation pattern, resulting in alterations to the full-length antibody-to-fragment ratio. The work strengthens the understanding of proteolytic cleavage of antibodies expressed in plants and presents a novel approach to stabilize full-length antibody by site-directed mutagenesis.—Hehle, V. K., Paul, M. J., Roberts, V. A., van Dolleweerd, C. J., Ma, J. K.-C. Site-targeted mutagenesis for stabilization of recombinant monoclonal antibody expressed in tobacco (Nicotiana tabacum) plants. PMID:26712217

Serial analysis of gene expression (SAGE) in normal human trabecular meshwork.

PubMed

Liu, Yutao; Munro, Drew; Layfield, David; Dellinger, Andrew; Walter, Jeffrey; Peterson, Katherine; Rickman, Catherine Bowes; Allingham, R Rand; Hauser, Michael A

2011-04-08

To identify the genes expressed in normal human trabecular meshwork tissue, a tissue critical to the pathogenesis of glaucoma. Total RNA was extracted from human trabecular meshwork (HTM) harvested from 3 different donors. Extracted RNA was used to synthesize individual SAGE (serial analysis of gene expression) libraries using the I-SAGE Long kit from Invitrogen. Libraries were analyzed using SAGE 2000 software to extract the 17 base pair sequence tags. The extracted sequence tags were mapped to the genome using SAGE Genie map. A total of 298,834 SAGE tags were identified from all HTM libraries (96,842, 88,126, and 113,866 tags, respectively). Collectively, there were 107,325 unique tags. There were 10,329 unique tags with a minimum of 2 counts from a single library. These tags were mapped to known unique Unigene clusters. Approximately 29% of the tags (orphan tags) did not map to a known Unigene cluster. Thirteen percent of the tags mapped to at least 2 Unigene clusters. Sequence tags from many glaucoma-related genes, including myocilin, optineurin, and WD repeat domain 36, were identified. This is the first time SAGE analysis has been used to characterize the gene expression profile in normal HTM. SAGE analysis provides an unbiased sampling of gene expression of the target tissue. These data will provide new and valuable information to improve understanding of the biology of human aqueous outflow.

Structural and functional conservation of CLEC-2 with the species-specific regulation of transcript expression in evolution.

PubMed

Wang, Lan; Ren, Shifang; Zhu, Haiyan; Zhang, Dongmei; Hao, Yuqing; Ruan, Yuanyuan; Zhou, Lei; Lee, Chiayu; Qiu, Lin; Yun, Xiaojing; Xie, Jianhui

2012-08-01

CLEC-2 was first identified by sequence similarity to C-type lectin-like molecules with immune functions and has been reported as a receptor for the platelet-aggregating snake venom toxin rhodocytin and the endogenous sialoglycoprotein podoplanin. Recent researches indicate that CLEC-2-deficient mice were lethal at the embryonic stage associated with disorganized and blood-filled lymphatic vessels and severe edema. In view of a necessary role of CLEC-2 in the individual development, it is of interest to investigate its phylogenetic homology and highly conserved functional regions. In this work, we reported that CLEC-2 from different species holds with an extraordinary conservation by sequence alignment and phylogenetic tree analysis. The functional structures including N-linked oligosaccharide sites and ligand-binding domain implement a structural and functional conservation in a variety of species. The glycosylation sites (N120 and N134) are necessary for the surface expression CLEC-2. CLEC-2 from different species possesses the binding activity of mouse podoplanin. Nevertheless, the expression of CLEC-2 is regulated with a species-specific manner. The alternative splicing of pre-mRNA, a regulatory mechanism of gene expression, and the binding sites on promoter for several key transcription factors vary between different species. Therefore, CLEC-2 shares high sequence homology and functional identity. However the transcript expression might be tightly regulated by different mechanisms in evolution.

Genome-Scale Transcriptome Analysis in Response to Nitric Oxide in Birch Cells: Implications of the Triterpene Biosynthetic Pathway

PubMed Central

Zeng, Fansuo; Sun, Fengkun; Li, Leilei; Liu, Kun; Zhan, Yaguang

2014-01-01

Evidence supporting nitric oxide (NO) as a mediator of plant biochemistry continues to grow, but its functions at the molecular level remains poorly understood and, in some cases, controversial. To study the role of NO at the transcriptional level in Betula platyphylla cells, we conducted a genome-scale transcriptome analysis of these cells. The transcriptome of untreated birch cells and those treated by sodium nitroprusside (SNP) were analyzed using the Solexa sequencing. Data were collected by sequencing cDNA libraries of birch cells, which had a long period to adapt to the suspension culture conditions before SNP-treated cells and untreated cells were sampled. Among the 34,100 UniGenes detected, BLASTX search revealed that 20,631 genes showed significant (E-values≤10−5) sequence similarity with proteins from the NR-database. Numerous expressed sequence tags (i.e., 1374) were identified as differentially expressed between the 12 h SNP-treated cells and control cells samples: 403 up-regulated and 971 down-regulated. From this, we specifically examined a core set of NO-related transcripts. The altered expression levels of several transcripts, as determined by transcriptome analysis, was confirmed by qRT-PCR. The results of transcriptome analysis, gene expression quantification, the content of triterpenoid and activities of defensive enzymes elucidated NO has a significant effect on many processes including triterpenoid production, carbohydrate metabolism and cell wall biosynthesis. PMID:25551661

Deep transcriptome sequencing provides new insights into the structural and functional organization of the wheat genome.

PubMed

Pingault, Lise; Choulet, Frédéric; Alberti, Adriana; Glover, Natasha; Wincker, Patrick; Feuillet, Catherine; Paux, Etienne

2015-02-10

Because of its size, allohexaploid nature, and high repeat content, the bread wheat genome is a good model to study the impact of the genome structure on gene organization, function, and regulation. However, because of the lack of a reference genome sequence, such studies have long been hampered and our knowledge of the wheat gene space is still limited. The access to the reference sequence of the wheat chromosome 3B provided us with an opportunity to study the wheat transcriptome and its relationships to genome and gene structure at a level that has never been reached before. By combining this sequence with RNA-seq data, we construct a fine transcriptome map of the chromosome 3B. More than 8,800 transcription sites are identified, that are distributed throughout the entire chromosome. Expression level, expression breadth, alternative splicing as well as several structural features of genes, including transcript length, number of exons, and cumulative intron length are investigated. Our analysis reveals a non-monotonic relationship between gene expression and structure and leads to the hypothesis that gene structure is determined by its function, whereas gene expression is subject to energetic cost. Moreover, we observe a recombination-based partitioning at the gene structure and function level. Our analysis provides new insights into the relationships between gene and genome structure and function. It reveals mechanisms conserved with other plant species as well as superimposed evolutionary forces that shaped the wheat gene space, likely participating in wheat adaptation.

GENE-Counter: A Computational Pipeline for the Analysis of RNA-Seq Data for Gene Expression Differences

PubMed Central

Di, Yanming; Schafer, Daniel W.; Wilhelm, Larry J.; Fox, Samuel E.; Sullivan, Christopher M.; Curzon, Aron D.; Carrington, James C.; Mockler, Todd C.; Chang, Jeff H.

2011-01-01

GENE-counter is a complete Perl-based computational pipeline for analyzing RNA-Sequencing (RNA-Seq) data for differential gene expression. In addition to its use in studying transcriptomes of eukaryotic model organisms, GENE-counter is applicable for prokaryotes and non-model organisms without an available genome reference sequence. For alignments, GENE-counter is configured for CASHX, Bowtie, and BWA, but an end user can use any Sequence Alignment/Map (SAM)-compliant program of preference. To analyze data for differential gene expression, GENE-counter can be run with any one of three statistics packages that are based on variations of the negative binomial distribution. The default method is a new and simple statistical test we developed based on an over-parameterized version of the negative binomial distribution. GENE-counter also includes three different methods for assessing differentially expressed features for enriched gene ontology (GO) terms. Results are transparent and data are systematically stored in a MySQL relational database to facilitate additional analyses as well as quality assessment. We used next generation sequencing to generate a small-scale RNA-Seq dataset derived from the heavily studied defense response of Arabidopsis thaliana and used GENE-counter to process the data. Collectively, the support from analysis of microarrays as well as the observed and substantial overlap in results from each of the three statistics packages demonstrates that GENE-counter is well suited for handling the unique characteristics of small sample sizes and high variability in gene counts. PMID:21998647

Molecular cloning and responsive expression to injury stimulus of a defender against cell death 1 (DAD1) gene from bay scallops Argopecten irradians.

PubMed

Zhu, Ling; Song, Linsheng; Zhang, Huan; Zhao, Jianmin; Li, Chenghua; Xu, Wei

2008-06-01

Apoptosis is an active process of cell death, which is an integral part of growth and development in multicellular organisms. The defender against cell death 1 (DAD1), the regulatory protein to inhibit the apoptosis process, was first cloned from the bay scallop Argopecten irradians by randomly sequencing a whole tissue cDNA library and rapid amplification of cDNA end (RACE). The full-length cDNA of the A. irradians DAD1 was 607 bp, consist of a 5'-terminal untranslated region (UTR) of 63 bp, a 3'-terminal UTR of 205 bp with a canonical polyadenylation signal sequence AATAAA and a poly (A) tail, and an open reading frame of 339 bp. The deduced amino acid sequence of the A. irradians DAD1 showed 75.5% identity to Araneus ventricosus, 74.5% to Drosophila melanogaster, and 73.6% to Homo sapiens, Sus scrofa, Mesocricetus auratus, Rattus norvegicus and Mus musculus. Excluding the Saccharomyces cerevisiae DAD1 homologue, all animal DAD1 including A. irradians DAD1 homologue formed a subgroup and all plant DAD1 proteins formed another subgroup in the phylogenetic analysis. The A. irradians DAD1 was expressed in all examined tissues including adductor muscle, mantle, gills, digestive gland, gonad and hemolymph, suggesting that A. irradians DAD1 is expressed in most body tissues. Furthermore, the mRNA expression levels of A. irradians DAD1 gene of hemolymph were particularly high after injury, suggesting that the gene is responsive to injury stimuli.

Highly specific gene silencing in a monocot species by artificial microRNAs derived from chimeric miRNA precursors

DOE PAGES

Carbonell, Alberto; Fahlgren, Noah; Mitchell, Skyler; ...

2015-05-20

Artificial microRNAs (amiRNAs) are used for selective gene silencing in plants. However, current methods to produce amiRNA constructs for silencing transcripts in monocot species are not suitable for simple, cost-effective and large-scale synthesis. Here, a series of expression vectors based on Oryza sativa MIR390 (OsMIR390) precursor was developed for high-throughput cloning and high expression of amiRNAs in monocots. Four different amiRNA sequences designed to target specifically endogenous genes and expressed from OsMIR390-based vectors were validated in transgenic Brachypodium distachyon plants. Surprisingly, amiRNAs accumulated to higher levels and were processed more accurately when expressed from chimeric OsMIR390-based precursors that include distalmore » stem-loop sequences from Arabidopsis thaliana MIR390a (AtMIR390a). In all cases, transgenic plants displayed the predicted phenotypes induced by target gene repression, and accumulated high levels of amiRNAs and low levels of the corresponding target transcripts. Genome-wide transcriptome profiling combined with 5-RLM-RACE analysis in transgenic plants confirmed that amiRNAs were highly specific. Finally, significance Statement A series of amiRNA vectors based on Oryza sativa MIR390 (OsMIR390) precursor were developed for simple, cost-effective and large-scale synthesis of amiRNA constructs to silence genes in monocots. Unexpectedly, amiRNAs produced from chimeric OsMIR390-based precursors including Arabidopsis thaliana MIR390a distal stem-loop sequences accumulated elevated levels of highly effective and specific amiRNAs in transgenic Brachypodium distachyon plants.« less

An Optimized Transient Dual Luciferase Assay for Quantifying MicroRNA Directed Repression of Targeted Sequences

PubMed Central

Moyle, Richard L.; Carvalhais, Lilia C.; Pretorius, Lara-Simone; Nowak, Ekaterina; Subramaniam, Gayathery; Dalton-Morgan, Jessica; Schenk, Peer M.

2017-01-01

Studies investigating the action of small RNAs on computationally predicted target genes require some form of experimental validation. Classical molecular methods of validating microRNA action on target genes are laborious, while approaches that tag predicted target sequences to qualitative reporter genes encounter technical limitations. The aim of this study was to address the challenge of experimentally validating large numbers of computationally predicted microRNA-target transcript interactions using an optimized, quantitative, cost-effective, and scalable approach. The presented method combines transient expression via agroinfiltration of Nicotiana benthamiana leaves with a quantitative dual luciferase reporter system, where firefly luciferase is used to report the microRNA-target sequence interaction and Renilla luciferase is used as an internal standard to normalize expression between replicates. We report the appropriate concentration of N. benthamiana leaf extracts and dilution factor to apply in order to avoid inhibition of firefly LUC activity. Furthermore, the optimal ratio of microRNA precursor expression construct to reporter construct and duration of the incubation period post-agroinfiltration were determined. The optimized dual luciferase assay provides an efficient, repeatable and scalable method to validate and quantify microRNA action on predicted target sequences. The optimized assay was used to validate five predicted targets of rice microRNA miR529b, with as few as six technical replicates. The assay can be extended to assess other small RNA-target sequence interactions, including assessing the functionality of an artificial miRNA or an RNAi construct on a targeted sequence. PMID:28979287

Sustained expression of MCP-1 by low wall shear stress loading concomitant with turbulent flow on endothelial cells of intracranial aneurysm.

PubMed

Aoki, Tomohiro; Yamamoto, Kimiko; Fukuda, Miyuki; Shimogonya, Yuji; Fukuda, Shunichi; Narumiya, Shuh

2016-05-09

Enlargement of a pre-existing intracranial aneurysm is a well-established risk factor of rupture. Excessive low wall shear stress concomitant with turbulent flow in the dome of an aneurysm may contribute to progression and rupture. However, how stress conditions regulate enlargement of a pre-existing aneurysm remains to be elucidated. Wall shear stress was calculated with 3D-computational fluid dynamics simulation using three cases of unruptured intracranial aneurysm. The resulting value, 0.017 Pa at the dome, was much lower than that in the parent artery. We loaded wall shear stress corresponding to the value and also turbulent flow to the primary culture of endothelial cells. We then obtained gene expression profiles by RNA sequence analysis. RNA sequence analysis detected hundreds of differentially expressed genes among groups. Gene ontology and pathway analysis identified signaling related with cell division/proliferation as overrepresented in the low wall shear stress-loaded group, which was further augmented by the addition of turbulent flow. Moreover, expression of some chemoattractants for inflammatory cells, including MCP-1, was upregulated under low wall shear stress with concomitant turbulent flow. We further examined the temporal sequence of expressions of factors identified in an in vitro study using a rat model. No proliferative cells were detected, but MCP-1 expression was induced and sustained in the endothelial cell layer. Low wall shear stress concomitant with turbulent flow contributes to sustained expression of MCP-1 in endothelial cells and presumably plays a role in facilitating macrophage infiltration and exacerbating inflammation, which leads to enlargement or rupture.

A novel cytochrome P450 gene (CYP4G25) of the silkmoth Antheraea yamamai: cloning and expression pattern in pharate first instar larvae in relation to diapause.

PubMed

Yang, Ping; Tanaka, Hiromasa; Kuwano, Eiichi; Suzuki, Koichi

2008-03-01

A new cytochrome P450 gene, CYP4G25, was identified as a differentially expressed gene between the diapausing and post-diapausing pharate first instar larvae of the wild silkmoth Antheraea yamamai, using subtractive cDNA hybridization. The cDNA sequence of CYP4G25 has an open reading frame of 1674 nucleotides encoding 557 amino acid residues. Sequence analysis of the putative CYP4G25 protein disclosed the motif FXXGXRXCXG that is essential for heme binding in P450 cytochromes. Hybridization in situ demonstrated predominant expression of CYP4G25 in the integument of pharate first instar larvae. Northern blotting analysis showed an intensive signal after the initiation of diapause and no or weak expression throughout the periods of pre-diapause and post-diapause, including larval development. These results indicate that CYP4G25 is strongly associated with diapause in pharate first instar larvae.

Cloning and expression of a novel lysophospholipase which structurally resembles lecithin cholesterol acyltransferase.

PubMed

Taniyama, Y; Shibata, S; Kita, S; Horikoshi, K; Fuse, H; Shirafuji, H; Sumino, Y; Fujino, M

1999-04-02

Lecithin cholesterol acyltransferase (LCAT) is the key enzyme in the esterification of plasma cholesterol and in the reverse cholesterol transport on high-density lipoprotein (HDL). We have found a novel LCAT-related gene among differentially expressed cDNA fragments between two types of foam cells derived from THP-1 cells, which are different in cholesterol efflux ability, using a subtractive PCR technique. The deduced 412-amino-acid sequence has 49% amino acid sequence similarity with human LCAT. In contrast to the liver-specific expression of LCAT, mRNA expression of the gene was observed mainly in peripheral tissues including kidney, placenta, pancreas, testis, spleen, heart, and skeletal muscle. The protein exists in human plasma and is probably associated with HDL. Moreover, we discovered that the recombinant protein hydrolyzed lysophosphatidylcholine (lysoPC), a proatherogenic lipid, to glycerophosphorylcholine and a free fatty acid. We have therefore named this novel enzyme LCAT-like lysophospholipase (LLPL), through which a new catabolic pathway for lysoPC on lipoproteins could be elucidated. Copyright 1999 Academic Press.

Natural Antisense Transcripts: Molecular Mechanisms and Implications in Breast Cancers

PubMed Central

Latgé, Guillaume; Poulet, Christophe; Bours, Vincent; Jerusalem, Guy

2018-01-01

Natural antisense transcripts are RNA sequences that can be transcribed from both DNA strands at the same locus but in the opposite direction from the gene transcript. Because strand-specific high-throughput sequencing of the antisense transcriptome has only been available for less than a decade, many natural antisense transcripts were first described as long non-coding RNAs. Although the precise biological roles of natural antisense transcripts are not known yet, an increasing number of studies report their implication in gene expression regulation. Their expression levels are altered in many physiological and pathological conditions, including breast cancers. Among the potential clinical utilities of the natural antisense transcripts, the non-coding|coding transcript pairs are of high interest for treatment. Indeed, these pairs can be targeted by antisense oligonucleotides to specifically tune the expression of the coding-gene. Here, we describe the current knowledge about natural antisense transcripts, their varying molecular mechanisms as gene expression regulators, and their potential as prognostic or predictive biomarkers in breast cancers. PMID:29301303

Natural Antisense Transcripts: Molecular Mechanisms and Implications in Breast Cancers.

PubMed

Latgé, Guillaume; Poulet, Christophe; Bours, Vincent; Josse, Claire; Jerusalem, Guy

2018-01-02

Natural antisense transcripts are RNA sequences that can be transcribed from both DNA strands at the same locus but in the opposite direction from the gene transcript. Because strand-specific high-throughput sequencing of the antisense transcriptome has only been available for less than a decade, many natural antisense transcripts were first described as long non-coding RNAs. Although the precise biological roles of natural antisense transcripts are not known yet, an increasing number of studies report their implication in gene expression regulation. Their expression levels are altered in many physiological and pathological conditions, including breast cancers. Among the potential clinical utilities of the natural antisense transcripts, the non-coding|coding transcript pairs are of high interest for treatment. Indeed, these pairs can be targeted by antisense oligonucleotides to specifically tune the expression of the coding-gene. Here, we describe the current knowledge about natural antisense transcripts, their varying molecular mechanisms as gene expression regulators, and their potential as prognostic or predictive biomarkers in breast cancers.

Genome-wide analyses of long noncoding RNA expression profiles correlated with radioresistance in nasopharyngeal carcinoma via next-generation deep sequencing.

PubMed

Li, Guo; Liu, Yong; Liu, Chao; Su, Zhongwu; Ren, Shuling; Wang, Yunyun; Deng, Tengbo; Huang, Donghai; Tian, Yongquan; Qiu, Yuanzheng

2016-09-06

Radioresistance is one of the major factors limiting the therapeutic efficacy and prognosis of patients with nasopharyngeal carcinoma (NPC). Accumulating evidence has suggested that aberrant expression of long noncoding RNAs (lncRNAs) contributes to cancer progression. Therefore, here we identified lncRNAs associated with radioresistance in NPC. The differential expression profiles of lncRNAs associated with NPC radioresistance were constructed by next-generation deep sequencing by comparing radioresistant NPC cells with their parental cells. LncRNA-related mRNAs were predicted and analyzed using bioinformatics algorithms compared with the mRNA profiles related to radioresistance obtained in our previous study. Several lncRNAs and associated mRNAs were validated in established NPC radioresistant cell models and NPC tissues. By comparison between radioresistant CNE-2-Rs and parental CNE-2 cells by next-generation deep sequencing, a total of 781 known lncRNAs and 2054 novel lncRNAs were annotated. The top five upregulated and downregulated known/novel lncRNAs were detected using quantitative real-time reverse transcription-polymerase chain reaction, and 7/10 known lncRNAs and 3/10 novel lncRNAs were demonstrated to have significant differential expression trends that were the same as those predicted by deep sequencing. From the prediction process, 13 pairs of lncRNAs and their associated genes were acquired, and the prediction trends of three pairs were validated in both radioresistant CNE-2-Rs and 6-10B-Rs cell lines, including lncRNA n373932 and SLITRK5, n409627 and PRSS12, and n386034 and RIMKLB. LncRNA n373932 and its related SLITRK5 showed dramatic expression changes in post-irradiation radioresistant cells and a negative expression correlation in NPC tissues (R = -0.595, p < 0.05). Our study provides an overview of the expression profiles of radioresistant lncRNAs and potentially related mRNAs, which will facilitate future investigations into the function of lncRNAs in NPC radioresistance.

Genome-wide discovery and differential regulation of conserved and novel microRNAs in chickpea via deep sequencing.

PubMed

Jain, Mukesh; Chevala, V V S Narayana; Garg, Rohini

2014-11-01

MicroRNAs (miRNAs) are essential components of complex gene regulatory networks that orchestrate plant development. Although several genomic resources have been developed for the legume crop chickpea, miRNAs have not been discovered until now. For genome-wide discovery of miRNAs in chickpea (Cicer arietinum), we sequenced the small RNA content from seven major tissues/organs employing Illumina technology. About 154 million reads were generated, which represented more than 20 million distinct small RNA sequences. We identified a total of 440 conserved miRNAs in chickpea based on sequence similarity with known miRNAs in other plants. In addition, 178 novel miRNAs were identified using a miRDeep pipeline with plant-specific scoring. Some of the conserved and novel miRNAs with significant sequence similarity were grouped into families. The chickpea miRNAs targeted a wide range of mRNAs involved in diverse cellular processes, including transcriptional regulation (transcription factors), protein modification and turnover, signal transduction, and metabolism. Our analysis revealed several miRNAs with differential spatial expression. Many of the chickpea miRNAs were expressed in a tissue-specific manner. The conserved and differential expression of members of the same miRNA family in different tissues was also observed. Some of the same family members were predicted to target different chickpea mRNAs, which suggested the specificity and complexity of miRNA-mediated developmental regulation. This study, for the first time, reveals a comprehensive set of conserved and novel miRNAs along with their expression patterns and putative targets in chickpea, and provides a framework for understanding regulation of developmental processes in legumes. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Genome-wide discovery of novel and conserved microRNAs in white shrimp (Litopenaeus vannamei).

PubMed

Xi, Qian-Yun; Xiong, Yuan-Yan; Wang, Yuan-Mei; Cheng, Xiao; Qi, Qi-En; Shu, Gang; Wang, Song-Bo; Wang, Li-Na; Gao, Ping; Zhu, Xiao-Tong; Jiang, Qing-Yan; Zhang, Yong-Liang; Liu, Li

2015-01-01

Of late years, a large amount of conserved and species-specific microRNAs (miRNAs) have been performed on identification from species which are economically important but lack a full genome sequence. In this study, Solexa deep sequencing and cross-species miRNA microarray were used to detect miRNAs in white shrimp. We identified 239 conserved miRNAs, 14 miRNA* sequences and 20 novel miRNAs by bioinformatics analysis from 7,561,406 high-quality reads representing 325,370 distinct sequences. The all 20 novel miRNAs were species-specific in white shrimp and not homologous in other species. Using the conserved miRNAs from the miRBase database as a query set to search for homologs from shrimp expressed sequence tags (ESTs), 32 conserved computationally predicted miRNAs were discovered in shrimp. In addition, using microarray analysis in the shrimp fed with Panax ginseng polysaccharide complex, 151 conserved miRNAs were identified, 18 of which were significant up-expression, while 49 miRNAs were significant down-expression. In particular, qRT-PCR analysis was also performed for nine miRNAs in three shrimp tissues such as muscle, gill and hepatopancreas. Results showed that these miRNAs expression are tissue specific. Combining results of the three methods, we detected 20 novel and 394 conserved miRNAs. Verification with quantitative reverse transcription (qRT-PCR) and Northern blot showed a high confidentiality of data. The study provides the first comprehensive specific miRNA profile of white shrimp, which includes useful information for future investigations into the function of miRNAs in regulation of shrimp development and immunology.

De novo sequencing and analysis of the cranberry fruit transcriptome to identify putative genes involved in flavonoid biosynthesis, transport and regulation.

PubMed

Sun, Haiyue; Liu, Yushan; Gai, Yuzhuo; Geng, Jinman; Chen, Li; Liu, Hongdi; Kang, Limin; Tian, Youwen; Li, Yadong

2015-09-02

Cranberries (Vaccinium macrocarpon Ait.), renowned for their excellent health benefits, are an important berry crop. Here, we performed transcriptome sequencing of one cranberry cultivar, from fruits at two different developmental stages, on the Illumina HiSeq 2000 platform. Our main goals were to identify putative genes for major metabolic pathways of bioactive compounds and compare the expression patterns between white fruit (W) and red fruit (R) in cranberry. In this study, two cDNA libraries of W and R were constructed. Approximately 119 million raw sequencing reads were generated and assembled de novo, yielding 57,331 high quality unigenes with an average length of 739 bp. Using BLASTx, 38,460 unigenes were identified as putative homologs of annotated sequences in public protein databases, including NCBI NR, NT, Swiss-Prot, KEGG, COG and GO. Of these, 21,898 unigenes mapped to 128 KEGG pathways, with the metabolic pathways, secondary metabolites, glycerophospholipid metabolism, ether lipid metabolism, starch and sucrose metabolism, purine metabolism, and pyrimidine metabolism being well represented. Among them, many candidate genes were involved in flavonoid biosynthesis, transport and regulation. Furthermore, digital gene expression (DEG) analysis identified 3,257 unigenes that were differentially expressed between the two fruit developmental stages. In addition, 14,473 simple sequence repeats (SSRs) were detected. Our results present comprehensive gene expression information about the cranberry fruit transcriptome that could facilitate our understanding of the molecular mechanisms of fruit development in cranberries. Although it will be necessary to validate the functions carried out by these genes, these results could be used to improve the quality of breeding programs for the cranberry and related species.

Evaluation of Gaussia luciferase and foot-and-mouth disease virus 2A translational interrupter chimeras as polycistronic reporters for transgene expression.

PubMed

Puckette, Michael; Burrage, Thomas; Neilan, John G; Rasmussen, Max

2017-06-12

The Gaussia princeps luciferase is used as a stand-alone reporter of transgene expression for in vitro and in vivo expression systems due to the rapid and easy monitoring of luciferase activity. We sought to simultaneously quantitate production of other recombinant proteins by transcriptionally linking the Gaussia princeps luciferase gene to other genes of interest through the foot-and-mouth disease virus 2A translational interrupter sequence. We produced six plasmids, each encoding a single open reading frame, with the foot-and-mouth disease virus 2A sequence placed either N-terminal or C-terminal to the Gaussia princeps luciferase gene. Two plasmids included novel Gaussia princeps luciferase variants with the position 1 methionine deleted. Placing a foot-and-mouth disease virus 2A translational interrupter sequence on either the N- or C-terminus of the Gaussia princeps luciferase gene did not prevent the secretion or luminescence of resulting chimeric luciferase proteins. We also measured the ability of another polycistronic plasmid vector with a 2A-luciferase sequence placed downstream of the foot-and-mouth disease virus P1 and 3C protease genes to produce of foot-and-mouth disease virus-like particles and luciferase activity from transfected cells. Incorporation of the 2A-luciferase sequence into a transgene encoding foot-and-mouth disease virus structural proteins retained luciferase activity and the ability to form virus-like particles. We demonstrated a mechanism for the near real-time, sequential, non-destructive quantitative monitoring of transcriptionally-linked recombinant proteins and a valuable method for monitoring transgene expression in recombinant vaccine constructs.

Genomic Sequence around Butterfly Wing Development Genes: Annotation and Comparative Analysis

PubMed Central

Conceição, Inês C.; Long, Anthony D.; Gruber, Jonathan D.; Beldade, Patrícia

2011-01-01

Background Analysis of genomic sequence allows characterization of genome content and organization, and access beyond gene-coding regions for identification of functional elements. BAC libraries, where relatively large genomic regions are made readily available, are especially useful for species without a fully sequenced genome and can increase genomic coverage of phylogenetic and biological diversity. For example, no butterfly genome is yet available despite the unique genetic and biological properties of this group, such as diversified wing color patterns. The evolution and development of these patterns is being studied in a few target species, including Bicyclus anynana, where a whole-genome BAC library allows targeted access to large genomic regions. Methodology/Principal Findings We characterize ∼1.3 Mb of genomic sequence around 11 selected genes expressed in B. anynana developing wings. Extensive manual curation of in silico predictions, also making use of a large dataset of expressed genes for this species, identified repetitive elements and protein coding sequence, and highlighted an expansion of Alcohol dehydrogenase genes. Comparative analysis with orthologous regions of the lepidopteran reference genome allowed assessment of conservation of fine-scale synteny (with detection of new inversions and translocations) and of DNA sequence (with detection of high levels of conservation of non-coding regions around some, but not all, developmental genes). Conclusions The general properties and organization of the available B. anynana genomic sequence are similar to the lepidopteran reference, despite the more than 140 MY divergence. Our results lay the groundwork for further studies of new interesting findings in relation to both coding and non-coding sequence: 1) the Alcohol dehydrogenase expansion with higher similarity between the five tandemly-repeated B. anynana paralogs than with the corresponding B. mori orthologs, and 2) the high conservation of non-coding sequence around the genes wingless and Ecdysone receptor, both involved in multiple developmental processes including wing pattern formation. PMID:21909358

Brain selective transgene expression in zebrafish using an NRSE derived motif

PubMed Central

Bergeron, Sadie A.; Hannan, Markus C.; Codore, Hiba; Fero, Kandice; Li, Grace H.; Moak, Zachary; Yokogawa, Tohei; Burgess, Harold A.

2012-01-01

Transgenic technologies enable the manipulation and observation of circuits controlling behavior by permitting expression of genetically encoded reporter genes in neurons. Frequently though, neuronal expression is accompanied by transgene expression in non-neuronal tissues, which may preclude key experimental manipulations, including assessment of the contribution of neurons to behavior by ablation. To better restrict transgene expression to the nervous system in zebrafish larvae, we have used DNA sequences derived from the neuron-restrictive silencing element (NRSE). We find that one such sequence, REx2, when used in conjunction with several basal promoters, robustly suppresses transgene expression in non-neuronal tissues. Both in transient transgenic experiments and in stable enhancer trap lines, suppression is achieved without compromising expression within the nervous system. Furthermore, in REx2 enhancer trap lines non-neuronal expression can be de-repressed by knocking down expression of the NRSE binding protein RE1-silencing transcription factor (Rest). In one line, we show that the resulting pattern of reporter gene expression coincides with that of the adjacent endogenous gene, hapln3. We demonstrate that three common basal promoters are susceptible to the effects of the REx2 element, suggesting that this method may be useful for confining expression from many other promoters to the nervous system. This technique enables neural specific targeting of reporter genes and thus will facilitate the use of transgenic methods to manipulate circuit function in freely behaving larvae. PMID:23293587

Cytochrome P450 20A1 in zebrafish: Cloning, regulation and potential involvement in hyperactivity disorders

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

Lemaire, Benjamin; Kubota, Akira; O'Meara, Conor M.

Cytochrome P450 (CYP) enzymes for which there is no functional information are considered “orphan” CYPs. Previous studies showed that CYP20A1, an orphan, is expressed in human hippocampus and substantia nigra, and in zebrafish (Danio rerio) CYP20A1 maternal transcript occurs in eggs, suggesting involvement in brain and in early development. Moreover, hyperactivity is reported in humans with chromosome 2 microdeletions including CYP20A1. We examined CYP20A1 in zebrafish, including impacts of chemical exposure on expression. Zebrafish CYP20A1 cDNA was cloned, sequenced, and aligned with cloned human CYP20A1 and predicted vertebrate orthologs. CYP20A1s share a highly conserved N-terminal region and unusual sequences inmore » the I-helix and the heme-binding CYP signature motifs. CYP20A1 mRNA expression was observed in adult zebrafish organs including the liver, heart, gonads, spleen and brain, as well as the eye and optic nerve. Putative binding sites in proximal promoter regions of CYP20A1s, and response of zebrafish CYP20A1 to selected nuclear and xenobiotic receptor agonists, point to up-regulation by agents involved in steroid hormone response, cholesterol and lipid metabolism. There also was a dose-dependent reduction of CYP20A1 expression in embryos exposed to environmentally relevant levels of methylmercury. Morpholino knockdown of CYP20A1 in developing zebrafish resulted in behavioral effects, including hyperactivity and a slowing of the optomotor response in larvae. The results suggest that altered expression of CYP20A1 might be part of a mechanism linking methylmercury exposure to neurobehavioral deficits. The expanded information on CYP20A1 brings us closer to “deorphanization”, that is, identifying CYP20A1 functions and its roles in health and disease. - Highlights: • The “orphan” CYP20A1 was cloned from zebrafish and its sequence analyzed. • Knockdown of CYP20A1 reduced an optomotor response and elicited bursts of activity. • Effects of knockdown resemble some features of a microdeletion of CYP20A1 in human. • Expression of CYP20A1 was downregulated by the neurotoxicant methylmercury. • CYP20A1 may be involved in neurobehavioral processes and effects of some chemicals.« less

Molecular characterization and expression analysis of a suite of cytochrome P450 enzymes implicated in insect hydrocarbon degradation in the entomopathogenic fungus Beauveria bassiana.

PubMed

Pedrini, Nicolás; Zhang, Shizhu; Juárez, M Patricia; Keyhani, Nemat O

2010-08-01

The insect epicuticle or waxy layer comprises a heterogeneous mixture of lipids that include abundant levels of long-chain alkanes, alkenes, wax esters and fatty acids. This structure represents the first barrier against microbial attack and for broad-host-range insect pathogens, such as Beauveria bassiana, it is the initial interface mediating the host-pathogen interaction, since these organisms do not require any specialized mode of entry and infect target hosts via the cuticle. B. bassiana is able to grow on straight chain alkanes up to n-C(33) as a sole source of carbon and energy. The cDNA and genomic sequences, including putative regulatory elements, for eight cytochrome P450 enzymes, postulated to be involved in alkane and insect epicuticle degradation, were isolated and characterized. Expression studies using a range of alkanes as well as an insect-derived epicuticular extract from the blood-sucking bug Triatomas infestans revealed a differential expression pattern for the P450 genes examined, and suggest that B. bassiana contains a series of hydrocarbon-assimilating enzymes with overlapping specificity in order to target the surface lipids of insect hosts. Phylogenetic analysis of the translated ORFs of the sequences revealed that the enzyme which displayed the highest levels of induction on both alkanes and the insect epicuticular extract represents the founding member of a new cytochrome P450 family, with three of the other sequences assigned as the first members of new P450 subfamilies. The remaining four proteins clustered with known P450 families whose members include alkane monooxygenases.

Comprehensive analysis of gene expression patterns in Friedreich's ataxia fibroblasts by RNA sequencing reveals altered levels of protein synthesis factors and solute carriers

PubMed Central

Li, Yanjie; Lu, Yue; Lin, Kevin; Hauser, Lauren A.; Lynch, David R.

2017-01-01

ABSTRACT Friedreich's ataxia (FRDA) is an autosomal recessive neurodegenerative disease usually caused by large homozygous expansions of GAA repeat sequences in intron 1 of the frataxin (FXN) gene. FRDA patients homozygous for GAA expansions have low FXN mRNA and protein levels when compared with heterozygous carriers or healthy controls. Frataxin is a mitochondrial protein involved in iron–sulfur cluster synthesis, and many FRDA phenotypes result from deficiencies in cellular metabolism due to lowered expression of FXN. Presently, there is no effective treatment for FRDA, and biomarkers to measure therapeutic trial outcomes and/or to gauge disease progression are lacking. Peripheral tissues, including blood cells, buccal cells and skin fibroblasts, can readily be isolated from FRDA patients and used to define molecular hallmarks of disease pathogenesis. For instance, FXN mRNA and protein levels as well as FXN GAA-repeat tract lengths are routinely determined using all of these cell types. However, because these tissues are not directly involved in disease pathogenesis, their relevance as models of the molecular aspects of the disease is yet to be decided. Herein, we conducted unbiased RNA sequencing to profile the transcriptomes of fibroblast cell lines derived from 18 FRDA patients and 17 unaffected control individuals. Bioinformatic analyses revealed significantly upregulated expression of genes encoding plasma membrane solute carrier proteins in FRDA fibroblasts. Conversely, the expression of genes encoding accessory factors and enzymes involved in cytoplasmic and mitochondrial protein synthesis was consistently decreased in FRDA fibroblasts. Finally, comparison of genes differentially expressed in FRDA fibroblasts to three previously published gene expression signatures defined for FRDA blood cells showed substantial overlap between the independent datasets, including correspondingly deficient expression of antioxidant defense genes. Together, these results indicate that gene expression profiling of cells derived from peripheral tissues can, in fact, consistently reveal novel molecular pathways of the disease. When performed on statistically meaningful sample group sizes, unbiased global profiling analyses utilizing peripheral tissues are critical for the discovery and validation of FRDA disease biomarkers. PMID:29125828

Molecular characterisation of Atlantic salmon paramyxovirus (ASPV): A novel paramyxovirus associated with proliferative gill inflammation

USGS Publications Warehouse

Falk, K.; Batts, W.N.; Kvellestad, A.; Kurath, G.; Wiik-Nielsen, J.; Winton, J.R.

2008-01-01

Atlantic salmon paramyxovirus (ASPV) was isolated in 1995 from gills of farmed Atlantic salmon suffering from proliferative gill inflammation. The complete genome sequence of ASPV was determined, revealing a genome 16,968 nucleotides in length consisting of six non-overlapping genes coding for the nucleo- (N), phospho- (P), matrix- (M), fusion- (F), haemagglutinin-neuraminidase- (HN) and large polymerase (L) proteins in the order 3???-N-P-M-F-HN-L-5???. The various conserved features related to virus replication found in most paramyxoviruses were also found in ASPV. These include: conserved and complementary leader and trailer sequences, tri-nucleotide intergenic regions and highly conserved transcription start and stop signal sequences. The P gene expression strategy of ASPV was like that of the respiro-, morbilli- and henipaviruses, which express the P and C proteins from the primary transcript and edit a portion of the mRNA to encode V and W proteins. Sequence similarities among various features related to virus replication, pairwise comparisons of all deduced ASPV protein sequences with homologous regions from other members of the family Paramyxoviridae, and phylogenetic analyses of these amino acid sequences suggested that ASPV was a novel member of the sub-family Paramyxovirinae, most closely related to the respiroviruses. ?? 2008 Elsevier B.V. All rights reserved.

Expressed sequence tags from the plant trypanosomatid Phytomonas serpens.

PubMed

Pappas, Georgios J; Benabdellah, Karim; Zingales, Bianca; González, Antonio

2005-08-01

We have generated 2190 expressed sequence tags (ESTs) from a cDNA library of the plant trypanosomatid Phytomonas serpens. Upon processing and clustering the set of 1893 accepted sequences was reduced to 697 clusters consisting of 452 singletons and 245 contigs. Functional categories were assigned based on BLAST searches against a database of the eukaryotic orthologous groups of proteins (KOG). Thirty six percent of the generated sequences showed no hits against the KOG database and 39.6% presented similarity to the KOG classes corresponding to translation, ribosomal structure and biogenesis. The most populated cluster contained 45 ESTs homologous to members of the glucose transporter family. This fact can be immediately correlated to the reported Phytomonas dependence on anaerobic glycolytic ATP production due to the lack of cytochrome-mediated respiratory chain. In this context, not only a number of enzymes of the glycolytic pathway were identified but also of the Krebs cycle as well as specific components of the respiratory chain. The data here reported, including a few hundred unique sequences and the description of tandemly repeated motifs and putative transcript stability motifs at untranslated mRNA ends, represent an initial approach to overcome the lack of information on the molecular biology of this organism.

Advanced colorectal adenoma related gene expression signature may predict prognostic for colorectal cancer patients with adenoma-carcinoma sequence.

PubMed

Li, Bing; Shi, Xiao-Yu; Liao, Dai-Xiang; Cao, Bang-Rong; Luo, Cheng-Hua; Cheng, Shu-Jun

2015-01-01

There are still no absolute parameters predicting progression of adenoma into cancer. The present study aimed to characterize functional differences on the multistep carcinogenetic process from the adenoma-carcinoma sequence. All samples were collected and mRNA expression profiling was performed by using Agilent Microarray high-throughput gene-chip technology. Then, the characteristics of mRNA expression profiles of adenoma-carcinoma sequence were described with bioinformatics software, and we analyzed the relationship between gene expression profiles of adenoma-adenocarcinoma sequence and clinical prognosis of colorectal cancer. The mRNA expressions of adenoma-carcinoma sequence were significantly different between high-grade intraepithelial neoplasia group and adenocarcinoma group. The biological process of gene ontology function enrichment analysis on differentially expressed genes between high-grade intraepithelial neoplasia group and adenocarcinoma group showed that genes enriched in the extracellular structure organization, skeletal system development, biological adhesion and itself regulated growth regulation, with the P value after FDR correction of less than 0.05. In addition, IPR-related protein mainly focused on the insulin-like growth factor binding proteins. The variable trends of gene expression profiles for adenoma-carcinoma sequence were mainly concentrated in high-grade intraepithelial neoplasia and adenocarcinoma. The differentially expressed genes are significantly correlated between high-grade intraepithelial neoplasia group and adenocarcinoma group. Bioinformatics analysis is an effective way to study the gene expression profiles in the adenoma-carcinoma sequence, and may provide an effective tool to involve colorectal cancer research strategy into colorectal adenoma or advanced adenoma.

Both positive and negative regulatory elements mediate expression of a photoregulated CAB gene from Nicotiana plumbaginifolia.

PubMed Central

Castresana, C; Garcia-Luque, I; Alonso, E; Malik, V S; Cashmore, A R

1988-01-01

We have analyzed promoter regulatory elements from a photoregulated CAB gene (Cab-E) isolated from Nicotiana plumbaginifolia. These studies have been performed by introducing chimeric gene constructs into tobacco cells via Agrobacterium tumefaciens-mediated transformation. Expression studies on the regenerated transgenic plants have allowed us to characterize three positive and one negative cis-acting elements that influence photoregulated expression of the Cab-E gene. Within the upstream sequences we have identified two positive regulatory elements (PRE1 and PRE2) which confer maximum levels of photoregulated expression. These sequences contain multiple repeated elements related to the sequence-ACCGGCCCACTT-. We have also identified within the upstream region a negative regulatory element (NRE) extremely rich in AT sequences, which reduces the level of gene expression in the light. We have defined a light regulatory element (LRE) within the promoter region extending from -396 to -186 bp which confers photoregulated expression when fused to a constitutive nopaline synthase ('nos') promoter. Within this region there is a 132-bp element, extending from -368 to -234 bp, which on deletion from the Cab-E promoter reduces gene expression from high levels to undetectable levels. Finally, we have demonstrated for a full length Cab-E promoter conferring high levels of photoregulated expression, that sequences proximal to the Cab-E TATA box are not replaceable by corresponding sequences from a 'nos' promoter. This contrasts with the apparent equivalence of these Cab-E and 'nos' TATA box-proximal sequences in truncated promoters conferring low levels of photoregulated expression. Images PMID:2901343

Decreased expression of cell adhesion genes in cancer stem-like cells isolated from primary oral squamous cell carcinomas.

PubMed

Mishra, Amrendra; Sriram, Harshini; Chandarana, Pinal; Tanavde, Vivek; Kumar, Rekha V; Gopinath, Ashok; Govindarajan, Raman; Ramaswamy, S; Sadasivam, Subhashini

2018-05-01

The goal of this study was to isolate cancer stem-like cells marked by high expression of CD44, a putative cancer stem cell marker, from primary oral squamous cell carcinomas and identify distinctive gene expression patterns in these cells. From 1 October 2013 to 4 September 2015, 76 stage III-IV primary oral squamous cell carcinoma of the gingivobuccal sulcus were resected. In all, 13 tumours were analysed by immunohistochemistry to visualise CD44-expressing cells. Expression of CD44 within The Cancer Genome Atlas-Head and Neck Squamous Cell Carcinoma RNA-sequencing data was also assessed. Seventy resected tumours were dissociated into single cells and stained with antibodies to CD44 as well as CD45 and CD31 (together referred as Lineage/Lin). From 45 of these, CD44 + Lin - and CD44 - Lin - subpopulations were successfully isolated using fluorescence-activated cell sorting, and good-quality RNA was obtained from 14 such sorted pairs. Libraries from five pairs were sequenced and the results analysed using bioinformatics tools. Reverse transcription quantitative polymerase chain reaction was performed to experimentally validate the differential expression of selected candidate genes identified from the transcriptome sequencing in the same 5 and an additional 9 tumours. CD44 was expressed on the surface of poorly differentiated tumour cells, and within the The Cancer Genome Atlas-Head and Neck Squamous Cell Carcinoma samples, its messenger RNA levels were higher in tumours compared to normal. Transcriptomics revealed that 102 genes were upregulated and 85 genes were downregulated in CD44 + Lin - compared to CD44 - Lin - cells in at least 3 of the 5 tumours sequenced. The upregulated genes included those involved in immune regulation, while the downregulated genes were enriched for genes involved in cell adhesion. Decreased expression of PCDH18, MGP, SPARCL1 and KRTDAP was confirmed by reverse transcription quantitative polymerase chain reaction. Lower expression of the cell-cell adhesion molecule PCDH18 correlated with poorer overall survival in the The Cancer Genome Atlas-Head and Neck Squamous Cell Carcinoma data highlighting it as a potential negative prognostic factor in this cancer.

Phenotypic and genotypic expression of self-incompatibility haplotypes in Arabidopsis lyrata suggests unique origin of alleles in different dominance classes.

PubMed

Prigoda, Nadia L; Nassuth, Annette; Mable, Barbara K

2005-07-01

The highly divergent alleles of the SRK gene in outcrossing Arabidopsis lyrata have provided important insights into the evolutionary history of self-incompatibility (SI) alleles and serve as an ideal model for studies of the evolutionary and molecular interactions between alleles in cell-cell recognition systems in general. One tantalizing question is how new specificities arise in systems that require coordination between male and female components. Allelic recruitment via gene conversion has been proposed as one possibility, based on the division of DNA sequences at the SRK locus into two distinctive groups: (1) sequences whose relationships are not well resolved and display the long branch lengths expected for a gene under balancing selection (Class A); and (2) sequences falling into a well-supported group with shorter branch lengths (Class B) that are closely related to an unlinked paralogous locus. The purpose of this study was to determine if differences in phenotype (site of expression assayed using allele-specific reverse transcription-polymerase chain reaction) or function (dominance relationships assayed through controlled pollinations) accompany the sequence-based classification. Expression of Class A alleles was restricted to floral tissues, as predicted for genes involved in the SI response. In contrast, Class B alleles, despite being tightly linked to the SI phenotype, were unexpectedly expressed in both leaves and floral tissues; the same pattern found for a related unlinked paralogous sequence. Whereas Class A included haplotypes in three different dominance classes, all Class B haplotypes were found to be recessive to all except one Class A haplotype. In addition, mapping of expression and dominance patterns onto an S-domain-based genealogy suggested that allelic dominance may be determined more by evolutionary history than by frequency-dependent selection for lowered dominance as some theories suggest. The possibility that interlocus gene conversion might have contributed to allelic diversity is discussed.

Synthetic versions of firefly luciferase and Renilla luciferase reporter genes that resist transgene silencing in sugarcane

PubMed Central

2014-01-01

Background Down-regulation or silencing of transgene expression can be a major hurdle to both molecular studies and biotechnology applications in many plant species. Sugarcane is particularly effective at silencing introduced transgenes, including reporter genes such as the firefly luciferase gene. Synthesizing transgene coding sequences optimized for usage in the host plant is one method of enhancing transgene expression and stability. Using specified design rules we have synthesised new coding sequences for both the firefly luciferase and Renilla luciferase reporter genes. We have tested these optimized versions for enhanced levels of luciferase activity and for increased steady state luciferase mRNA levels in sugarcane. Results The synthetic firefly luciferase (luc*) and Renilla luciferase (Renluc*) coding sequences have elevated G + C contents in line with sugarcane codon usage, but maintain 75% identity to the native firefly or Renilla luciferase nucleotide sequences and 100% identity to the protein coding sequences. Under the control of the maize pUbi promoter, the synthetic luc* and Renluc* genes yielded 60x and 15x higher luciferase activity respectively, over the native firefly and Renilla luciferase genes in transient assays on sugarcane suspension cell cultures. Using a novel transient assay in sugarcane suspension cells combining co-bombardment and qRT-PCR, we showed that synthetic luc* and Renluc* genes generate increased transcript levels compared to the native firefly and Renilla luciferase genes. In stable transgenic lines, the luc* transgene generated significantly higher levels of expression than the native firefly luciferase transgene. The fold difference in expression was highest in the youngest tissues. Conclusions We developed synthetic versions of both the firefly and Renilla luciferase reporter genes that resist transgene silencing in sugarcane. These transgenes will be particularly useful for evaluating the expression patterns conferred by existing and newly isolated promoters in sugarcane tissues. The strategies used to design the synthetic luciferase transgenes could be applied to other transgenes that are aggressively silenced in sugarcane. PMID:24708613

Synthetic versions of firefly luciferase and Renilla luciferase reporter genes that resist transgene silencing in sugarcane.

PubMed

Chou, Ting-Chun; Moyle, Richard L

2014-04-08

Down-regulation or silencing of transgene expression can be a major hurdle to both molecular studies and biotechnology applications in many plant species. Sugarcane is particularly effective at silencing introduced transgenes, including reporter genes such as the firefly luciferase gene.Synthesizing transgene coding sequences optimized for usage in the host plant is one method of enhancing transgene expression and stability. Using specified design rules we have synthesised new coding sequences for both the firefly luciferase and Renilla luciferase reporter genes. We have tested these optimized versions for enhanced levels of luciferase activity and for increased steady state luciferase mRNA levels in sugarcane. The synthetic firefly luciferase (luc*) and Renilla luciferase (Renluc*) coding sequences have elevated G + C contents in line with sugarcane codon usage, but maintain 75% identity to the native firefly or Renilla luciferase nucleotide sequences and 100% identity to the protein coding sequences.Under the control of the maize pUbi promoter, the synthetic luc* and Renluc* genes yielded 60x and 15x higher luciferase activity respectively, over the native firefly and Renilla luciferase genes in transient assays on sugarcane suspension cell cultures.Using a novel transient assay in sugarcane suspension cells combining co-bombardment and qRT-PCR, we showed that synthetic luc* and Renluc* genes generate increased transcript levels compared to the native firefly and Renilla luciferase genes.In stable transgenic lines, the luc* transgene generated significantly higher levels of expression than the native firefly luciferase transgene. The fold difference in expression was highest in the youngest tissues. We developed synthetic versions of both the firefly and Renilla luciferase reporter genes that resist transgene silencing in sugarcane. These transgenes will be particularly useful for evaluating the expression patterns conferred by existing and newly isolated promoters in sugarcane tissues. The strategies used to design the synthetic luciferase transgenes could be applied to other transgenes that are aggressively silenced in sugarcane.

Selective forces and mutational biases drive stop codon usage in the human genome: a comparison with sense codon usage.

PubMed

Trotta, Edoardo

2016-05-17

The three stop codons UAA, UAG, and UGA signal the termination of mRNA translation. As a result of a mechanism that is not adequately understood, they are normally used with unequal frequencies. In this work, we showed that selective forces and mutational biases drive stop codon usage in the human genome. We found that, in respect to sense codons, stop codon usage was affected by stronger selective forces but was less influenced by neutral mutational biases. UGA is the most frequent termination codon in human genome. However, UAA was the preferred stop codon in genes with high breadth of expression, high level of expression, AT-rich coding sequences, housekeeping functions, and in gene ontology categories with the largest deviation from expected stop codon usage. Selective forces associated with the breadth and the level of expression favoured AT-rich sequences in the mRNA region including the stop site and its proximal 3'-UTR, but acted with scarce effects on sense codons, generating two regions, upstream and downstream of the stop codon, with strongly different base composition. By favouring low levels of GC-content, selection promoted labile local secondary structures at the stop site and its proximal 3'-UTR. The compositional and structural context favoured by selection was surprisingly emphasized in the class of ribosomal proteins and was consistent with sequence elements that increase the efficiency of translational termination. Stop codons were also heterogeneously distributed among chromosomes by a mechanism that was strongly correlated with the GC-content of coding sequences. In human genome, the nucleotide composition and the thermodynamic stability of stop codon site and its proximal 3'-UTR are correlated with the GC-content of coding sequences and with the breadth and the level of gene expression. In highly expressed genes stop codon usage is compositionally and structurally consistent with highly efficient translation termination signals.

ccrABEnt serine recombinase genes are widely distributed in the Enterococcus faecium and Enterococcus casseliflavus species groups and are expressed in E. faecium

PubMed Central

Bjørkeng, Eva Katrin; Tessema, Girum Tadesse; Lundblad, Eirik Wasmuth; Butaye, Patrick; Willems, Rob; Sollid, Johanna Ericsson; Sundsfjord, Arnfinn; Hegstad, Kristin

2010-01-01

The presence, distribution and expression of cassette chromosome recombinase (ccr) genes, which are homologous to the staphylococcal ccrAB genes and are designated ccrABEnt genes, were examined in enterococcal isolates (n=421) representing 13 different species. A total of 118 (28 %) isolates were positive for ccrABEnt genes by PCR, and a number of these were confirmed by Southern hybridization with a ccrAEnt probe (n=76) and partial DNA sequencing of ccrAEnt and ccrBEnt genes (n=38). ccrABEnt genes were present in Enterococcus faecium (58/216, 27 %), Enterococcus durans (31/38, 82 %), Enterococcus hirae (27/52, 50 %), Enterococcus casseliflavus (1/4, 25 %) and Enterococcus gallinarum (1/2, 50 %). In the eight other species tested, including Enterococcus faecalis (n=94), ccrABEnt genes were not found. Thirty-eight sequenced ccrABEnt genes from five different enterococcal species showed 94–100 % nucleotide sequence identity and linkage PCRs showed heterogeneity in the ccrABEnt flanking chromosomal genes. Expression analysis of ccrABEnt genes from the E. faecium DO strain showed constitutive expression as a bicistronic mRNA. The ccrABEnt mRNA levels were lower during log phase than stationary phase in relation to total mRNA. Multilocus sequence typing was performed on 39 isolates. ccrABEnt genes were detected in both hospital-related (10/29, 34 %) and non-hospital (4/10, 40 %) strains of E. faecium. Various sequence types were represented by both ccrABEnt positive and negative isolates, suggesting acquisition or loss of ccrABEnt in E. faecium. In summary, ccrABEnt genes, potentially involved in genome plasticity, are expressed in E. faecium and are widely distributed in the E. faecium and E. casseliflavus species groups. PMID:20817645

Oligo Design: a computer program for development of probes for oligonucleotide microarrays.

PubMed

Herold, Keith E; Rasooly, Avraham

2003-12-01

Oligonucleotide microarrays have demonstrated potential for the analysis of gene expression, genotyping, and mutational analysis. Our work focuses primarily on the detection and identification of bacteria based on known short sequences of DNA. Oligo Design, the software described here, automates several design aspects that enable the improved selection of oligonucleotides for use with microarrays for these applications. Two major features of the program are: (i) a tiling algorithm for the design of short overlapping temperature-matched oligonucleotides of variable length, which are useful for the analysis of single nucleotide polymorphisms and (ii) a set of tools for the analysis of multiple alignments of gene families and related short DNA sequences, which allow for the identification of conserved DNA sequences for PCR primer selection and variable DNA sequences for the selection of unique probes for identification. Note that the program does not address the full genome perspective but, instead, is focused on the genetic analysis of short segments of DNA. The program is Internet-enabled and includes a built-in browser and the automated ability to download sequences from GenBank by specifying the GI number. The program also includes several utilities, including audio recital of a DNA sequence (useful for verifying sequences against a written document), a random sequence generator that provides insight into the relationship between melting temperature and GC content, and a PCR calculator.

A novel variant of aquaporin 3 is expressed in killifish (Fundulus heteroclitus) intestine

PubMed Central

Jung, Dawoon; Adamo, Meredith A.; Lehman, Rebecca M.; Barnaby, Roxanna; Jackson, Craig E.; Jackson, Brian P.; Shaw, Joseph R.; Stanton, Bruce A.

2015-01-01

Killifish (Fundulus heteroclitus) are euryhaline teleosts that are widely used in environmental and toxicological studies, and they are tolerant to arsenic, in part due to very low assimilation of arsenic from the environment. The mechanism of arsenic uptake by the intestine, a major route of arsenic uptake in humans is unknown. Thus, the goal of this study was to determine if aquaglyceroporins (AQP), which transport water and other small molecules including arsenite across cell membranes, are expressed in the killifish intestine, and whether AQP expression is affected by osmotic stress. Through RT-PCR and sequence analysis of PCR amplicons, we demonstrated that the intestine expresses kfAQP3a and kfAQP3b, two previously identified variants, and also identified a novel variant of killifish AQP3 (kfAQP3c) in the intestine. The variants likely represent alternate splice forms. A BLAST search of the F. heteroclitus reference genome revealed that the AQP3 gene resides on a single locus, while an alignment of the AQP3 sequence among 384 individuals from eight population ranging from Rhode Island to North Carolina revealed that its coding sequence was remarkably conserved with no fixed polymorphism residing in the region that distinguishes these variants. We further demonstrate that the novel variant transports arsenite into HEK293T cells. Whereas kfAQP3a, which does not transport arsenite, was expressed in both freshwater (FW) and saltwater (SW) acclimated fish, kfAQP3b, an arsenic transporter, was expressed only in FW acclimated fish, and kfAQP3c was expressed only in SW acclimated fish. Thus, we have identified a novel, putative splice variant of kfAQP3, kfAQP3c, which transports arsenic and is expressed only in SW acclimated fish. PMID:25766383

Serotonin 1A receptor (5-HT1A) of the sea lamprey: cDNA cloning and expression in the central nervous system.

PubMed

Cornide-Petronio, María Eugenia; Anadón, Ramón; Barreiro-Iglesias, Antón; Rodicio, María Celina

2013-09-01

Serotonergic cells are among the earliest neurons to be born in the developing central nervous system and serotonin is known to regulate the development of the nervous system. One of the major targets of the activity of serotonergic cells is the serotonin 1A receptor (5-HT1A), an ancestral archetypical serotonin receptor. In this study, we cloned and characterized the 3D structure of the sea lamprey 5-HT1A, and studied the expression of its transcript in the central nervous system by means of in situ hybridization. In phylogenetic analyses, the sea lamprey 5-HT1A sequence clustered together with 5-HT1A sequences of vertebrates and emerged as an outgroup to all gnathostome sequences. In situ hybridization analysis during prolarval, larval and adult stages showed a widespread expression of the lamprey 5-ht1a transcript. In P1 prolarvae 5-ht1a mRNA expression was observed in diencephalic nuclei, the rhombencephalon and rostral spinal cord. At P2 prolarval stage the 5-ht1a expression extended to other brain areas including telencephalic regions. 5-ht1a expression in larvae was observed throughout almost all the main brain regions with the strongest expression in the olfactory bulbs, lateral pallium, striatum, preoptic region, habenula, prethalamus, thalamus, pretectum, hypothalamus, rhombencephalic reticular area, dorsal column nucleus and rostral spinal cord. In adults, the 5-ht1a transcript was also observed in cells of the subcommissural organ. Comparison of the expression of 5-ht1a between the sea lamprey and other vertebrates reveals a conserved pattern in most of the brain regions, likely reflecting the ancestral vertebrate condition.

Phage-mediated Delivery of Targeted sRNA Constructs to Knock Down Gene Expression in E. coli.

PubMed

Bernheim, Aude G; Libis, Vincent K; Lindner, Ariel B; Wintermute, Edwin H

2016-03-20

RNA-mediated knockdowns are widely used to control gene expression. This versatile family of techniques makes use of short RNA (sRNA) that can be synthesized with any sequence and designed to complement any gene targeted for silencing. Because sRNA constructs can be introduced to many cell types directly or using a variety of vectors, gene expression can be repressed in living cells without laborious genetic modification. The most common RNA knockdown technology, RNA interference (RNAi), makes use of the endogenous RNA-induced silencing complex (RISC) to mediate sequence recognition and cleavage of the target mRNA. Applications of this technique are therefore limited to RISC-expressing organisms, primarily eukaryotes. Recently, a new generation of RNA biotechnologists have developed alternative mechanisms for controlling gene expression through RNA, and so made possible RNA-mediated gene knockdowns in bacteria. Here we describe a method for silencing gene expression in E. coli that functionally resembles RNAi. In this system a synthetic phagemid is designed to express sRNA, which may designed to target any sequence. The expression construct is delivered to a population of E. coli cells with non-lytic M13 phage, after which it is able to stably replicate as a plasmid. Antisense recognition and silencing of the target mRNA is mediated by the Hfq protein, endogenous to E. coli. This protocol includes methods for designing the antisense sRNA, constructing the phagemid vector, packaging the phagemid into M13 bacteriophage, preparing a live cell population for infection, and performing the infection itself. The fluorescent protein mKate2 and the antibiotic resistance gene chloramphenicol acetyltransferase (CAT) are targeted to generate representative data and to quantify knockdown effectiveness.

A landscape of circular RNA expression in the human heart.

PubMed

Tan, Wilson L W; Lim, Benson T S; Anene-Nzelu, Chukwuemeka G O; Ackers-Johnson, Matthew; Dashi, Albert; See, Kelvin; Tiang, Zenia; Lee, Dominic Paul; Chua, Wee Woon; Luu, Tuan D A; Li, Peter Y Q; Richards, Arthur Mark; Foo, Roger S Y

2017-03-01

Circular RNA (circRNA) is a newly validated class of single-stranded RNA, ubiquitously expressed in mammalian tissues and possessing key functions including acting as microRNA sponges and as transcriptional regulators by binding to RNA-binding proteins. While independent studies confirm the expression of circRNA in various tissue types, genome-wide circRNA expression in the heart has yet to be described in detail. We performed deep RNA-sequencing on ribosomal-depleted RNA isolated from 12 human hearts, 25 mouse hearts and across a 28-day differentiation time-course of human embryonic stem cell-derived cardiomyocytes. Using purpose-designed bioinformatics tools, we uncovered a total of 15 318 and 3017 cardiac circRNA within human and mouse, respectively. Their abundance generally correlates with the abundance of their cognate linear RNA, but selected circRNAs exist at disproportionately higher abundance. Top highly expressed circRNA corresponded to key cardiac genes including Titin (TTN), RYR2, and DMD. The most abundant cardiac-expressed circRNA is a cytoplasmic localized single-exon circSLC8A1-1. The longest human transcript TTN alone generates up to 415 different exonic circRNA isoforms, the majority (83%) of which originates from the I-band domain. Finally, we confirmed the expression of selected cardiac circRNA by RT-PCR, Sanger sequencing and single molecule RNA-fluorescence in situ hybridization. Our data provide a detailed circRNA expression landscape in hearts. There is a high-abundance of specific cardiac-expressed circRNA. These findings open up a new avenue for future investigation into this emerging class of RNA. Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2016. For Permissions, please email: journals.permissions@oup.com.

Expressed Sequence Reference Standards for Evaluating Stage-specific Gene Expression in Southern Green Lacewings, Chrysoperla rufilabris

USDA-ARS?s Scientific Manuscript database

Five developmental stages of Chrysoperla rufilabris were tested using nine primer pairs. Three sequences were highly expressed at all life stages and six were differentially expressed. These primer pairs may be used as standards to quantitate functional gene expression associated with physiological ...

Identification and characterization of calcium transporter gene family in finger millet in relation to grain calcium content.

PubMed

Singh, Uma M; Metwal, Mamta; Singh, Manoj; Taj, Gohar; Kumar, Anil

2015-07-15

Calcium (Ca) is an essential mineral for proper growth and development of plants as well as animals. In plants including cereals, calcium is deposited in seed during its development which is mediated by specialized Ca transporters. Common cereal seeds contain very low amounts of Ca while the finger millet (Eleusine coracana) contains exceptionally high amounts of Ca in seed. In order to understand the role of Ca transporters in grain Ca accumulation, developing seed transcriptome of two finger millet genotypes (GP-1, low Ca and GP-45 high Ca) differing in seed Ca content was sequenced using Illumina paired-end sequencing technology and members of Ca transporter gene family were identified. Out of 109,218 and 120,130 contigs, 86 and 81 contigs encoding Ca transporters were identified in GP-1 and GP-45, respectively. After removal of redundant sequences, a total of 19 sequences were confirmed as Ca transporter genes, which includes 11 Ca(2+) ATPases, 07 Ca(2+)/cation exchangers and 01 Ca(2+) channel. The differential expressions of all genes were analyzed from transcriptome data and it was observed that 9 and 3 genes were highly expressed in GP-45 and GP-1 genotypes respectively. Validation of transcriptome expression data of selected Ca transporter genes was performed on different stages of developing spikes of both genotypes grown under different concentrations of exogenous Ca. In both genotypes, significant correlation was observed between the expression of these genes, especially EcCaX3, and on the amount of Ca accumulated in seed. The positive correlation of seed mass with the amount of Ca concentration was also observed. The efficient Ca transport property and responsiveness of EcCAX3 towards exogenous Ca could be utilized in future biofortification program. Copyright © 2015 Elsevier B.V. All rights reserved.

Identification and expression analysis of cDNA encoding insulin-like growth factor 2 in horses

PubMed Central

KIKUCHI, Kohta; SASAKI, Keisuke; AKIZAWA, Hiroki; TSUKAHARA, Hayato; BAI, Hanako; TAKAHASHI, Masashi; NAMBO, Yasuo; HATA, Hiroshi; KAWAHARA, Manabu

2017-01-01

Insulin-like growth factor 2 (IGF2) is responsible for a broad range of physiological processes during fetal development and adulthood, but genomic analyses of IGF2 containing the 5ʹ- and 3ʹ-untranslated regions (UTRs) in equines have been limited. In this study, we characterized the IGF2 mRNA containing the UTRs, and determined its expression pattern in the fetal tissues of horses. The complete equine IGF2 mRNA sequence harboring another exon approximately 2.8 kb upstream from the canonical transcription start site was identified as a new transcript variant. As this upstream exon did not contain the start codon, the amino acid sequence was identical to the canonical variant. Analysis of the deduced amino acid sequence revealed that the protein possessed two major domains, IlGF and IGF2_C, and analysis of IGF2 sequence polymorphism in fetal tissues of Hokkaido native horse and Thoroughbreds revealed a single nucleotide polymorphism (T to C transition) at position 398 in Thoroughbreds, which caused an amino acid substitution at position 133 in the IGF2 sequence. Furthermore, the expression pattern of the IGF2 mRNA in the fetal tissues of horses was determined for the first time, and was found to be consistent with those of other species. Taken together, these results suggested that the transcriptional and translational products of the IGF2 gene have conserved functions in the fetal development of mammals, including horses. PMID:29151450

MAGIC database and interfaces: an integrated package for gene discovery and expression.

PubMed

Cordonnier-Pratt, Marie-Michèle; Liang, Chun; Wang, Haiming; Kolychev, Dmitri S; Sun, Feng; Freeman, Robert; Sullivan, Robert; Pratt, Lee H

2004-01-01

The rapidly increasing rate at which biological data is being produced requires a corresponding growth in relational databases and associated tools that can help laboratories contend with that data. With this need in mind, we describe here a Modular Approach to a Genomic, Integrated and Comprehensive (MAGIC) Database. This Oracle 9i database derives from an initial focus in our laboratory on gene discovery via production and analysis of expressed sequence tags (ESTs), and subsequently on gene expression as assessed by both EST clustering and microarrays. The MAGIC Gene Discovery portion of the database focuses on information derived from DNA sequences and on its biological relevance. In addition to MAGIC SEQ-LIMS, which is designed to support activities in the laboratory, it contains several additional subschemas. The latter include MAGIC Admin for database administration, MAGIC Sequence for sequence processing as well as sequence and clone attributes, MAGIC Cluster for the results of EST clustering, MAGIC Polymorphism in support of microsatellite and single-nucleotide-polymorphism discovery, and MAGIC Annotation for electronic annotation by BLAST and BLAT. The MAGIC Microarray portion is a MIAME-compliant database with two components at present. These are MAGIC Array-LIMS, which makes possible remote entry of all information into the database, and MAGIC Array Analysis, which provides data mining and visualization. Because all aspects of interaction with the MAGIC Database are via a web browser, it is ideally suited not only for individual research laboratories but also for core facilities that serve clients at any distance.

Positive Selection Underlies Faster-Z Evolution of Gene Expression in Birds

PubMed Central

Dean, Rebecca; Harrison, Peter W.; Wright, Alison E.; Zimmer, Fabian; Mank, Judith E.

2015-01-01

The elevated rate of evolution for genes on sex chromosomes compared with autosomes (Fast-X or Fast-Z evolution) can result either from positive selection in the heterogametic sex or from nonadaptive consequences of reduced relative effective population size. Recent work in birds suggests that Fast-Z of coding sequence is primarily due to relaxed purifying selection resulting from reduced relative effective population size. However, gene sequence and gene expression are often subject to distinct evolutionary pressures; therefore, we tested for Fast-Z in gene expression using next-generation RNA-sequencing data from multiple avian species. Similar to studies of Fast-Z in coding sequence, we recover clear signatures of Fast-Z in gene expression; however, in contrast to coding sequence, our data indicate that Fast-Z in expression is due to positive selection acting primarily in females. In the soma, where gene expression is highly correlated between the sexes, we detected Fast-Z in both sexes, although at a higher rate in females, suggesting that many positively selected expression changes in females are also expressed in males. In the gonad, where intersexual correlations in expression are much lower, we detected Fast-Z for female gene expression, but crucially, not males. This suggests that a large amount of expression variation is sex-specific in its effects within the gonad. Taken together, our results indicate that Fast-Z evolution of gene expression is the product of positive selection acting on recessive beneficial alleles in the heterogametic sex. More broadly, our analysis suggests that the adaptive potential of Z chromosome gene expression may be much greater than that of gene sequence, results which have important implications for the role of sex chromosomes in speciation and sexual selection. PMID:26067773

Ankyrin-binding activity of nervous system cell adhesion molecules expressed in adult brain.

PubMed

Davis, J Q; Bennett, V

1993-01-01

A family of ankyrin-binding glycoproteins have been identified in adult rat brain that include alternatively spliced products of the same pre-mRNA. A composite sequence of ankyrin-binding glycoprotein (ABGP) shares 72% amino acid sequence identity with chicken neurofascin, a membrane-spanning neural cell adhesion molecule in the Ig super-family expressed in embryonic brain. ABGP polypeptides and ankyrin associate as pure proteins in a 1:1 molar stoichiometry at a site located in the predicted cytoplasmic domain. ABGP polypeptides are expressed late in postnatal development to approximately the same levels as ankyrin, and comprise a significant fraction of brain membrane proteins. Immunofluorescence studies have shown that ABGP polypeptides are co-localized with ankyrinB. Major differences in developmental expression have been reported for neurofascin in embryos compared with the late postnatal expression of ABGP, suggesting that ABGP and neurofascin represent products of gene duplication events that have subsequently evolved in parallel with distinct roles. Predicted cytoplasmic domains of rat ABGP and chicken neurofascin are nearly identical to each other and closely related to a group of nervous system cell adhesion molecules with variable extracellular domains, including L1, Nr-CAM and Ng-CAM of vertebrates, and neuroglian of Drosophila. A hypothesis to be evaluated is that ankyrin-binding activity is shared by all of these proteins.

Differentially-Expressed Genes Associated with Faster Growth of the Pacific Abalone, Haliotis discus hannai

PubMed Central

Choi, Mi-Jin; Kim, Gun-Do; Kim, Jong-Myoung; Lim, Han Kyu

2015-01-01

The Pacific abalone Haliotis discus hannai is used for commercial aquaculture in Korea. We examined the transcriptome of Pacific abalone Haliotis discus hannai siblings using NGS technology to identify genes associated with high growth rates. Pacific abalones grown for 200 days post-fertilization were divided into small-, medium-, and large-size groups with mean weights of 0.26 ± 0.09 g, 1.43 ± 0.405 g, and 5.24 ± 1.09 g, respectively. RNA isolated from the soft tissues of each group was subjected to RNA sequencing. Approximately 1%–3% of the transcripts were differentially expressed in abalones, depending on the growth rate. RT-PCR was carried out on thirty four genes selected to confirm the relative differences in expression detected by RNA sequencing. Six differentially-expressed genes were identified as associated with faster growth of the Pacific abalone. These include five up-regulated genes (including one specific to females) encoding transcripts homologous to incilarin A, perlucin, transforming growth factor-beta-induced protein immunoglobulin-heavy chain 3 (ig-h3), vitelline envelope zona pellucida domain 4, and defensin, and one down-regulated gene encoding tomoregulin in large abalones. Most of the transcripts were expressed predominantly in the hepatopancreas. The genes identified in this study will lead to development of markers for identification of high-growth-rate abalones and female abalones. PMID:26593905

Differentially-Expressed Genes Associated with Faster Growth of the Pacific Abalone, Haliotis discus hannai.

PubMed

Choi, Mi-Jin; Kim, Gun-Do; Kim, Jong-Myoung; Lim, Han Kyu

2015-11-18

The Pacific abalone Haliotis discus hannai is used for commercial aquaculture in Korea. We examined the transcriptome of Pacific abalone Haliotis discus hannai siblings using NGS technology to identify genes associated with high growth rates. Pacific abalones grown for 200 days post-fertilization were divided into small-, medium-, and large-size groups with mean weights of 0.26 ± 0.09 g, 1.43 ± 0.405 g, and 5.24 ± 1.09 g, respectively. RNA isolated from the soft tissues of each group was subjected to RNA sequencing. Approximately 1%-3% of the transcripts were differentially expressed in abalones, depending on the growth rate. RT-PCR was carried out on thirty four genes selected to confirm the relative differences in expression detected by RNA sequencing. Six differentially-expressed genes were identified as associated with faster growth of the Pacific abalone. These include five up-regulated genes (including one specific to females) encoding transcripts homologous to incilarin A, perlucin, transforming growth factor-beta-induced protein immunoglobulin-heavy chain 3 (ig-h3), vitelline envelope zona pellucida domain 4, and defensin, and one down-regulated gene encoding tomoregulin in large abalones. Most of the transcripts were expressed predominantly in the hepatopancreas. The genes identified in this study will lead to development of markers for identification of high-growth-rate abalones and female abalones.

The NLP toxin family in Phytophthora sojae includes rapidly evolving groups that lack necrosis-inducing activity.

PubMed

Dong, Suomeng; Kong, Guanghui; Qutob, Dinah; Yu, Xiaoli; Tang, Junli; Kang, Jixiong; Dai, Tingting; Wang, Hai; Gijzen, Mark; Wang, Yuanchao

2012-07-01

Necrosis- and ethylene-inducing-like proteins (NLP) are widely distributed in eukaryotic and prokaryotic plant pathogens and are considered to be important virulence factors. We identified, in total, 70 potential Phytophthora sojae NLP genes but 37 were designated as pseudogenes. Sequence alignment of the remaining 33 NLP delineated six groups. Three of these groups include proteins with an intact heptapeptide (Gly-His-Arg-His-Asp-Trp-Glu) motif, which is important for necrosis-inducing activity, whereas the motif is not conserved in the other groups. In total, 19 representative NLP genes were assessed for necrosis-inducing activity by heterologous expression in Nicotiana benthamiana. Surprisingly, only eight genes triggered cell death. The expression of the NLP genes in P. sojae was examined, distinguishing 20 expressed and 13 nonexpressed NLP genes. Real-time reverse-transcriptase polymerase chain reaction results indicate that most NLP are highly expressed during cyst germination and infection stages. Amino acid substitution ratios (Ka/Ks) of 33 NLP sequences from four different P. sojae strains resulted in identification of positive selection sites in a distinct NLP group. Overall, our study indicates that expansion and pseudogenization of the P. sojae NLP family results from an ongoing birth-and-death process, and that varying patterns of expression, necrosis-inducing activity, and positive selection suggest that NLP have diversified in function.

Screening differentially expressed genes in an amphipod (Hyalella azteca) exposed to fungicide vinclozolin by suppression subtractive hybridization.

PubMed

Wu, Yun H; Wu, Tsung M; Hong, Chwan Y; Wang, Yei S; Yen, Jui H

2014-01-01

Vinclozolin, a dicarboximide fungicide, is an endocrine disrupting chemical that competes with an androgenic endocrine disruptor compound. Most research has focused on the epigenetic effect of vinclozolin in humans. In terms of ecotoxicology, understanding the effect of vinclozolin on non-target organisms is important. The expression profile of a comprehensive set of genes in the amphipod Hyalella azteca exposed to vinclozolin was examined. The expressed sequence tags in low-dose vinclozolin-treated and -untreated amphipods were isolated and identified by suppression subtractive hybridization. DNA dot blotting was used to confirm the results and establish a subtracted cDNA library for comparing all differentially expressed sequences with and without vinclozolin treatment. In total, 494 differentially expressed genes, including hemocyanin, heatshock protein, cytochrome, cytochrome oxidase and NADH dehydrogenase were detected. Hemocyanin was the most abundant gene. DNA dot blotting revealed 55 genes with significant differential expression. These genes included larval serum protein 1 alpha, E3 ubiquitin-protein ligase, mitochondrial cytochrome c oxidase, mitochondrial protein, proteasome inhibitor, hemocyanin, zinc-finger-containing protein, mitochondrial NADH-ubiquinone oxidoreductase and epididymal sperm-binding protein. Vinclozolin appears to upregulate stress-related genes and hemocyanin, related to immunity. Moreover, vinclozolin downregulated NADH dehydrogenase, related to respiration. Thus, even a non-lethal concentration of vinclozolin still has an effect at the genetic level in H. azteca and presents a potential risk, especially as it would affect non-target organism hormone metabolism.

Apoptosis-inducing signal sequence mutation in carbonic anhydrase IV identified in patients with the RP17 form of retinitis pigmentosa

PubMed Central

Rebello, George; Ramesar, Rajkumar; Vorster, Alvera; Roberts, Lisa; Ehrenreich, Liezle; Oppon, Ekow; Gama, Dumisani; Bardien, Soraya; Greenberg, Jacquie; Bonapace, Giuseppe; Waheed, Abdul; Shah, Gul N.; Sly, William S.

2004-01-01

Genetic and physical mapping of the RP17 locus on 17q identified a 3.6-megabase candidate region that includes the gene encoding carbonic anhydrase IV (CA4), a glycosylphosphatidylinositol-anchored protein that is highly expressed in the choriocapillaris of the human eye. By sequencing candidate genes in this region, we identified a mutation that causes replacement of an arginine with a tryptophan (R14W) in the signal sequence of the CA4 gene at position -5 relative to the signal sequence cleavage site. This mutation was found to cosegregate with the disease phenotype in two large families and was not found in 36 unaffected family members or 100 controls. Expression of the mutant cDNA in COS-7 cells produced several findings, suggesting a mechanism by which the mutation can explain the autosomal dominant disease. In transfected COS-7 cells, the R14W mutation (i) reduced the steady-state level of carbonic anhydrase IV activity expressed by 28% due to a combination of decreased synthesis and accelerated turnover; (ii) led to up-regulation of immunoglobulin-binding protein, double-stranded RNA-regulated protein kinase-like ER kinase, and CCAAT/enhancer-binding protein homologous protein, markers of the unfolded protein response and endoplasmic reticulum stress; and (iii) induced apoptosis, as evidenced by annexin V binding and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling staining, in most cells expressing the mutant, but not the WT, protein. We suggest that a high level of expression of the mutant allele in the endothelial cells of the choriocapillaris leads to apoptosis, leading in turn to ischemia in the overlying retina and producing autosomal dominant retinitis pigmentosa. PMID:15090652

Mining genes involved in insecticide resistance of Liposcelis bostrychophila Badonnel by transcriptome and expression profile analysis.

PubMed

Dou, Wei; Shen, Guang-Mao; Niu, Jin-Zhi; Ding, Tian-Bo; Wei, Dan-Dan; Wang, Jin-Jun

2013-01-01

Recent studies indicate that infestations of psocids pose a new risk for global food security. Among the psocids species, Liposcelis bostrychophila Badonnel has gained recognition in importance because of its parthenogenic reproduction, rapid adaptation, and increased worldwide distribution. To date, the molecular data available for L. bostrychophila is largely limited to genes identified through homology. Also, no transcriptome data relevant to psocids infection is available. In this study, we generated de novo assembly of L. bostrychophila transcriptome performed through the short read sequencing technology (Illumina). In a single run, we obtained more than 51 million sequencing reads that were assembled into 60,012 unigenes (mean size = 711 bp) by Trinity. The transcriptome sequences from different developmental stages of L. bostrychophila including egg, nymph and adult were annotated with non-redundant (Nr) protein database, gene ontology (GO), cluster of orthologous groups of proteins (COG), and KEGG orthology (KO). The analysis revealed three major enzyme families involved in insecticide metabolism as differentially expressed in the L. bostrychophila transcriptome. A total of 49 P450-, 31 GST- and 21 CES-specific genes representing the three enzyme families were identified. Besides, 16 transcripts were identified to contain target site sequences of resistance genes. Furthermore, we profiled gene expression patterns upon insecticide (malathion and deltamethrin) exposure using the tag-based digital gene expression (DGE) method. The L. bostrychophila transcriptome and DGE data provide gene expression data that would further our understanding of molecular mechanisms in psocids. In particular, the findings of this investigation will facilitate identification of genes involved in insecticide resistance and designing of new compounds for control of psocids.

Mining Genes Involved in Insecticide Resistance of Liposcelis bostrychophila Badonnel by Transcriptome and Expression Profile Analysis

PubMed Central

Dou, Wei; Shen, Guang-Mao; Niu, Jin-Zhi; Ding, Tian-Bo; Wei, Dan-Dan; Wang, Jin-Jun

2013-01-01

Background Recent studies indicate that infestations of psocids pose a new risk for global food security. Among the psocids species, Liposcelis bostrychophila Badonnel has gained recognition in importance because of its parthenogenic reproduction, rapid adaptation, and increased worldwide distribution. To date, the molecular data available for L. bostrychophila is largely limited to genes identified through homology. Also, no transcriptome data relevant to psocids infection is available. Methodology and Principal Findings In this study, we generated de novo assembly of L. bostrychophila transcriptome performed through the short read sequencing technology (Illumina). In a single run, we obtained more than 51 million sequencing reads that were assembled into 60,012 unigenes (mean size = 711 bp) by Trinity. The transcriptome sequences from different developmental stages of L. bostrychophila including egg, nymph and adult were annotated with non-redundant (Nr) protein database, gene ontology (GO), cluster of orthologous groups of proteins (COG), and KEGG orthology (KO). The analysis revealed three major enzyme families involved in insecticide metabolism as differentially expressed in the L. bostrychophila transcriptome. A total of 49 P450-, 31 GST- and 21 CES-specific genes representing the three enzyme families were identified. Besides, 16 transcripts were identified to contain target site sequences of resistance genes. Furthermore, we profiled gene expression patterns upon insecticide (malathion and deltamethrin) exposure using the tag-based digital gene expression (DGE) method. Conclusion The L. bostrychophila transcriptome and DGE data provide gene expression data that would further our understanding of molecular mechanisms in psocids. In particular, the findings of this investigation will facilitate identification of genes involved in insecticide resistance and designing of new compounds for control of psocids. PMID:24278202

Sequence and expression variation in SUPPRESSOR of OVEREXPRESSION of CONSTANS 1 (SOC1): homeolog evolution in Indian Brassicas.

PubMed

Sri, Tanu; Mayee, Pratiksha; Singh, Anandita

2015-09-01

Whole genome sequence analyses allow unravelling such evolutionary consequences of meso-triplication event in Brassicaceae (∼14-20 million years ago (MYA)) as differential gene fractionation and diversification in homeologous sub-genomes. This study presents a simple gene-centric approach involving microsynteny and natural genetic variation analysis for understanding SUPPRESSOR of OVEREXPRESSION of CONSTANS 1 (SOC1) homeolog evolution in Brassica. Analysis of microsynteny in Brassica rapa homeologous regions containing SOC1 revealed differential gene fractionation correlating to reported fractionation status of sub-genomes of origin, viz. least fractionated (LF), moderately fractionated 1 (MF1) and most fractionated (MF2), respectively. Screening 18 cultivars of 6 Brassica species led to the identification of 8 genomic and 27 transcript variants of SOC1, including splice-forms. Co-occurrence of both interrupted and intronless SOC1 genes was detected in few Brassica species. In silico analysis characterised Brassica SOC1 as MADS intervening, K-box, C-terminal (MIKC(C)) transcription factor, with highly conserved MADS and I domains relative to K-box and C-terminal domain. Phylogenetic analyses and multiple sequence alignments depicting shared pattern of silent/non-silent mutations assigned Brassica SOC1 homologs into groups based on shared diploid base genome. In addition, a sub-genome structure in uncharacterised Brassica genomes was inferred. Expression analysis of putative MF2 and LF (Brassica diploid base genome A (AA)) sub-genome-specific SOC1 homeologs of Brassica juncea revealed near identical expression pattern. However, MF2-specific homeolog exhibited significantly higher expression implying regulatory diversification. In conclusion, evidence for polyploidy-induced sequence and regulatory evolution in Brassica SOC1 is being presented wherein differential homeolog expression is implied in functional diversification.

An Epstein-Barr virus immediate-early gene product trans-activates gene expression from the human immunodeficiency virus long terminal repeat.

PubMed Central

Kenney, S; Kamine, J; Markovitz, D; Fenrick, R; Pagano, J

1988-01-01

Acquired immunodeficiency syndrome patients are frequently coinfected with Epstein-Barr virus (EBV). In this report, we demonstrate that an EBV immediate-early gene product, BamHI MLF1, stimulates expression of the bacterial chloramphenicol acetyltransferase (CAT) gene linked to the human immunodeficiency virus (HIV) promoter. The HIV promoter sequences necessary for trans-activation by EBV do not include the tat-responsive sequences. In addition, in contrast to the other herpesvirus trans-activators previously studied, the EBV BamHI MLF1 gene product appears to function in part by a posttranscriptional mechanism, since it increases pHIV-CAT protein activity more than it increases HIV-CAT mRNA. This ability of an EBV gene product to activate HIV gene expression may have biologic consequences in persons coinfected with both viruses. Images PMID:2830625

Epstein-Barr virus immediate-early gene product trans-activates gene expression from the human immunodeficiency virus long terminal repeat

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

Kenney, S.; Kamine, J.; Markovitz, D.

Acquired immunodeficiency syndrome patients are frequently coinfected with Epstein-Barr virus (EBV). In this report, the authors demonstrate that an EBV immediate-early gene product, BamHI MLF1, stimulates expression of the bacterial chloramphenicol acetyltransferase (CAT) gene linked to the human immunodeficiency virus (HIV) promoter. The HIV promoter sequences necessary for trans-activation by EBV do not include the tat-responsive sequences. In addition, in contrast to the other herpesvirus trans-activators previously studied, the EBV BamHI MLF1 gene product appears to function in part by a posttranscriptional mechanism, since it increases pHIV-CAT protein activity more than it increases HIV-CAT mRNA. This ability of an EBVmore » gene product to activate HIV gene expression may have biologic consequences in persons coinfected with both viruses.« less

"De-novo" amino acid sequence elucidation of protein G'e by combined "top-down" and "bottom-up" mass spectrometry.

PubMed

Yefremova, Yelena; Al-Majdoub, Mahmoud; Opuni, Kwabena F M; Koy, Cornelia; Cui, Weidong; Yan, Yuetian; Gross, Michael L; Glocker, Michael O

2015-03-01

Mass spectrometric de-novo sequencing was applied to review the amino acid sequence of a commercially available recombinant protein G´ with great scientific and economic importance. Substantial deviations to the published amino acid sequence (Uniprot Q54181) were found by the presence of 46 additional amino acids at the N-terminus, including a so-called "His-tag" as well as an N-terminal partial α-N-gluconoylation and α-N-phosphogluconoylation, respectively. The unexpected amino acid sequence of the commercial protein G' comprised 241 amino acids and resulted in a molecular mass of 25,998.9 ± 0.2 Da for the unmodified protein. Due to the higher mass that is caused by its extended amino acid sequence compared with the original protein G' (185 amino acids), we named this protein "protein G'e." By means of mass spectrometric peptide mapping, the suggested amino acid sequence, as well as the N-terminal partial α-N-gluconoylations, was confirmed with 100% sequence coverage. After the protein G'e sequence was determined, we were able to determine the expression vector pET-28b from Novagen with the Xho I restriction enzyme cleavage site as the best option that was used for cloning and expressing the recombinant protein G'e in E. coli. A dissociation constant (K(d)) value of 9.4 nM for protein G'e was determined thermophoretically, showing that the N-terminal flanking sequence extension did not cause significant changes in the binding affinity to immunoglobulins.

ORFer--retrieval of protein sequences and open reading frames from GenBank and storage into relational databases or text files.

PubMed

Büssow, Konrad; Hoffmann, Steve; Sievert, Volker

2002-12-19

Functional genomics involves the parallel experimentation with large sets of proteins. This requires management of large sets of open reading frames as a prerequisite of the cloning and recombinant expression of these proteins. A Java program was developed for retrieval of protein and nucleic acid sequences and annotations from NCBI GenBank, using the XML sequence format. Annotations retrieved by ORFer include sequence name, organism and also the completeness of the sequence. The program has a graphical user interface, although it can be used in a non-interactive mode. For protein sequences, the program also extracts the open reading frame sequence, if available, and checks its correct translation. ORFer accepts user input in the form of single or lists of GenBank GI identifiers or accession numbers. It can be used to extract complete sets of open reading frames and protein sequences from any kind of GenBank sequence entry, including complete genomes or chromosomes. Sequences are either stored with their features in a relational database or can be exported as text files in Fasta or tabulator delimited format. The ORFer program is freely available at http://www.proteinstrukturfabrik.de/orfer. The ORFer program allows for fast retrieval of DNA sequences, protein sequences and their open reading frames and sequence annotations from GenBank. Furthermore, storage of sequences and features in a relational database is supported. Such a database can supplement a laboratory information system (LIMS) with appropriate sequence information.

Functional role of SETD2, BAP1, PARP-3 and PBRM1 candidate genes on the regulation of hTERT gene expression

PubMed Central

Linne, Hannah; Yasaei, Hemad; Marriott, Alison; Harvey, Amanda; Mokbel, Kefah; Newbold, Robert; Roberts, Terry

2017-01-01

Narrowing the search for the critical hTERT repressor sequence(s) has identified three regions on chromosome 3p (3p12-p21.1, 3p21.2 and 3p21.3-p22). However, the precise location and identity of the sequence(s) responsible for hTERT transcriptional repression remains elusive. In order to identify critical hTERT repressor sequences located within human chromosome 3p12-p22, we investigated hTERT transcriptional activity within 21NT microcell hybrid clones containing chromosome 3 fragments. Mapping of chromosome 3 structure in a single hTERT-repressed 21NT-#3fragment hybrid clone, revealed a 490kb region of deletion localised to 3p21.3 and encompassing the histone H3, lysine 36 (H3K36) trimethyltransferase enzyme SETD2; a putative tumour suppressor gene in breast cancer. Three additional genes, BAP1, PARP-3 and PBRM1, were also selected for further investigation based on their location within the 3p21.1-p21.3 region, together with their documented role in the epigenetic regulation of target gene expression or hTERT regulation. All four genes (SETD2, BAP1, PARP-3 and PBRM1) were found to be expressed at low levels in 21NT. Gene copy number variation (CNV) analysis of SETD2, BAP1, PARP-3 and PBRM1 within a panel of nine breast cancer cell lines demonstrated single copy number loss of all candidate genes within five (56%) cell lines (including 21NT cells). Stable, forced overexpression of BAP1, but not PARP2, SETD2 or PBRM1, within 21NT cells was associated with a significant reduction in hTERT expression levels relative to wild-type controls. We propose that at least two sequences exist on human chromosome 3p, that function to regulate hTERT transcription within human breast cancer cells. PMID:28977912

Functional role of SETD2, BAP1, PARP-3 and PBRM1 candidate genes on the regulation of hTERT gene expression.

PubMed

Linne, Hannah; Yasaei, Hemad; Marriott, Alison; Harvey, Amanda; Mokbel, Kefah; Newbold, Robert; Roberts, Terry

2017-09-22

Narrowing the search for the critical hTERT repressor sequence(s) has identified three regions on chromosome 3p (3p12-p21.1, 3p21.2 and 3p21.3-p22). However, the precise location and identity of the sequence(s) responsible for hTERT transcriptional repression remains elusive. In order to identify critical hTERT repressor sequences located within human chromosome 3p12-p22, we investigated hTERT transcriptional activity within 21NT microcell hybrid clones containing chromosome 3 fragments. Mapping of chromosome 3 structure in a single hTERT- repressed 21NT-#3fragment hybrid clone, revealed a 490kb region of deletion localised to 3p21.3 and encompassing the histone H3, lysine 36 (H3K36) trimethyltransferase enzyme SETD2; a putative tumour suppressor gene in breast cancer. Three additional genes, BAP1, PARP-3 and PBRM1, were also selected for further investigation based on their location within the 3p21.1-p21.3 region, together with their documented role in the epigenetic regulation of target gene expression or hTERT regulation. All four genes (SETD2, BAP1, PARP-3 and PBRM1) were found to be expressed at low levels in 21NT. Gene copy number variation (CNV) analysis of SETD2, BAP1, PARP-3 and PBRM1 within a panel of nine breast cancer cell lines demonstrated single copy number loss of all candidate genes within five (56%) cell lines (including 21NT cells). Stable, forced overexpression of BAP1, but not PARP2, SETD2 or PBRM1, within 21NT cells was associated with a significant reduction in hTERT expression levels relative to wild-type controls. We propose that at least two sequences exist on human chromosome 3p, that function to regulate hTERT transcription within human breast cancer cells.

Expressed sequence tag analysis of guinea pig (Cavia porcellus) eye tissues for NEIBank

PubMed Central

Simpanya, Mukoma F.; Wistow, Graeme; Gao, James; David, Larry L.; Giblin, Frank J.

2008-01-01

Purpose To characterize gene expression patterns in guinea pig ocular tissues and identify orthologs of human genes from NEIBank expressed sequence tags. Methods RNA was extracted from dissected eye tissues of 2.5-month-old guinea pigs to make three unamplified and unnormalized cDNA libraries in the pCMVSport-6 vector for the lens, retina, and eye minus lens and retina. Over 4,000 clones were sequenced from each library and were analyzed using GRIST for clustering and gene identification. Lens crystallin EST data were validated using two-dimensional electrophoresis (2-DE), matrix assisted laser desorption (MALDI), and electrospray ionization mass spectrometry (ESIMS). Results Combined data from the three libraries generated a total of 6,694 distinctive gene clusters, with each library having between 1,000 and 3,000 clusters. Approximately 60% of the total gene clusters were novel cDNA sequences and had significant homologies to other mammalian sequences in GenBank. Complete cDNA sequences were obtained for many guinea pig lens proteins, including αA/αAinsert-, γN-, and γS-crystallins, lengsin and GRIFIN. The ratio of αA- to αB-crystallin on 2-DE gels was 8: 1 in the lens nucleus and 6.5: 1 in the cortex. Analysis of ESTs, genome sequence, and proteins (by MALDI), did not reveal any evidence for the presence of γD-, γE-, and γF-crystallin in the guinea pig. Predicted masses of many guinea pig lens crystallins were confirmed by ESIMS analysis. For the retina, orthologs of human phototransduction genes were found, such as Rhodopsin, S-antigen (Sag, Arrestin), and Transducin. The guinea-pig ortholog of NRL, a key rod photoreceptor-specific transcription factor, was also represented in EST data. In the ‘rest-of-eye’ library, the most abundant transcripts included decorin and keratin 12, representative of the cornea. Conclusions Genomic analysis of guinea pig eye tissues provides sequence-verified clones for future studies. Guinea pig orthologs of many human eye specific genes were identified. Guinea pig gene structures were similar to their human and rodent gene counterparts. Surprisingly, no orthologs of γD-, γE-, and γF-crystallin were found in EST, proteomic, or the current guinea pig genome data. PMID:19104676

mESAdb: microRNA Expression and Sequence Analysis Database

PubMed Central

Kaya, Koray D.; Karakülah, Gökhan; Yakıcıer, Cengiz M.; Acar, Aybar C.; Konu, Özlen

2011-01-01

microRNA expression and sequence analysis database (http://konulab.fen.bilkent.edu.tr/mirna/) (mESAdb) is a regularly updated database for the multivariate analysis of sequences and expression of microRNAs from multiple taxa. mESAdb is modular and has a user interface implemented in PHP and JavaScript and coupled with statistical analysis and visualization packages written for the R language. The database primarily comprises mature microRNA sequences and their target data, along with selected human, mouse and zebrafish expression data sets. mESAdb analysis modules allow (i) mining of microRNA expression data sets for subsets of microRNAs selected manually or by motif; (ii) pair-wise multivariate analysis of expression data sets within and between taxa; and (iii) association of microRNA subsets with annotation databases, HUGE Navigator, KEGG and GO. The use of existing and customized R packages facilitates future addition of data sets and analysis tools. Furthermore, the ability to upload and analyze user-specified data sets makes mESAdb an interactive and expandable analysis tool for microRNA sequence and expression data. PMID:21177657

mESAdb: microRNA expression and sequence analysis database.

PubMed

Kaya, Koray D; Karakülah, Gökhan; Yakicier, Cengiz M; Acar, Aybar C; Konu, Ozlen

2011-01-01

microRNA expression and sequence analysis database (http://konulab.fen.bilkent.edu.tr/mirna/) (mESAdb) is a regularly updated database for the multivariate analysis of sequences and expression of microRNAs from multiple taxa. mESAdb is modular and has a user interface implemented in PHP and JavaScript and coupled with statistical analysis and visualization packages written for the R language. The database primarily comprises mature microRNA sequences and their target data, along with selected human, mouse and zebrafish expression data sets. mESAdb analysis modules allow (i) mining of microRNA expression data sets for subsets of microRNAs selected manually or by motif; (ii) pair-wise multivariate analysis of expression data sets within and between taxa; and (iii) association of microRNA subsets with annotation databases, HUGE Navigator, KEGG and GO. The use of existing and customized R packages facilitates future addition of data sets and analysis tools. Furthermore, the ability to upload and analyze user-specified data sets makes mESAdb an interactive and expandable analysis tool for microRNA sequence and expression data.

A regulatory sequence from the retinoid X receptor γ gene directs expression to horizontal cells and photoreceptors in the embryonic chicken retina.

PubMed

Blixt, Maria K E; Hallböök, Finn

2016-01-01

Combining techniques of episomal vector gene-specific Cre expression and genomic integration using the piggyBac transposon system enables studies of gene expression-specific cell lineage tracing in the chicken retina. In this work, we aimed to target the retinal horizontal cell progenitors. A 208 bp gene regulatory sequence from the chicken retinoid X receptor γ gene (RXRγ208) was used to drive Cre expression. RXRγ is expressed in progenitors and photoreceptors during development. The vector was combined with a piggyBac "donor" vector containing a floxed STOP sequence followed by enhanced green fluorescent protein (EGFP), as well as a piggyBac helper vector for efficient integration into the host cell genome. The vectors were introduced into the embryonic chicken retina with in ovo electroporation. Tissue electroporation targets specific developmental time points and in specific structures. Cells that drove Cre expression from the regulatory RXRγ208 sequence excised the floxed STOP-sequence and expressed GFP. The approach generated a stable lineage with robust expression of GFP in retinal cells that have activated transcription from the RXRγ208 sequence. Furthermore, GFP was expressed in cells that express horizontal or photoreceptor markers when electroporation was performed between developmental stages 22 and 28. Electroporation of a stage 12 optic cup gave multiple cell types in accordance with RXRγ gene expression in the early retina. In this study, we describe an easy, cost-effective, and time-efficient method for testing regulatory sequences in general. More specifically, our results open up the possibility for further studies of the RXRγ-gene regulatory network governing the formation of photoreceptor and horizontal cells. In addition, the method presents approaches to target the expression of effector genes, such as regulators of cell fate or cell cycle progression, to these cells and their progenitor.

Single-Cell RNA-Sequencing: Assessment of Differential Expression Analysis Methods.

PubMed

Dal Molin, Alessandra; Baruzzo, Giacomo; Di Camillo, Barbara

2017-01-01

The sequencing of the transcriptomes of single-cells, or single-cell RNA-sequencing, has now become the dominant technology for the identification of novel cell types and for the study of stochastic gene expression. In recent years, various tools for analyzing single-cell RNA-sequencing data have been proposed, many of them with the purpose of performing differentially expression analysis. In this work, we compare four different tools for single-cell RNA-sequencing differential expression, together with two popular methods originally developed for the analysis of bulk RNA-sequencing data, but largely applied to single-cell data. We discuss results obtained on two real and one synthetic dataset, along with considerations about the perspectives of single-cell differential expression analysis. In particular, we explore the methods performance in four different scenarios, mimicking different unimodal or bimodal distributions of the data, as characteristic of single-cell transcriptomics. We observed marked differences between the selected methods in terms of precision and recall, the number of detected differentially expressed genes and the overall performance. Globally, the results obtained in our study suggest that is difficult to identify a best performing tool and that efforts are needed to improve the methodologies for single-cell RNA-sequencing data analysis and gain better accuracy of results.

Identification of human chromosome 22 transcribed sequences with ORF expressed sequence tags

PubMed Central

de Souza, Sandro J.; Camargo, Anamaria A.; Briones, Marcelo R. S.; Costa, Fernando F.; Nagai, Maria Aparecida; Verjovski-Almeida, Sergio; Zago, Marco A.; Andrade, Luis Eduardo C.; Carrer, Helaine; El-Dorry, Hamza F. A.; Espreafico, Enilza M.; Habr-Gama, Angelita; Giannella-Neto, Daniel; Goldman, Gustavo H.; Gruber, Arthur; Hackel, Christine; Kimura, Edna T.; Maciel, Rui M. B.; Marie, Suely K. N.; Martins, Elizabeth A. L.; Nóbrega, Marina P.; Paçó-Larson, Maria Luisa; Pardini, Maria Inês M. C.; Pereira, Gonçalo G.; Pesquero, João Bosco; Rodrigues, Vanderlei; Rogatto, Silvia R.; da Silva, Ismael D. C. G.; Sogayar, Mari C.; de Fátima Sonati, Maria; Tajara, Eloiza H.; Valentini, Sandro R.; Acencio, Marcio; Alberto, Fernando L.; Amaral, Maria Elisabete J.; Aneas, Ivy; Bengtson, Mário Henrique; Carraro, Dirce M.; Carvalho, Alex F.; Carvalho, Lúcia Helena; Cerutti, Janete M.; Corrêa, Maria Lucia C.; Costa, Maria Cristina R.; Curcio, Cyntia; Gushiken, Tsieko; Ho, Paulo L.; Kimura, Elza; Leite, Luciana C. C.; Maia, Gustavo; Majumder, Paromita; Marins, Mozart; Matsukuma, Adriana; Melo, Analy S. A.; Mestriner, Carlos Alberto; Miracca, Elisabete C.; Miranda, Daniela C.; Nascimento, Ana Lucia T. O.; Nóbrega, Francisco G.; Ojopi, Élida P. B.; Pandolfi, José Rodrigo C.; Pessoa, Luciana Gilbert; Rahal, Paula; Rainho, Claudia A.; da Ro's, Nancy; de Sá, Renata G.; Sales, Magaly M.; da Silva, Neusa P.; Silva, Tereza C.; da Silva, Wilson; Simão, Daniel F.; Sousa, Josane F.; Stecconi, Daniella; Tsukumo, Fernando; Valente, Valéria; Zalcberg, Heloisa; Brentani, Ricardo R.; Reis, Luis F. L.; Dias-Neto, Emmanuel; Simpson, Andrew J. G.

2000-01-01

Transcribed sequences in the human genome can be identified with confidence only by alignment with sequences derived from cDNAs synthesized from naturally occurring mRNAs. We constructed a set of 250,000 cDNAs that represent partial expressed gene sequences and that are biased toward the central coding regions of the resulting transcripts. They are termed ORF expressed sequence tags (ORESTES). The 250,000 ORESTES were assembled into 81,429 contigs. Of these, 1,181 (1.45%) were found to match sequences in chromosome 22 with at least one ORESTES contig for 162 (65.6%) of the 247 known genes, for 67 (44.6%) of the 150 related genes, and for 45 of the 148 (30.4%) EST-predicted genes on this chromosome. Using a set of stringent criteria to validate our sequences, we identified a further 219 previously unannotated transcribed sequences on chromosome 22. Of these, 171 were in fact also defined by EST or full length cDNA sequences available in GenBank but not utilized in the initial annotation of the first human chromosome sequence. Thus despite representing less than 15% of all expressed human sequences in the public databases at the time of the present analysis, ORESTES sequences defined 48 transcribed sequences on chromosome 22 not defined by other sequences. All of the transcribed sequences defined by ORESTES coincided with DNA regions predicted as encoding exons by genscan. (http://genes.mit.edu/GENSCAN.html). PMID:11070084

Genetic divergence between freshwater and marine morphs of alewife (Alosa pseudoharengus): a 'next-generation' sequencing analysis.

PubMed

Czesny, Sergiusz; Epifanio, John; Michalak, Pawel

2012-01-01

Alewife Alosa pseudoharengus, a small clupeid fish native to Atlantic Ocean, has recently (∼150 years ago) invaded the North American Great Lakes and despite challenges of freshwater environment its populations exploded and disrupted local food web structures. This range expansion has been accompanied by dramatic changes at all levels of organization. Growth rates, size at maturation, or fecundity are only a few of the most distinct morphological and life history traits that contrast the two alewife morphs. A question arises to what extent these rapidly evolving differences between marine and freshwater varieties result from regulatory (including phenotypic plasticity) or structural mutations. To gain insights into expression changes and sequence divergence between marine and freshwater alewives, we sequenced transcriptomes of individuals from Lake Michigan and Atlantic Ocean. Population specific single nucleotide polymorphisms were rare but interestingly occurred in sequences of genes that also tended to show large differences in expression. Our results show that the striking phenotypic divergence between anadromous and lake alewives can be attributed to massive regulatory modifications rather than coding changes.

Genetic Divergence between Freshwater and Marine Morphs of Alewife (Alosa pseudoharengus): A ‘Next-Generation’ Sequencing Analysis

PubMed Central

Czesny, Sergiusz; Epifanio, John; Michalak, Pawel

2012-01-01

Alewife Alosa pseudoharengus, a small clupeid fish native to Atlantic Ocean, has recently (∼150 years ago) invaded the North American Great Lakes and despite challenges of freshwater environment its populations exploded and disrupted local food web structures. This range expansion has been accompanied by dramatic changes at all levels of organization. Growth rates, size at maturation, or fecundity are only a few of the most distinct morphological and life history traits that contrast the two alewife morphs. A question arises to what extent these rapidly evolving differences between marine and freshwater varieties result from regulatory (including phenotypic plasticity) or structural mutations. To gain insights into expression changes and sequence divergence between marine and freshwater alewives, we sequenced transcriptomes of individuals from Lake Michigan and Atlantic Ocean. Population specific single nucleotide polymorphisms were rare but interestingly occurred in sequences of genes that also tended to show large differences in expression. Our results show that the striking phenotypic divergence between anadromous and lake alewives can be attributed to massive regulatory modifications rather than coding changes. PMID:22438868

The foldase CYPB is a component of the secretory pathway of Aspergillus niger and contains the endoplasmic reticulum retention signal HEEL.

PubMed

Derkx, P M; Madrid, S M

2001-12-01

Here we report the isolation and characterization of the cypB gene from Aspergillus niger. The cypB gene encodes a protein with a predicted molecular weight of 20.7 kDa, which shows a high degree of identity to the cyclophilin family of peptidyl prolyl cis-trans isomerases (PPIases) from other eukaryotes. The 5' untranslated region of cypB includes three sequences resembling UPREs (unfolded protein response elements). The expression of cypB is upregulated by tunicamycin and DTT, suggesting that at least one UPRE is functional. The CYPB protein also has a 23-amino acid sequence which serves to target the protein to the endoplasmic reticulum (ER), and the ER retention sequence HEEL. CYPB-(His)(6) was expressed in Escherichia coli; the purified protein is capable of isomerizing a substrate peptide in vitro. This is also the first report to show that C-terminal addition of the sequence HEEL is sufficient to ensure retention of the green fluorescent protein (GFP) within the ER.

Comprehensive analysis of single molecule sequencing-derived complete genome and whole transcriptome of Hyposidra talaca nuclear polyhedrosis virus.

PubMed

Nguyen, Thong T; Suryamohan, Kushal; Kuriakose, Boney; Janakiraman, Vasantharajan; Reichelt, Mike; Chaudhuri, Subhra; Guillory, Joseph; Divakaran, Neethu; Rabins, P E; Goel, Ridhi; Deka, Bhabesh; Sarkar, Suman; Ekka, Preety; Tsai, Yu-Chih; Vargas, Derek; Santhosh, Sam; Mohan, Sangeetha; Chin, Chen-Shan; Korlach, Jonas; Thomas, George; Babu, Azariah; Seshagiri, Somasekar

2018-06-12

We sequenced the Hyposidra talaca NPV (HytaNPV) double stranded circular DNA genome using PacBio single molecule sequencing technology. We found that the HytaNPV genome is 139,089 bp long with a GC content of 39.6%. It encodes 141 open reading frames (ORFs) including the 37 baculovirus core genes, 25 genes conserved among lepidopteran baculoviruses, 72 genes known in baculovirus, and 7 genes unique to the HytaNPV genome. It is a group II alphabaculovirus that codes for the F protein and lacks the gp64 gene found in group I alphabaculovirus viruses. Using RNA-seq, we confirmed the expression of the ORFs identified in the HytaNPV genome. Phylogenetic analysis showed HytaNPV to be closest to BusuNPV, SujuNPV and EcobNPV that infect other tea pests, Buzura suppressaria, Sucra jujuba, and Ectropis oblique, respectively. We identified repeat elements and a conserved non-coding baculovirus element in the genome. Analysis of the putative promoter sequences identified motif consistent with the temporal expression of the genes observed in the RNA-seq data.

Step-by-Step Construction of Gene Co-expression Networks from High-Throughput Arabidopsis RNA Sequencing Data.

PubMed

Contreras-López, Orlando; Moyano, Tomás C; Soto, Daniela C; Gutiérrez, Rodrigo A

2018-01-01

The rapid increase in the availability of transcriptomics data generated by RNA sequencing represents both a challenge and an opportunity for biologists without bioinformatics training. The challenge is handling, integrating, and interpreting these data sets. The opportunity is to use this information to generate testable hypothesis to understand molecular mechanisms controlling gene expression and biological processes (Fig. 1). A successful strategy to generate tractable hypotheses from transcriptomics data has been to build undirected network graphs based on patterns of gene co-expression. Many examples of new hypothesis derived from network analyses can be found in the literature, spanning different organisms including plants and specific fields such as root developmental biology.In order to make the process of constructing a gene co-expression network more accessible to biologists, here we provide step-by-step instructions using published RNA-seq experimental data obtained from a public database. Similar strategies have been used in previous studies to advance root developmental biology. This guide includes basic instructions for the operation of widely used open source platforms such as Bio-Linux, R, and Cytoscape. Even though the data we used in this example was obtained from Arabidopsis thaliana, the workflow developed in this guide can be easily adapted to work with RNA-seq data from any organism.

Variations in endothelin receptor B subtype 2 (EDNRB2) coding sequences and mRNA expression levels in 4 Muscovy duck plumage colour phenotypes.

PubMed

Wu, N; Qin, H; Wang, M; Bian, Y; Dong, B; Sun, G; Zhao, W; Chang, G; Xu, Q; Chen, G

2017-04-01

1. Endothelin receptor B subtype 2 (EDNRB2) is a paralog of EDNRB, which encodes a 7-transmembrane G-protein coupled receptor. Previous studies reported that EDNRB was essential for melanoblast migration in mammals and ducks. 2. Muscovy ducks have different plumage colour phenotypes. Variations in EDNRB2 coding sequences (CDSs) and mRNA expression levels were investigated in 4 different Muscovy duck plumage colour phenotypes, including black, black mutant, silver and white head. 3. The EDNRB2 gene from Muscovy duck was cloned; it had a length of 6435 bp and encoded 437 amino acids. The coding region was screened and potential single nucleotide polymorphisms were identified. Eight mutations were obtained, including one missense variant (c.64C > T) and 7 synonymous substitutions. The substitutions were associated with plumage colour phenotypes. 4. The EDNRB2 mRNA expression levels were compared between feather pulp from black birds and black mutant birds. The results indicated that EDNRB2 transcripts in feather pulp were significantly higher in black feathers than in white feathers. 5. The results determined the variation of EDNRB2 CDS and mRNA expression in Muscovy ducks of various plumage colours.

EXONSAMPLER: a computer program for genome-wide and candidate gene exon sampling for targeted next-generation sequencing.

PubMed

Cosart, Ted; Beja-Pereira, Albano; Luikart, Gordon

2014-11-01

The computer program EXONSAMPLER automates the sampling of thousands of exon sequences from publicly available reference genome sequences and gene annotation databases. It was designed to provide exon sequences for the efficient, next-generation gene sequencing method called exon capture. The exon sequences can be sampled by a list of gene name abbreviations (e.g. IFNG, TLR1), or by sampling exons from genes spaced evenly across chromosomes. It provides a list of genomic coordinates (a bed file), as well as a set of sequences in fasta format. User-adjustable parameters for collecting exon sequences include a minimum and maximum acceptable exon length, maximum number of exonic base pairs (bp) to sample per gene, and maximum total bp for the entire collection. It allows for partial sampling of very large exons. It can preferentially sample upstream (5 prime) exons, downstream (3 prime) exons, both external exons, or all internal exons. It is written in the Python programming language using its free libraries. We describe the use of EXONSAMPLER to collect exon sequences from the domestic cow (Bos taurus) genome for the design of an exon-capture microarray to sequence exons from related species, including the zebu cow and wild bison. We collected ~10% of the exome (~3 million bp), including 155 candidate genes, and ~16,000 exons evenly spaced genomewide. We prioritized the collection of 5 prime exons to facilitate discovery and genotyping of SNPs near upstream gene regulatory DNA sequences, which control gene expression and are often under natural selection. © 2014 John Wiley & Sons Ltd.

Human HOXA5 homeodomain enhances protein transduction and its application to vascular inflammation

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

Lee, Ji Young; Park, Kyoung sook; Cho, Eun Jung

2011-07-01

Highlights: {yields} We have developed an E. coli protein expression vector including human specific gene sequences for protein cellular delivery. {yields} The plasmid was generated by ligation the nucleotides 770-817 of the homeobox A5 mRNA sequence. {yields} HOXA5-APE1/Ref-1 inhibited TNF-alpha-induced monocyte adhesion to endothelial cells. {yields} Human HOXA5-PTD vector provides a powerful research tools for uncovering cellular functions of proteins or for the generation of human PTD-containing proteins. -- Abstract: Cellular protein delivery is an emerging technique by which exogenous recombinant proteins are delivered into mammalian cells across the membrane. We have developed an Escherichia coli expression vector including humanmore » specific gene sequences for protein cellular delivery. The plasmid was generated by ligation the nucleotides 770-817 of the homeobox A5 mRNA sequence which was matched with protein transduction domain (PTD) of homeodomain protein A5 (HOXA5) into pET expression vector. The cellular uptake of HOXA5-PTD-EGFP was detected in 1 min and its transduction reached a maximum at 1 h within cell lysates. The cellular uptake of HOXA5-EGFP at 37 {sup o}C was greater than in 4 {sup o}C. For study for the functional role of human HOXA5-PTD, we purified HOXA5-APE1/Ref-1 and applied it on monocyte adhesion. Pretreatment with HOXA5-APE1/Ref-1 (100 nM) inhibited TNF-{alpha}-induced monocyte adhesion to endothelial cells, compared with HOXA5-EGFP. Taken together, our data suggested that human HOXA5-PTD vector provides a powerful research tools for uncovering cellular functions of proteins or for the generation of human PTD-containing proteins.« less

An ovary transcriptome for all maturational stages of the striped bass (Morone saxatilis), a highly advanced perciform fish.

PubMed

Reading, Benjamin J; Chapman, Robert W; Schaff, Jennifer E; Scholl, Elizabeth H; Opperman, Charles H; Sullivan, Craig V

2012-02-21

The striped bass and its relatives (genus Morone) are important fisheries and aquaculture species native to estuaries and rivers of the Atlantic coast and Gulf of Mexico in North America. To open avenues of gene expression research on reproduction and breeding of striped bass, we generated a collection of expressed sequence tags (ESTs) from a complementary DNA (cDNA) library representative of their ovarian transcriptome. Sequences of a total of 230,151 ESTs (51,259,448 bp) were acquired by Roche 454 pyrosequencing of cDNA pooled from ovarian tissues obtained at all stages of oocyte growth, at ovulation (eggs), and during preovulatory atresia. Quality filtering of ESTs allowed assembly of 11,208 high-quality contigs ≥ 100 bp, including 2,984 contigs 500 bp or longer (average length 895 bp). Blastx comparisons revealed 5,482 gene orthologues (E-value < 10-3), of which 4,120 (36.7% of total contigs) were annotated with Gene Ontology terms (E-value < 10-6). There were 5,726 remaining unknown unique sequences (51.1% of total contigs). All of the high-quality EST sequences are available in the National Center for Biotechnology Information (NCBI) Short Read Archive (GenBank: SRX007394). Informative contigs were considered to be abundant if they were assembled from groups of ESTs comprising ≥ 0.15% of the total short read sequences (≥ 345 reads/contig). Approximately 52.5% of these abundant contigs were predicted to have predominant ovary expression through digital differential display in silico comparisons to zebrafish (Danio rerio) UniGene orthologues. Over 1,300 Gene Ontology terms from Biological Process classes of Reproduction, Reproductive process, and Developmental process were assigned to this collection of annotated contigs. This first large reference sequence database available for the ecologically and economically important temperate basses (genus Morone) provides a foundation for gene expression studies in these species. The predicted predominance of ovary gene expression and assignment of directly relevant Gene Ontology classes suggests a powerful utility of this dataset for analysis of ovarian gene expression related to fundamental questions of oogenesis. Additionally, a high definition Agilent 60-mer oligo ovary 'UniClone' microarray with 8 × 15,000 probe format has been designed based on this striped bass transcriptome (eArray Group: Striper Group, Design ID: 029004).

Transcriptome analysis of Cymbidium sinense and its application to the identification of genes associated with floral development

PubMed Central

2013-01-01

Background Cymbidium sinense belongs to the Orchidaceae, which is one of the most abundant angiosperm families. C. sinense, a high-grade traditional potted flower, is most prevalent in China and some Southeast Asian countries. The control of flowering time is a major bottleneck in the industrialized development of C. sinense. Little is known about the mechanisms responsible for floral development in this orchid. Moreover, genome references for entire transcriptome sequences do not currently exist for C. sinense. Thus, transcriptome and expression profiling data for this species are needed as an important resource to identify genes and to better understand the biological mechanisms of floral development in C. sinense. Results In this study, de novo transcriptome assembly and gene expression analysis using Illumina sequencing technology were performed. Transcriptome analysis assembles gene-related information related to vegetative and reproductive growth of C. sinense. Illumina sequencing generated 54,248,006 high quality reads that were assembled into 83,580 unigenes with an average sequence length of 612 base pairs, including 13,315 clusters and 70,265 singletons. A total of 41,687 (49.88%) unique sequences were annotated, 23,092 of which were assigned to specific metabolic pathways by the Kyoto Encyclopedia of Genes and Genomes (KEGG). Gene Ontology (GO) analysis of the annotated unigenes revealed that the majority of sequenced genes were associated with metabolic and cellular processes, cell and cell parts, catalytic activity and binding. Furthermore, 120 flowering-associated unigenes, 73 MADS-box unigenes and 28 CONSTANS-LIKE (COL) unigenes were identified from our collection. In addition, three digital gene expression (DGE) libraries were constructed for the vegetative phase (VP), floral differentiation phase (FDP) and reproductive phase (RP). The specific expression of many genes in the three development phases was also identified. 32 genes among three sub-libraries with high differential expression were selected as candidates connected with flower development. Conclusion RNA-seq and DGE profiling data provided comprehensive gene expression information at the transcriptional level that could facilitate our understanding of the molecular mechanisms of floral development at three development phases of C. sinense. This data could be used as an important resource for investigating the genetics of the flowering pathway and various biological mechanisms in this orchid. PMID:23617896

Transcriptome analysis of Cymbidium sinense and its application to the identification of genes associated with floral development.

PubMed

Zhang, Jianxia; Wu, Kunlin; Zeng, Songjun; Teixeira da Silva, Jaime A; Zhao, Xiaolan; Tian, Chang-En; Xia, Haoqiang; Duan, Jun

2013-04-24

Cymbidium sinense belongs to the Orchidaceae, which is one of the most abundant angiosperm families. C. sinense, a high-grade traditional potted flower, is most prevalent in China and some Southeast Asian countries. The control of flowering time is a major bottleneck in the industrialized development of C. sinense. Little is known about the mechanisms responsible for floral development in this orchid. Moreover, genome references for entire transcriptome sequences do not currently exist for C. sinense. Thus, transcriptome and expression profiling data for this species are needed as an important resource to identify genes and to better understand the biological mechanisms of floral development in C. sinense. In this study, de novo transcriptome assembly and gene expression analysis using Illumina sequencing technology were performed. Transcriptome analysis assembles gene-related information related to vegetative and reproductive growth of C. sinense. Illumina sequencing generated 54,248,006 high quality reads that were assembled into 83,580 unigenes with an average sequence length of 612 base pairs, including 13,315 clusters and 70,265 singletons. A total of 41,687 (49.88%) unique sequences were annotated, 23,092 of which were assigned to specific metabolic pathways by the Kyoto Encyclopedia of Genes and Genomes (KEGG). Gene Ontology (GO) analysis of the annotated unigenes revealed that the majority of sequenced genes were associated with metabolic and cellular processes, cell and cell parts, catalytic activity and binding. Furthermore, 120 flowering-associated unigenes, 73 MADS-box unigenes and 28 CONSTANS-LIKE (COL) unigenes were identified from our collection. In addition, three digital gene expression (DGE) libraries were constructed for the vegetative phase (VP), floral differentiation phase (FDP) and reproductive phase (RP). The specific expression of many genes in the three development phases was also identified. 32 genes among three sub-libraries with high differential expression were selected as candidates connected with flower development. RNA-seq and DGE profiling data provided comprehensive gene expression information at the transcriptional level that could facilitate our understanding of the molecular mechanisms of floral development at three development phases of C. sinense. This data could be used as an important resource for investigating the genetics of the flowering pathway and various biological mechanisms in this orchid.

Comparative venom gland transcriptomics of Naja kaouthia (monocled cobra) from Malaysia and Thailand: elucidating geographical venom variation and insights into sequence novelty

PubMed Central

Chanhome, Lawan; Tan, Nget Hong

2017-01-01

Background The monocled cobra (Naja kaouthia) is a medically important venomous snake in Southeast Asia. Its venom has been shown to vary geographically in relation to venom composition and neurotoxic activity, indicating vast diversity of the toxin genes within the species. To investigate the polygenic trait of the venom and its locale-specific variation, we profiled and compared the venom gland transcriptomes of N. kaouthia from Malaysia (NK-M) and Thailand (NK-T) applying next-generation sequencing (NGS) technology. Methods The transcriptomes were sequenced on the Illumina HiSeq platform, assembled and followed by transcript clustering and annotations for gene expression and function. Pairwise or multiple sequence alignments were conducted on the toxin genes expressed. Substitution rates were studied for the major toxins co-expressed in NK-M and NK-T. Results and discussion The toxin transcripts showed high redundancy (41–82% of the total mRNA expression) and comprised 23 gene families expressed in NK-M and NK-T, respectively (22 gene families were co-expressed). Among the venom genes, three-finger toxins (3FTxs) predominated in the expression, with multiple sequences noted. Comparative analysis and selection study revealed that 3FTxs are genetically conserved between the geographical specimens whilst demonstrating distinct differential expression patterns, implying gene up-regulation for selected principal toxins, or alternatively, enhanced transcript degradation or lack of transcription of certain traits. One of the striking features that elucidates the inter-geographical venom variation is the up-regulation of α-neurotoxins (constitutes ∼80.0% of toxin’s fragments per kilobase of exon model per million mapped reads (FPKM)), particularly the long-chain α-elapitoxin-Nk2a (48.3%) in NK-T but only 1.7% was noted in NK-M. Instead, short neurotoxin isoforms were up-regulated in NK-M (46.4%). Another distinct transcriptional pattern observed is the exclusively and abundantly expressed cytotoxin CTX-3 in NK-T. The findings suggested correlation with the geographical variation in proteome and toxicity of the venom, and support the call for optimising antivenom production and use in the region. Besides, the current study uncovered full and partial sequences of numerous toxin genes from N. kaouthia which have not been reported hitherto; these include N. kaouthia-specific l-amino acid oxidase (LAAO), snake venom serine protease (SVSP), cystatin, acetylcholinesterase (AChE), hyaluronidase (HYA), waprin, phospholipase B (PLB), aminopeptidase (AP), neprilysin, etc. Taken together, the findings further enrich the snake toxin database and provide deeper insights into the genetic diversity of cobra venom toxins. PMID:28392982

Classification and Weakly Supervised Pain Localization using Multiple Segment Representation.

PubMed

Sikka, Karan; Dhall, Abhinav; Bartlett, Marian Stewart

2014-10-01

Automatic pain recognition from videos is a vital clinical application and, owing to its spontaneous nature, poses interesting challenges to automatic facial expression recognition (AFER) research. Previous pain vs no-pain systems have highlighted two major challenges: (1) ground truth is provided for the sequence, but the presence or absence of the target expression for a given frame is unknown, and (2) the time point and the duration of the pain expression event(s) in each video are unknown. To address these issues we propose a novel framework (referred to as MS-MIL) where each sequence is represented as a bag containing multiple segments, and multiple instance learning (MIL) is employed to handle this weakly labeled data in the form of sequence level ground-truth. These segments are generated via multiple clustering of a sequence or running a multi-scale temporal scanning window, and are represented using a state-of-the-art Bag of Words (BoW) representation. This work extends the idea of detecting facial expressions through 'concept frames' to 'concept segments' and argues through extensive experiments that algorithms such as MIL are needed to reap the benefits of such representation. The key advantages of our approach are: (1) joint detection and localization of painful frames using only sequence-level ground-truth, (2) incorporation of temporal dynamics by representing the data not as individual frames but as segments, and (3) extraction of multiple segments, which is well suited to signals with uncertain temporal location and duration in the video. Extensive experiments on UNBC-McMaster Shoulder Pain dataset highlight the effectiveness of the approach by achieving competitive results on both tasks of pain classification and localization in videos. We also empirically evaluate the contributions of different components of MS-MIL. The paper also includes the visualization of discriminative facial patches, important for pain detection, as discovered by our algorithm and relates them to Action Units that have been associated with pain expression. We conclude the paper by demonstrating that MS-MIL yields a significant improvement on another spontaneous facial expression dataset, the FEEDTUM dataset.

Application of Cydia pomonella expressed sequence tags: identification and expression of three general odorant binding proteins in codling moth

USDA-ARS?s Scientific Manuscript database

The codling moth, Cydia pomonella, is one of the most important pests of pome fruits in the world, yet the molecular genetics and physiology of this insect remains poorly understood. A combined assembly of 8340 expressed sequence tags (ESTs) was generated from Roche 454 GS-FLX sequencing of 8 tissu...

A reassessment of IgM memory subsets in humans

PubMed Central

Bagnara, Davide; Squillario, Margherita; Kipling, David; Mora, Thierry; Walczak, Aleksandra M.; Da Silva, Lucie; Weller, Sandra; Dunn-Walters, Deborah K.; Weill, Jean-Claude; Reynaud, Claude-Agnès

2015-01-01

From paired blood and spleen samples from three adult donors we performed high-throughput V-h sequencing of human B-cell subsets defined by IgD and CD27 expression: IgD+CD27+ (“MZ”), IgD−CD27+(“memory”, including IgM (“IgM-only”), IgG and IgA) and IgD−CD27− cells (“double-negative”, including IgM, IgG and IgA). 91,294 unique sequences clustered in 42,670 clones, revealing major clonal expansions in each of these subsets. Among these clones, we further analyzed those shared sequences from different subsets or tissues for Vh-gene mutation, H-CDR3-length, and Vh/Jh usage, comparing these different characteristics with all sequences from their subset of origin, for which these parameters constitute a distinct signature. The IgM-only repertoire profile differed notably from that of MZ B cells by a higher mutation frequency, and lower Vh4 and higher Jh6 gene usage. Strikingly, IgM sequences from clones shared between the MZ and the memory IgG/IgA compartments showed a mutation and repertoire profile of IgM-only and not of MZ B cells. Similarly, all IgM clonal relationships (between MZ, IgM-only, and double-negative compartments) involved sequences with the characteristics of IgM-only B cells. Finally, clonal relationships between tissues suggested distinct recirculation characteristics between MZ and switched B cells. The “IgM-only” subset (including cells with its repertoire signature but higher IgD or lower CD27 expression levels) thus appear as the only subset showing precursor-product relationships with CD27+ switched memory B cells, indicating that they represent germinal center-derived IgM memory B cells, and that IgM memory and MZ B cells constitute two distinct entities. PMID:26355154

A Reassessment of IgM Memory Subsets in Humans.

PubMed

Bagnara, Davide; Squillario, Margherita; Kipling, David; Mora, Thierry; Walczak, Aleksandra M; Da Silva, Lucie; Weller, Sandra; Dunn-Walters, Deborah K; Weill, Jean-Claude; Reynaud, Claude-Agnès

2015-10-15

From paired blood and spleen samples from three adult donors, we performed high-throughput VH sequencing of human B cell subsets defined by IgD and CD27 expression: IgD(+)CD27(+) ("marginal zone [MZ]"), IgD(-)CD27(+) ("memory," including IgM ["IgM-only"], IgG and IgA) and IgD(-)CD27(-) cells ("double-negative," including IgM, IgG, and IgA). A total of 91,294 unique sequences clustered in 42,670 clones, revealing major clonal expansions in each of these subsets. Among these clones, we further analyzed those shared sequences from different subsets or tissues for VH gene mutation, H-CDR3-length, and VH/JH usage, comparing these different characteristics with all sequences from their subset of origin for which these parameters constitute a distinct signature. The IgM-only repertoire profile differed notably from that of MZ B cells by a higher mutation frequency and lower VH4 and higher JH6 gene usage. Strikingly, IgM sequences from clones shared between the MZ and the memory IgG/IgA compartments showed a mutation and repertoire profile of IgM-only and not of MZ B cells. Similarly, all IgM clonal relationships (among MZ, IgM-only, and double-negative compartments) involved sequences with the characteristics of IgM-only B cells. Finally, clonal relationships between tissues suggested distinct recirculation characteristics between MZ and switched B cells. The "IgM-only" subset (including cells with its repertoire signature but higher IgD or lower CD27 expression levels) thus appear as the only subset showing precursor-product relationships with CD27(+) switched memory B cells, indicating that they represent germinal center-derived IgM memory B cells and that IgM memory and MZ B cells constitute two distinct entities. Copyright © 2015 by The American Association of Immunologists, Inc.

The use of a viral 2A sequence for the simultaneous over-expression of both the vgf gene and enhanced green fluorescent protein (eGFP) in vitro and in vivo

PubMed Central

Lewis, Jo E.; Brameld, John M.; Hill, Phil; Barrett, Perry; Ebling, Francis J.P.; Jethwa, Preeti H.

2015-01-01

Introduction The viral 2A sequence has become an attractive alternative to the traditional internal ribosomal entry site (IRES) for simultaneous over-expression of two genes and in combination with recombinant adeno-associated viruses (rAAV) has been used to manipulate gene expression in vitro. New method To develop a rAAV construct in combination with the viral 2A sequence to allow long-term over-expression of the vgf gene and fluorescent marker gene for tracking of the transfected neurones in vivo. Results Transient transfection of the AAV plasmid containing the vgf gene, viral 2A sequence and eGFP into SH-SY5Y cells resulted in eGFP fluorescence comparable to a commercially available reporter construct. This increase in fluorescent cells was accompanied by an increase in VGF mRNA expression. Infusion of the rAAV vector containing the vgf gene, viral 2A sequence and eGFP resulted in eGFP fluorescence in the hypothalamus of both mice and Siberian hamsters, 32 weeks post infusion. In situ hybridisation confirmed that the location of VGF mRNA expression in the hypothalamus corresponded to the eGFP pattern of fluorescence. Comparison with old method The viral 2A sequence is much smaller than the traditional IRES and therefore allowed over-expression of the vgf gene with fluorescent tracking without compromising viral capacity. Conclusion The use of the viral 2A sequence in the AAV plasmid allowed the simultaneous expression of both genes in vitro. When used in combination with rAAV it resulted in long-term over-expression of both genes at equivalent locations in the hypothalamus of both Siberian hamsters and mice, without any adverse effects. PMID:26300182

Distinct profiles of expressed sequence tags during intestinal regeneration in the sea cucumber Holothuria glaberrima

PubMed Central

Rojas-Cartagena, Carmencita; Ortíz-Pineda, Pablo; Ramírez-Gómez, Francisco; Suárez-Castillo, Edna C.; Matos-Cruz, Vanessa; Rodríguez, Carlos; Ortíz-Zuazaga, Humberto; García-Arrarás, José E.

2010-01-01

Repair and regeneration are key processes for tissue maintenance, and their disruption may lead to disease states. Little is known about the molecular mechanisms that underline the repair and regeneration of the digestive tract. The sea cucumber Holothuria glaberrima represents an excellent model to dissect and characterize the molecular events during intestinal regeneration. To study the gene expression profile, cDNA libraries were constructed from normal, 3-day, and 7-day regenerating intestines of H. glaberrima. Clones were randomly sequenced and queried against the nonredundant protein database at the National Center for Biotechnology Information. RT-PCR analyses were made of several genes to determine their expression profile during intestinal regeneration. A total of 5,173 sequences from three cDNA libraries were obtained. About 46.2, 35.6, and 26.2% of the sequences for the normal, 3-days, and 7-days cDNA libraries, respectively, shared significant similarity with known sequences in the protein database of GenBank but only present 10% of similarity among them. Analysis of the libraries in terms of functional processes, protein domains, and most common sequences suggests that a differential expression profile is taking place during the regeneration process. Further examination of the expressed sequence tag dataset revealed that 12 putative genes are differentially expressed at significant level (R > 6). Experimental validation by RT-PCR analysis reveals that at least three genes (unknown C-4677-1, melanotransferrin, and centaurin) present a differential expression during regeneration. These findings strongly suggest that the gene expression profile varies among regeneration stages and provide evidence for the existence of differential gene expression. PMID:17579180

Transcriptome profiling in imipenem-selected Acinetobacter baumannii.

PubMed

Chang, Kai-Chih; Kuo, Han-Yueh; Tang, Chuan Yi; Chang, Cheng-Wei; Lu, Chia-Wei; Liu, Chih-Chin; Lin, Huei-Ru; Chen, Kuan-Hsueh; Liou, Ming-Li

2014-09-26

Carbapenem-resistance in Acinetobacter baumannii has gradually become a global challenge. To identify the genes involved in carbapenem resistance in A. baumannii, the transcriptomic responses of the completely sequenced strain ATCC 17978 selected with 0.5 mg/L (IPM-2 m) and 2 mg/L (IPM-8 m) imipenem were investigated using RNA-sequencing to identify differences in the gene expression patterns. A total of 88 and 68 genes were differentially expressed in response to IPM-2 m and IPM-8 m selection, respectively. Among the expressed genes, 50 genes were highly expressed in IPM-2 m, 30 genes were highly expressed in IPM-8 m, and 38 genes were expressed common in both strains. Six groups of genes were simultaneously expressed in IPM-2 m and IPM-8 m mutants. The three gene groups involved in DNA recombination were up-regulated, including recombinase, transposase and DNA repair, and beta-lactamase OXA-95 and homologous recombination. The remaining gene groups involved in biofilm formation were down-regulated, including quorum sensing, secretion systems, and the csu operon. The antibiotic resistance determinants, including RND efflux transporters and multidrug resistance pumps, were over-expressed in response to IPM-2 m selection, followed by a decrease in response to IPM-8 m selection. Among the genes over-expressed in both strains, blaOXA-95, previously clustered with the blaOXA-51-like family, showed 14-fold (IPM-2 m) to 330-fold (IPM-8 m) over-expression. The expression of blaOXA-95 in IPM-2 m and IPM-8 m cells was positively correlated with the rate of imipenem hydrolysis, as demonstrated through Liquid Chromatography-Mass Spectrometry/Mass Spectrometry, suggesting that blaOXA-95 plays a critical role in conferring carbapenem resistance. In addition, A. baumannii shows an inverse relationship between carbapenem resistance and biofilm production. Gene recombination and blaOXA-95 play critical roles in carbapenem resistance in A. baumannii. Taken together, the results of the present study provide a foundation for future studies of the network systems associated with carbapenem resistance.

De novo assembly and characterization of the Trichuris trichiura adult worm transcriptome using Ion Torrent sequencing.

PubMed

Santos, Leonardo N; Silva, Eduardo S; Santos, André S; De Sá, Pablo H; Ramos, Rommel T; Silva, Artur; Cooper, Philip J; Barreto, Maurício L; Loureiro, Sebastião; Pinheiro, Carina S; Alcantara-Neves, Neuza M; Pacheco, Luis G C

2016-07-01

Infection with helminthic parasites, including the soil-transmitted helminth Trichuris trichiura (human whipworm), has been shown to modulate host immune responses and, consequently, to have an impact on the development and manifestation of chronic human inflammatory diseases. De novo derivation of helminth proteomes from sequencing of transcriptomes will provide valuable data to aid identification of parasite proteins that could be evaluated as potential immunotherapeutic molecules in near future. Herein, we characterized the transcriptome of the adult stage of the human whipworm T. trichiura, using next-generation sequencing technology and a de novo assembly strategy. Nearly 17.6 million high-quality clean reads were assembled into 6414 contiguous sequences, with an N50 of 1606bp. In total, 5673 protein-encoding sequences were confidentially identified in the T. trichiura adult worm transcriptome; of these, 1013 sequences represent potential newly discovered proteins for the species, most of which presenting orthologs already annotated in the related species T. suis. A number of transcripts representing probable novel non-coding transcripts for the species T. trichiura were also identified. Among the most abundant transcripts, we found sequences that code for proteins involved in lipid transport, such as vitellogenins, and several chitin-binding proteins. Through a cross-species expression analysis of gene orthologs shared by T. trichiura and the closely related parasites T. suis and T. muris it was possible to find twenty-six protein-encoding genes that are consistently highly expressed in the adult stages of the three helminth species. Additionally, twenty transcripts could be identified that code for proteins previously detected by mass spectrometry analysis of protein fractions of the whipworm somatic extract that present immunomodulatory activities. Five of these transcripts were amongst the most highly expressed protein-encoding sequences in the T. trichiura adult worm. Besides, orthologs of proteins demonstrated to have potent immunomodulatory properties in related parasitic helminths were also predicted from the T. trichiura de novo assembled transcriptome. Copyright © 2016. Published by Elsevier B.V.

Cold Urticaria, Immunodeficiency, and Autoimmunity Related to PLCG2 Deletions

PubMed Central

Ombrello, Michael J.; Remmers, Elaine F.; Sun, Guangping; Freeman, Alexandra F.; Datta, Shrimati; Torabi-Parizi, Parizad; Subramanian, Naeha; Bunney, Tom D.; Baxendale, Rhona W.; Martins, Marta S.; Romberg, Neil; Komarow, Hirsh; Aksentijevich, Ivona; Kim, Hun Sik; Ho, Jason; Cruse, Glenn; Jung, Mi-Yeon; Gilfillan, Alasdair M.; Metcalfe, Dean D.; Nelson, Celeste; O'Brien, Michelle; Wisch, Laura; Stone, Kelly; Douek, Daniel C.; Gandhi, Chhavi; Wanderer, Alan A.; Lee, Hane; Nelson, Stanley F.; Shianna, Kevin V.; Cirulli, Elizabeth T.; Goldstein, David B.; Long, Eric O.; Moir, Susan; Meffre, Eric; Holland, Steven M.; Kastner, Daniel L.; Katan, Matilda; Hoffman, Hal M.; Milner, Joshua D.

2012-01-01

Background Mendelian analysis of disorders of immune regulation can provide insight into molecular pathways associated with host defense and immune tolerance. Methods We identified three families with a dominantly inherited complex of cold-induced urticaria, antibody deficiency, and susceptibility to infection and autoimmunity. Immunophenotyping methods included flow cytometry, analysis of serum immunoglobulins and autoantibodies, lymphocyte stimulation, and enzymatic assays. Genetic studies included linkage analysis, targeted Sanger sequencing, and next-generation whole-genome sequencing. Results Cold urticaria occurred in all affected subjects. Other, variable manifestations included atopy, granulomatous rash, autoimmune thyroiditis, the presence of antinuclear antibodies, sinopulmonary infections, and common variable immunodeficiency. Levels of serum IgM and IgA and circulating natural killer cells and class-switched memory B cells were reduced. Linkage analysis showed a 7-Mb candidate interval on chromosome 16q in one family, overlapping by 3.5 Mb a disease-associated haplotype in a smaller family. This interval includes PLCG2, encoding phospholipase Cγ2 (PLCγ2), a signaling molecule expressed in B cells, natural killer cells, and mast cells. Sequencing of complementary DNA revealed heterozygous transcripts lacking exon 19 in two families and lacking exons 20 through 22 in a third family. Genomic sequencing identified three distinct in-frame deletions that cosegregated with disease. These deletions, located within a region encoding an autoinhibitory domain, result in protein products with constitutive phospholipase activity. PLCG2-expressing cells had diminished cellular signaling at 37°C but enhanced signaling at subphysiologic temperatures. Conclusions Genomic deletions in PLCG2 cause gain of PLCγ2 function, leading to signaling abnormalities in multiple leukocyte subsets and a phenotype encompassing both excessive and deficient immune function. (Funded by the National Institutes of Health Intramural Research Programs and others.) PMID:22236196

Generation of 2A-linked multicistronic cassettes by recombinant PCR.

PubMed

Szymczak-Workman, Andrea L; Vignali, Kate M; Vignali, Dario A A

2012-02-01

The need for reliable, multicistronic vectors for multigene delivery is at the forefront of biomedical technology. It is now possible to express multiple proteins from a single open reading frame (ORF) using 2A peptide-linked multicistronic vectors. These small sequences, when cloned between genes, allow for efficient, stoichiometric production of discrete protein products within a single vector through a novel "cleavage" event within the 2A peptide sequence. Expression of more than two genes using conventional approaches has several limitations, most notably imbalanced protein expression and large size. The use of 2A peptide sequences alleviates these concerns. They are small (18-22 amino acids) and have divergent amino-terminal sequences, which minimizes the chance for homologous recombination and allows for multiple, different 2A peptide sequences to be used within a single vector. Importantly, separation of genes placed between 2A peptide sequences is nearly 100%, which allows for stoichiometric and concordant expression of the genes, regardless of the order of placement within the vector. This protocol describes the use of recombinant polymerase chain reaction (PCR) to connect multiple 2A-linked protein sequences. The final construct is subcloned into an expression vector.

Bovine mammary gene expression profiling during the onset of lactation.

PubMed

Gao, Yuanyuan; Lin, Xueyan; Shi, Kerong; Yan, Zhengui; Wang, Zhonghua

2013-01-01

Lactogenesis includes two stages. Stage I begins a few weeks before parturition. Stage II is initiated around the time of parturition and extends for several days afterwards. To better understand the molecular events underlying these changes, genome-wide gene expression profiling was conducted using digital gene expression (DGE) on bovine mammary tissue at three time points (on approximately day 35 before parturition (-35 d), day 7 before parturition (-7 d) and day 3 after parturition (+3 d)). Approximately 6.2 million (M), 5.8 million (M) and 6.1 million (M) 21-nt cDNA tags were sequenced in the three cDNA libraries (-35 d, -7 d and +3 d), respectively. After aligning to the reference sequences, the three cDNA libraries included 8,662, 8,363 and 8,359 genes, respectively. With a fold change cutoff criteria of ≥ 2 or ≤-2 and a false discovery rate (FDR) of ≤ 0.001, a total of 812 genes were significantly differentially expressed at -7 d compared with -35 d (stage I). Gene ontology analysis showed that those significantly differentially expressed genes were mainly associated with cell cycle, lipid metabolism, immune response and biological adhesion. A total of 1,189 genes were significantly differentially expressed at +3 d compared with -7 d (stage II), and these genes were mainly associated with the immune response and cell cycle. Moreover, there were 1,672 genes significantly differentially expressed at +3 d compared with -35 d. Gene ontology analysis showed that the main differentially expressed genes were those associated with metabolic processes. The results suggest that the mammary gland begins to lactate not only by a gain of function but also by a broad suppression of function to effectively push most of the cell's resources towards lactation.

Heterogeneous conservation of Dlx paralog co-expression in jawed vertebrates.

PubMed

Debiais-Thibaud, Mélanie; Metcalfe, Cushla J; Pollack, Jacob; Germon, Isabelle; Ekker, Marc; Depew, Michael; Laurenti, Patrick; Borday-Birraux, Véronique; Casane, Didier

2013-01-01

The Dlx gene family encodes transcription factors involved in the development of a wide variety of morphological innovations that first evolved at the origins of vertebrates or of the jawed vertebrates. This gene family expanded with the two rounds of genome duplications that occurred before jawed vertebrates diversified. It includes at least three bigene pairs sharing conserved regulatory sequences in tetrapods and teleost fish, but has been only partially characterized in chondrichthyans, the third major group of jawed vertebrates. Here we take advantage of developmental and molecular tools applied to the shark Scyliorhinus canicula to fill in the gap and provide an overview of the evolution of the Dlx family in the jawed vertebrates. These results are analyzed in the theoretical framework of the DDC (Duplication-Degeneration-Complementation) model. The genomic organisation of the catshark Dlx genes is similar to that previously described for tetrapods. Conserved non-coding elements identified in bony fish were also identified in catshark Dlx clusters and showed regulatory activity in transgenic zebrafish. Gene expression patterns in the catshark showed that there are some expression sites with high conservation of the expressed paralog(s) and other expression sites with events of paralog sub-functionalization during jawed vertebrate diversification, resulting in a wide variety of evolutionary scenarios within this gene family. Dlx gene expression patterns in the catshark show that there has been little neo-functionalization in Dlx genes over gnathostome evolution. In most cases, one tandem duplication and two rounds of vertebrate genome duplication have led to at least six Dlx coding sequences with redundant expression patterns followed by some instances of paralog sub-functionalization. Regulatory constraints such as shared enhancers, and functional constraints including gene pleiotropy, may have contributed to the evolutionary inertia leading to high redundancy between gene expression patterns.

Differential Expression of miRNAs in the Respiratory Tree of the Sea Cucumber Apostichopus japonicus Under Hypoxia Stress.

PubMed

Huo, Da; Sun, Lina; Li, Xiaoni; Ru, Xiaoshang; Liu, Shilin; Zhang, Libin; Xing, Lili; Yang, Hongsheng

2017-11-06

The sea cucumber, an important economic species, has encountered high mortality since 2013 in northern China because of seasonal environmental stress such as hypoxia, high temperature, and low salinity. MicroRNAs (miRNAs) are important in regulating gene expression in marine organisms in response to environmental change. In this study, high-throughput sequencing was used to investigate alterations in miRNA expression in the sea cucumber under different levels of dissolved oxygen (DO). Nine small RNA libraries were constructed from the sea cucumber respiratory trees. A total of 26 differentially expressed miRNAs, including 12 upregulated and 14 downregulated miRNAs, were observed in severe hypoxia (DO 2 mg/L) compared with mild hypoxia (DO 4 mg/L) and normoxic conditions (DO 8 mg/L). Twelve differentially expressed miRNAs were clustered in severe hypoxia. In addition, real-time PCR revealed that 14 randomly selected differentially expressed miRNAs showed significantly increased expressions in severe hypoxia and the expressions of nine miRNAs, including key miRNAs such as Aja-miR-1, Aja-miR-2008, and Aja-miR-184, were consistent with the sequencing results. Moreover, gene ontology and pathway analyses of putative target genes suggest that these miRNAs are important in redox, transport, transcription, and hydrolysis under hypoxia stress. Notably, novel-miR-1, novel-miR-2, and novel-miR-3 were specifically clustered and upregulated in severe hypoxia, which may provide new insights into novel "hypoxamiR" identification. These results will provide a basis for future studies of miRNA regulation and molecular adaptive mechanisms in sea cucumbers under hypoxia stress. Copyright © 2017 Huo et al.

Music performance and the perception of key.

PubMed

Thompson, W F; Cuddy, L L

1997-02-01

The effect of music performance on perceived key movement was examined. Listeners judged key movement in sequences presented without performance expression (mechanical) in Experiment 1 and with performance expression in Experiment 2. Modulation distance varied. Judgments corresponded to predictions based on the cycle of fifths and toroidal models of key relatedness, with the highest correspondence for performed versions with the toroidal model. In Experiment 3, listeners compared mechanical sequences with either performed sequences or modifications of performed sequences. Modifications preserved expressive differences between chords, but not between voices. Predictions from Experiments 1 and 2 held only for performed sequences, suggesting that differences between voices are informative of key movement. Experiment 4 confirmed that modifications did not disrupt musicality. Analyses of performances further suggested a link between performance expression and key.

Large-scale transcriptome analysis in chickpea (Cicer arietinum L.), an orphan legume crop of the semi-arid tropics of Asia and Africa.

PubMed

Hiremath, Pavana J; Farmer, Andrew; Cannon, Steven B; Woodward, Jimmy; Kudapa, Himabindu; Tuteja, Reetu; Kumar, Ashish; Bhanuprakash, Amindala; Mulaosmanovic, Benjamin; Gujaria, Neha; Krishnamurthy, Laxmanan; Gaur, Pooran M; Kavikishor, Polavarapu B; Shah, Trushar; Srinivasan, Ramamurthy; Lohse, Marc; Xiao, Yongli; Town, Christopher D; Cook, Douglas R; May, Gregory D; Varshney, Rajeev K

2011-10-01

Chickpea (Cicer arietinum L.) is an important legume crop in the semi-arid regions of Asia and Africa. Gains in crop productivity have been low however, particularly because of biotic and abiotic stresses. To help enhance crop productivity using molecular breeding techniques, next generation sequencing technologies such as Roche/454 and Illumina/Solexa were used to determine the sequence of most gene transcripts and to identify drought-responsive genes and gene-based molecular markers. A total of 103,215 tentative unique sequences (TUSs) have been produced from 435,018 Roche/454 reads and 21,491 Sanger expressed sequence tags (ESTs). Putative functions were determined for 49,437 (47.8%) of the TUSs, and gene ontology assignments were determined for 20,634 (41.7%) of the TUSs. Comparison of the chickpea TUSs with the Medicago truncatula genome assembly (Mt 3.5.1 build) resulted in 42,141 aligned TUSs with putative gene structures (including 39,281 predicted intron/splice junctions). Alignment of ∼37 million Illumina/Solexa tags generated from drought-challenged root tissues of two chickpea genotypes against the TUSs identified 44,639 differentially expressed TUSs. The TUSs were also used to identify a diverse set of markers, including 728 simple sequence repeats (SSRs), 495 single nucleotide polymorphisms (SNPs), 387 conserved orthologous sequence (COS) markers, and 2088 intron-spanning region (ISR) markers. This resource will be useful for basic and applied research for genome analysis and crop improvement in chickpea. Plant Biotechnology Journal © 2011 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd. No claim to original US government works.

High-resolution mapping of the 11q13 amplicon and identification of a gene, TAOS1, that is amplified and overexpressed in oral cancer cells

PubMed Central

Huang, Xin; Gollin, Susanne M.; Raja, Siva; Godfrey, Tony E.

2002-01-01

Amplification of chromosomal band 11q13 is a common event in human cancer. It has been reported in about 45% of head and neck carcinomas and in other cancers including esophageal, breast, liver, lung, and bladder cancer. To understand the mechanism of 11q13 amplification and to identify the potential oncogene(s) driving it, we have fine-mapped the structure of the amplicon in oral squamous cell carcinoma cell lines and localized the proximal and distal breakpoints. A 5-Mb physical map of the region has been prepared from which sequence is available. We quantified copy number of sequence-tagged site markers at 42–550 kb intervals along the length of the amplicon and defined the amplicon core and breakpoints by using TaqMan-based quantitative microsatellite analysis. The core of the amplicon maps to a 1.5-Mb region. The proximal breakpoint localizes to two intervals between sequence-tagged site markers, 550 kb and 160 kb in size, and the distal breakpoint maps to a 250 kb interval. The cyclin D1 gene maps to the amplicon core, as do two new expressed sequence tag clusters. We have analyzed one of these expressed sequence tag clusters and now report that it contains a previously uncharacterized gene, TAOS1 (tumor amplified and overexpressed sequence 1), which is both amplified and overexpressed in oral cancer cells. The data suggest that TAOS1 may be an amplification-dependent candidate oncogene with a role in the development and/or progression of human tumors, including oral squamous cell carcinomas. The approach described here should be useful for characterizing amplified genomic regions in a wide variety of tumors. PMID:12172009

Differential effects of simple repeating DNA sequences on gene expression from the SV40 early promoter.

PubMed

Amirhaeri, S; Wohlrab, F; Wells, R D

1995-02-17

The influence of simple repeat sequences, cloned into different positions relative to the SV40 early promoter/enhancer, on the transient expression of the chloramphenicol acetyltransferase (CAT) gene was investigated. Insertion of (G)29.(C)29 in either orientation into the 5'-untranslated region of the CAT gene reduced expression in CV-1 cells 50-100 fold when compared with controls with random sequence inserts. Analysis of CAT-specific mRNA levels demonstrated that the effect was due to a reduction of CAT mRNA production rather than to posttranscriptional events. In contrast, insertion of the same insert in either orientation upstream of the promoter-enhancer or downstream of the gene stimulated gene expression 2-3-fold. These effects could be reversed by cotransfection of a competitor plasmid carrying (G)25.(C)25 sequences. The results suggest that a G.C-binding transcription factor modulates gene expression in this system and that promoter strength can be regulated by providing protein-binding sites in trans. Although constructs containing longer tracts of alternating (C-G), (T-G), or (A-T) sequences inhibited CAT expression when inserted in the 5'-untranslated region of the CAT gene, the amount of CAT mRNA was unaffected. Hence, these inhibitions must be due to posttranscriptional events, presumably at the level of translation. These effects of microsatellite sequences on gene expression are discussed with respect to recent data on related simple repeat sequences which cause several human genetic diseases.

Cooperative action of multiple cis-acting elements is required for N-myc expression in branchial arches: specific contribution of GATA3.

PubMed

Potvin, Eric; Beuret, Laurent; Cadrin-Girard, Jean-François; Carter, Marcelle; Roy, Sophie; Tremblay, Michel; Charron, Jean

2010-11-01

The precise expression of the N-myc proto-oncogene is essential for normal mammalian development, whereas altered N-myc gene regulation is known to be a determinant factor in tumor formation. Using transgenic mouse embryos, we show that N-myc sequences from kb -8.7 to kb +7.2 are sufficient to reproduce the N-myc embryonic expression profile in developing branchial arches and limb buds. These sequences encompass several regulatory elements dispersed throughout the N-myc locus, including an upstream limb bud enhancer, a downstream somite enhancer, a branchial arch enhancer in the second intron, and a negative regulatory element in the first intron. N-myc expression in the limb buds is under the dominant control of the limb bud enhancer. The expression in the branchial arches necessitates the interplay of three regulatory domains. The branchial arch enhancer cooperates with the somite enhancer region to prevent an inhibitory activity contained in the first intron. The characterization of the branchial arch enhancer has revealed a specific role of the transcription factor GATA3 in the regulation of N-myc expression. Together, these data demonstrate that correct N-myc developmental expression is achieved via cooperation of multiple positive and negative regulatory elements.

Global Analysis of Gene Expression Profiles in Developing Physic Nut (Jatropha curcas L.) Seeds

PubMed Central

Jiang, Huawu; Wu, Pingzhi; Zhang, Sheng; Song, Chi; Chen, Yaping; Li, Meiru; Jia, Yongxia; Fang, Xiaohua; Chen, Fan; Wu, Guojiang

2012-01-01

Background Physic nut (Jatropha curcas L.) is an oilseed plant species with high potential utility as a biofuel. Furthermore, following recent sequencing of its genome and the availability of expressed sequence tag (EST) libraries, it is a valuable model plant for studying carbon assimilation in endosperms of oilseed plants. There have been several transcriptomic analyses of developing physic nut seeds using ESTs, but they have provided limited information on the accumulation of stored resources in the seeds. Methodology/Principal Findings We applied next-generation Illumina sequencing technology to analyze global gene expression profiles of developing physic nut seeds 14, 19, 25, 29, 35, 41, and 45 days after pollination (DAP). The acquired profiles reveal the key genes, and their expression timeframes, involved in major metabolic processes including: carbon flow, starch metabolism, and synthesis of storage lipids and proteins in the developing seeds. The main period of storage reserves synthesis in the seeds appears to be 29–41 DAP, and the fatty acid composition of the developing seeds is consistent with relative expression levels of different isoforms of acyl-ACP thioesterase and fatty acid desaturase genes. Several transcription factor genes whose expression coincides with storage reserve deposition correspond to those known to regulate the process in Arabidopsis. Conclusions/Significance The results will facilitate searches for genes that influence de novo lipid synthesis, accumulation and their regulatory networks in developing physic nut seeds, and other oil seeds. Thus, they will be helpful in attempts to modify these plants for efficient biofuel production. PMID:22574177

Transcriptome sequencing of rhizome tissue of Sinopodophyllum hexandrum at two temperatures.

PubMed

Kumari, Anita; Singh, Heikham Russiachand; Jha, Ashwani; Swarnkar, Mohit Kumar; Shankar, Ravi; Kumar, Sanjay

2014-10-07

Sinopodophyllum hexandrum is an endangered medicinal herb, which is commonly present in elevations ranging between 2,400-4,500 m and is sensitive to temperature. Medicinal property of the species is attributed to the presence of podophyllotoxin in the rhizome tissue. The present work analyzed transcriptome of rhizome tissue of S. hexandrum exposed to 15°C and 25°C to understand the temperature mediated molecular responses including those associated with podophyllotoxin biosynthesis. Deep sequencing of transcriptome with an average coverage of 88.34X yielded 60,089 assembled transcript sequences representing 20,387 unique genes having homology to known genes. Fragments per kilobase of exon per million fragments mapped (FPKM) based expression analysis revealed genes related to growth and development were over-expressed at 15°C, whereas genes involved in stress response were over-expressed at 25°C. There was a decreasing trend of podophyllotoxin accumulation at 25°C; data was well supported by the expression of corresponding genes of the pathway. FPKM data was validated by quantitative real-time polymerase chain reaction data using a total of thirty four genes and a positive correlation between the two platforms of gene expression was obtained. Also, detailed analyses yielded cytochrome P450s, methyltransferases and glycosyltransferases which could be the potential candidate hitherto unidentified genes of podophyllotoxin biosynthesis pathway. The present work revealed temperature responsive transcriptome of S. hexandrum on Illumina platform. Data suggested expression of genes for growth and development and podophyllotoxin biosynthesis at 15°C, and prevalence of those associated with stress response at 25°C.

Gene Expression Profiles in Paired Gingival Biopsies from Periodontitis-Affected and Healthy Tissues Revealed by Massively Parallel Sequencing

PubMed Central

Båge, Tove; Lagervall, Maria; Jansson, Leif; Lundeberg, Joakim; Yucel-Lindberg, Tülay

2012-01-01

Periodontitis is a chronic inflammatory disease affecting the soft tissue and bone that surrounds the teeth. Despite extensive research, distinctive genes responsible for the disease have not been identified. The objective of this study was to elucidate transcriptome changes in periodontitis, by investigating gene expression profiles in gingival tissue obtained from periodontitis-affected and healthy gingiva from the same patient, using RNA-sequencing. Gingival biopsies were obtained from a disease-affected and a healthy site from each of 10 individuals diagnosed with periodontitis. Enrichment analysis performed among uniquely expressed genes for the periodontitis-affected and healthy tissues revealed several regulated pathways indicative of inflammation for the periodontitis-affected condition. Hierarchical clustering of the sequenced biopsies demonstrated clustering according to the degree of inflammation, as observed histologically in the biopsies, rather than clustering at the individual level. Among the top 50 upregulated genes in periodontitis-affected tissues, we investigated two genes which have not previously been demonstrated to be involved in periodontitis. These included interferon regulatory factor 4 and chemokine (C-C motif) ligand 18, which were also expressed at the protein level in gingival biopsies from patients with periodontitis. In conclusion, this study provides a first step towards a quantitative comprehensive insight into the transcriptome changes in periodontitis. We demonstrate for the first time site-specific local variation in gene expression profiles of periodontitis-affected and healthy tissues obtained from patients with periodontitis, using RNA-seq. Further, we have identified novel genes expressed in periodontitis tissues, which may constitute potential therapeutic targets for future treatment strategies of periodontitis. PMID:23029519

Changes in rat spinal cord gene expression after inflammatory hyperalgesia of the joint and manual therapy.

PubMed

Ruhlen, Rachel L; Singh, Vineet K; Pazdernik, Vanessa K; Towns, Lex C; Snider, Eric J; Sargentini, Neil J; Degenhardt, Brian F

2014-10-01

Mobilization of a joint affects local tissue directly but may also have other effects that are mediated through the central nervous system. To identify differential gene expression in the spinal cords of rats with or without inflammatory joint injury after manual therapy or no treatment. Rats were randomly assigned to 1 of 4 treatment groups: no injury and no touch (NI/NT), injury and no touch (I/NT), no injury and manual therapy (NI/MT), and injury and manual therapy (I/MT). We induced acute inflammatory joint injury in the rats by injecting carrageenan into an ankle. Rats in the no-injury groups did not receive carrageenan injection. One day after injury, rats received manual therapy to the knee of the injured limb. Rats in the no-touch groups were anesthetized without receiving manual therapy. Spinal cords were harvested 30 minutes after therapy or no touch, and spinal cord gene expression was analyzed by microarray for 3 comparisons: NI/NT vs I/NT, I/MT vs I/NT, and NI/NT vs NI/MT. Three rats were assigned to each group. Of 38,875 expressed sequence tags, 755 were differentially expressed in the NI/NT vs I/NT comparison. For the other comparisons, no expressed sequence tags were differentially expressed. Cluster analysis revealed that the differentially expressed sequence tags were over-represented in several categories, including ion homeostasis (enrichment score, 2.29), transmembrane (enrichment score, 1.55), and disulfide bond (enrichment score, 2.04). An inflammatory injury to the ankle of rats caused differential expression of genes in the spinal cord. Consistent with other studies, genes involved in ion transport were among those affected. However, manual therapy to the knees of injured limbs or to rats without injury did not alter gene expression in the spinal cord. Thus, evidence for central nervous system mediation of manual therapy was not observed. © 2014 The American Osteopathic Association.

A-WINGS: an integrated genome database for Pleurocybella porrigens (Angel's wing oyster mushroom, Sugihiratake).

PubMed

Yamamoto, Naoki; Suzuki, Tomohiro; Kobayashi, Masaaki; Dohra, Hideo; Sasaki, Yohei; Hirai, Hirofumi; Yokoyama, Koji; Kawagishi, Hirokazu; Yano, Kentaro

2014-12-03

The angel's wing oyster mushroom (Pleurocybella porrigens, Sugihiratake) is a well-known delicacy. However, its potential risk in acute encephalopathy was recently revealed by a food poisoning incident. To disclose the genes underlying the accident and provide mechanistic insight, we seek to develop an information infrastructure containing omics data. In our previous work, we sequenced the genome and transcriptome using next-generation sequencing techniques. The next step in achieving our goal is to develop a web database to facilitate the efficient mining of large-scale omics data and identification of genes specifically expressed in the mushroom. This paper introduces a web database A-WINGS (http://bioinf.mind.meiji.ac.jp/a-wings/) that provides integrated genomic and transcriptomic information for the angel's wing oyster mushroom. The database contains structure and functional annotations of transcripts and gene expressions. Functional annotations contain information on homologous sequences from NCBI nr and UniProt, Gene Ontology, and KEGG Orthology. Digital gene expression profiles were derived from RNA sequencing (RNA-seq) analysis in the fruiting bodies and mycelia. The omics information stored in the database is freely accessible through interactive and graphical interfaces by search functions that include 'GO TREE VIEW' browsing, keyword searches, and BLAST searches. The A-WINGS database will accelerate omics studies on specific aspects of the angel's wing oyster mushroom and the family Tricholomataceae.

Strong positive selection and recombination drive the antigenic variation of the PilE protein of the human pathogen Neisseria meningitidis.

PubMed

Andrews, T Daniel; Gojobori, Takashi

2004-01-01

The PilE protein is the major component of the Neisseria meningitidis pilus, which is encoded by the pilE/pilS locus that includes an expressed gene and eight homologous silent fragments. The silent gene fragments have been shown to recombine through gene conversion with the expressed gene and thereby provide a means by which novel antigenic variants of the PilE protein can be generated. We have analyzed the evolutionary rate of the pilE gene using the nucleotide sequence of two complete pilE/pilS loci. The very high rate of evolution displayed by the PilE protein appears driven by both recombination and positive selection. Within the semivariable region of the pilE and pilS genes, recombination appears to occur within multiple small sequence blocks that lie between conserved sequence elements. Within the hypervariable region, positive selection was identified from comparison of the silent and expressed genes. The unusual gene conversion mechanism that operates at the pilE/pilS locus is a strategy employed by N. meningitidis to enhance mutation of certain regions of the PilE protein. The silent copies of the gene effectively allow "parallelized" evolution of pilE, thus enabling the encoded protein to rapidly explore a large area of sequence space in an effort to find novel antigenic variants.

Transcriptome sequencing and differential gene expression analysis in Viola yedoensis Makino (Fam. Violaceae) responsive to cadmium (Cd) pollution

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

Gao, Jian; Luo, Mao; Zhu, Ye

2015-03-27

Viola yedoensis Makino is an important Chinese traditional medicine plant adapted to cadmium (Cd) pollution regions. Illumina sequencing technology was used to sequence the transcriptome of V. yedoensis Makino. We sequenced Cd-treated (VIYCd) and untreated (VIYCK) samples of V. yedoensis, and obtained 100,410,834 and 83,587,676 high quality reads, respectively. After de novo assembly and quantitative assessment, 109,800 unigenes were finally generated with an average length of 661 bp. We then obtained functional annotations by aligning unigenes with public protein databases including NR, NT, SwissProt, KEGG and COG. In addition, 892 differentially expressed genes (DEGs) were investigated between the two libraries ofmore » untreated (VIYCK) and Cd-treated (VIYCd) plants. Moreover, 15 randomly selected DEGs were further validated with qRT-PCR and the results were highly accordant with the Solexa analysis. This study firstly generated a successful global analysis of the V. yedoensis transcriptome and it will provide for further studies on gene expression, genomics, and functional genomics in Violaceae. - Highlights: • A de novo assembly generated 109,800 unigenes and 5,4479 of them were annotated. • 31,285 could be classified into 26 COG categories. • 263 biosynthesis pathways were predicted and classified into five categories. • 892 DEGs were detected and 15 of them were validated by qRT-PCR.« less

Improved Annotation of 3′ Untranslated Regions and Complex Loci by Combination of Strand-Specific Direct RNA Sequencing, RNA-Seq and ESTs

PubMed Central

Song, Junfang; Duc, Céline; Storey, Kate G.; McLean, W. H. Irwin; Brown, Sara J.; Simpson, Gordon G.; Barton, Geoffrey J.

2014-01-01

The reference annotations made for a genome sequence provide the framework for all subsequent analyses of the genome. Correct and complete annotation in addition to the underlying genomic sequence is particularly important when interpreting the results of RNA-seq experiments where short sequence reads are mapped against the genome and assigned to genes according to the annotation. Inconsistencies in annotations between the reference and the experimental system can lead to incorrect interpretation of the effect on RNA expression of an experimental treatment or mutation in the system under study. Until recently, the genome-wide annotation of 3′ untranslated regions received less attention than coding regions and the delineation of intron/exon boundaries. In this paper, data produced for samples in Human, Chicken and A. thaliana by the novel single-molecule, strand-specific, Direct RNA Sequencing technology from Helicos Biosciences which locates 3′ polyadenylation sites to within +/− 2 nt, were combined with archival EST and RNA-Seq data. Nine examples are illustrated where this combination of data allowed: (1) gene and 3′ UTR re-annotation (including extension of one 3′ UTR by 5.9 kb); (2) disentangling of gene expression in complex regions; (3) clearer interpretation of small RNA expression and (4) identification of novel genes. While the specific examples displayed here may become obsolete as genome sequences and their annotations are refined, the principles laid out in this paper will be of general use both to those annotating genomes and those seeking to interpret existing publically available annotations in the context of their own experimental data. PMID:24722185

Cloning and expression of a nuclear encoded plastid specific 33 kDa ribonucleoprotein gene (33RNP) from pea that is light stimulated.

PubMed

Reddy, M K; Nair, S; Singh, B N; Mudgil, Y; Tewari, K K; Sopory, S K

2001-01-24

We report the cloning and sequencing of both cDNA and genomic DNA of a 33 kDa chloroplast ribonucleoprotein (33RNP) from pea. The analysis of the predicted amino acid sequence of the cDNA clone revealed that the encoded protein contains two RNA binding domains, including the conserved consensus ribonucleoprotein sequences CS-RNP1 and CS-RNP2, on the C-terminus half and the presence of a putative transit peptide sequence in the N-terminus region. The phylogenetic and multiple sequence alignment analysis of pea chloroplast RNP along with RNPs reported from the other plant sources revealed that the pea 33RNP is very closely related to Nicotiana sylvestris 31RNP and 28RNP and also to 31RNP and 28RNP of Arabidopsis and spinach, respectively. The pea 33RNP was expressed in Escherichia coli and purified to homogeneity. The in vitro import of precursor protein into chloroplasts confirmed that the N-terminus putative transit peptide is a bona fide transit peptide and 33RNP is localized in the chloroplast. The nucleic acid-binding properties of the recombinant protein, as revealed by South-Western analysis, showed that 33RNP has higher binding affinity for poly (U) and oligo dT than for ssDNA and dsDNA. The steady state transcript level was higher in leaves than in roots and the expression of this gene is light stimulated. Sequence analysis of the genomic clone revealed that the gene contains four exons and three introns. We have also isolated and analyzed the 5' flanking region of the pea 33RNP gene.

Two Paralogous Families of a Two-Gene Subtilisin Operon Are Widely Distributed in Oral Treponemes

PubMed Central

Correia, Frederick F.; Plummer, Alvin R.; Ellen, Richard P.; Wyss, Chris; Boches, Susan K.; Galvin, Jamie L.; Paster, Bruce J.; Dewhirst, Floyd E.

2003-01-01

Certain oral treponemes express a highly proteolytic phenotype and have been associated with periodontal diseases. The periodontal pathogen Treponema denticola produces dentilisin, a serine protease of the subtilisin family. The two-gene operon prcA-prtP is required for expression of active dentilisin (PrtP), a putative lipoprotein attached to the treponeme's outer membrane or sheath. The purpose of this study was to examine the diversity and structure of treponemal subtilisin-like proteases in order to better understand their distribution and function. The complete sequences of five prcA-prtP operons were determined for Treponema lecithinolyticum, “Treponema vincentii,” and two canine species. Partial operon sequences were obtained for T. socranskii subsp. 04 as well as 450- to 1,000-base fragments of prtP genes from four additional treponeme strains. Phylogenetic analysis demonstrated that the sequences fall into two paralogous families. The first family includes the sequence from T. denticola. Treponemes possessing this operon family express chymotrypsin-like protease activity and can cleave the substrate N-succinyl-alanyl-alanyl-prolyl-phenylalanine-p-nitroanilide (SAAPFNA). Treponemes possessing the second paralog family do not possess chymotrypsin-like activity or cleave SAAPFNA. Despite examination of a range of protein and peptide substrates, the specificity of the second protease family remains unknown. Each of the fully sequenced prcA and prtP genes contains a 5′ hydrophobic leader sequence with a treponeme lipobox. The two paralogous families of treponeme subtilisins represent a new subgroup within the subtilisin family of proteases and are the only subtilisin lipoprotein family. The present study demonstrated that the subtilisin paralogs comprising a two-gene operon are widely distributed among treponemes. PMID:14617650

Analysis of 10,000 ESTs from lymphocytes of the cynomolgus monkey to improve our understanding of its immune system

PubMed Central

Chen, Wei-Hua; Wang, Xue-Xia; Lin, Wei; He, Xiao-Wei; Wu, Zhen-Qiang; Lin, Ying; Hu, Song-Nian; Wang, Xiao-Ning

2006-01-01

Background The cynomolgus monkey (Macaca fascicularis) is one of the most widely used surrogate animal models for an increasing number of human diseases and vaccines, especially immune-system-related ones. Towards a better understanding of the gene expression background upon its immunogenetics, we constructed a cDNA library from Epstein-Barr virus (EBV)-transformed B lymphocytes of a cynomolgus monkey and sequenced 10,000 randomly picked clones. Results After processing, 8,312 high-quality expressed sequence tags (ESTs) were generated and assembled into 3,728 unigenes. Annotations of these uniquely expressed transcripts demonstrated that out of the 2,524 open reading frame (ORF) positive unigenes (mitochondrial and ribosomal sequences were not included), 98.8% shared significant similarities (E-value less than 1e-10) with the NCBI nucleotide (nt) database, while only 67.7% (E-value less than 1e-5) did so with the NCBI non-redundant protein (nr) database. Further analysis revealed that 90.0% of the unigenes that shared no similarities to the nr database could be assigned to human chromosomes, in which 75 did not match significantly to any cynomolgus monkey and human ESTs. The mapping regions to known human genes on the human genome were described in detail. The protein family and domain analysis revealed that the first, second and fourth of the most abundantly expressed protein families were all assigned to immunoglobulin and major histocompatibility complex (MHC)-related proteins. The expression profiles of these genes were compared with that of homologous genes in human blood, lymph nodes and a RAMOS cell line, which demonstrated expression changes after transformation with EBV. The degree of sequence similarity of the MHC class I and II genes to the human reference sequences was evaluated. The results indicated that class I molecules showed weak amino acid identities (<90%), while class II showed slightly higher ones. Conclusion These results indicated that the genes expressed in the cynomolgus monkey could be used to identify novel protein-coding genes and revise those incomplete or incorrect annotations in the human genome by comparative methods, since the old world monkeys and humans share high similarities at the molecular level, especially within coding regions. The identification of multiple genes involved in the immune response, their sequence variations to the human homologues, and their responses to EBV infection could provide useful information to improve our understanding of the cynomolgus monkey immune system. PMID:16618371

omiRas: a Web server for differential expression analysis of miRNAs derived from small RNA-Seq data.

PubMed

Müller, Sören; Rycak, Lukas; Winter, Peter; Kahl, Günter; Koch, Ina; Rotter, Björn

2013-10-15

Small RNA deep sequencing is widely used to characterize non-coding RNAs (ncRNAs) differentially expressed between two conditions, e.g. healthy and diseased individuals and to reveal insights into molecular mechanisms underlying condition-specific phenotypic traits. The ncRNAome is composed of a multitude of RNAs, such as transfer RNA, small nucleolar RNA and microRNA (miRNA), to name few. Here we present omiRas, a Web server for the annotation, comparison and visualization of interaction networks of ncRNAs derived from next-generation sequencing experiments of two different conditions. The Web tool allows the user to submit raw sequencing data and results are presented as: (i) static annotation results including length distribution, mapping statistics, alignments and quantification tables for each library as well as lists of differentially expressed ncRNAs between conditions and (ii) an interactive network visualization of user-selected miRNAs and their target genes based on the combination of several miRNA-mRNA interaction databases. The omiRas Web server is implemented in Python, PostgreSQL, R and can be accessed at: http://tools.genxpro.net/omiras/.

Stable zymomonas mobilis xylose and arabinose fermenting strains

DOEpatents

Zhang, Min [Lakewood, CO; Chou, Yat-Chen [Taipei, TW

2008-04-08

The present invention briefly includes a transposon for stable insertion of foreign genes into a bacterial genome, comprising at least one operon having structural genes encoding enzymes selected from the group consisting of xylAxylB, araBAD and tal/tkt, and at least one promoter for expression of the structural genes in the bacterium, a pair of inverted insertion sequences, the operons contained inside the insertion sequences, and a transposase gene located outside of the insertion sequences. A plasmid shuttle vector for transformation of foreign genes into a bacterial genome, comprising at least one operon having structural genes encoding enzymes selected from the group consisting of xylAxylB, araBAD and tal/tkt, at least one promoter for expression of the structural genes in the bacterium, and at least two DNA fragments having homology with a gene in the bacterial genome to be transformed, is also provided.The transposon and shuttle vectors are useful in constructing significantly different Zymomonas mobilis strains, according to the present invention, which are useful in the conversion of the cellulose derived pentose sugars into fuels and chemicals, using traditional fermentation technology, because they are stable for expression in a non-selection medium.

Cloning and expression in Escherichia coli of isopenicillin N synthetase genes from Streptomyces lipmanii and Aspergillus nidulans.

PubMed Central

Weigel, B J; Burgett, S G; Chen, V J; Skatrud, P L; Frolik, C A; Queener, S W; Ingolia, T D

1988-01-01

beta-Lactam antibiotics such as penicillins and cephalosporins are synthesized by a wide variety of microbes, including procaryotes and eucaryotes. Isopenicillin N synthetase catalyzes a key reaction in the biosynthetic pathway of penicillins and cephalosporins. The genes encoding this protein have previously been cloned from the filamentous fungi Cephalosporium acremonium and Penicillium chrysogenum and characterized. We have extended our analysis to the isopenicillin N synthetase genes from the fungus Aspergillus nidulans and the gram-positive procaryote Streptomyces lipmanii. The isopenicillin N synthetase genes from these organisms have been cloned and sequenced, and the proteins encoded by the open reading frames were expressed in Escherichia coli. Active isopenicillin N synthetase enzyme was recovered from extracts of E. coli cells prepared from cells containing each of the genes in expression vectors. The four isopenicillin N synthetase genes studied are closely related. Pairwise comparison of the DNA sequences showed between 62.5 and 75.7% identity; comparison of the predicted amino acid sequences showed between 53.9 and 80.6% identity. The close homology of the procaryotic and eucaryotic isopenicillin N synthetase genes suggests horizontal transfer of the genes during evolution. Images PMID:3045077

The identification and functional annotation of RNA structures conserved in vertebrates

PubMed Central

Seemann, Stefan E.; Mirza, Aashiq H.; Hansen, Claus; Bang-Berthelsen, Claus H.; Garde, Christian; Christensen-Dalsgaard, Mikkel; Torarinsson, Elfar; Yao, Zizhen; Workman, Christopher T.; Pociot, Flemming; Nielsen, Henrik; Tommerup, Niels; Ruzzo, Walter L.; Gorodkin, Jan

2017-01-01

Structured elements of RNA molecules are essential in, e.g., RNA stabilization, localization, and protein interaction, and their conservation across species suggests a common functional role. We computationally screened vertebrate genomes for conserved RNA structures (CRSs), leveraging structure-based, rather than sequence-based, alignments. After careful correction for sequence identity and GC content, we predict ∼516,000 human genomic regions containing CRSs. We find that a substantial fraction of human–mouse CRS regions (1) colocalize consistently with binding sites of the same RNA binding proteins (RBPs) or (2) are transcribed in corresponding tissues. Additionally, a CaptureSeq experiment revealed expression of many of our CRS regions in human fetal brain, including 662 novel ones. For selected human and mouse candidate pairs, qRT-PCR and in vitro RNA structure probing supported both shared expression and shared structure despite low abundance and low sequence identity. About 30,000 CRS regions are located near coding or long noncoding RNA genes or within enhancers. Structured (CRS overlapping) enhancer RNAs and extended 3′ ends have significantly increased expression levels over their nonstructured counterparts. Our findings of transcribed uncharacterized regulatory regions that contain CRSs support their RNA-mediated functionality. PMID:28487280

Transcriptome analysis and gene expression profiling of abortive and developing ovules during fruit development in hazelnut.

PubMed

Cheng, Yunqing; Liu, Jianfeng; Zhang, Huidi; Wang, Ju; Zhao, Yixin; Geng, Wanting

2015-01-01

A high ratio of blank fruit in hazelnut (Corylus heterophylla Fisch) is a very common phenomenon that causes serious yield losses in northeast China. The development of blank fruit in the Corylus genus is known to be associated with embryo abortion. However, little is known about the molecular mechanisms responsible for embryo abortion during the nut development stage. Genomic information for C. heterophylla Fisch is not available; therefore, data related to transcriptome and gene expression profiling of developing and abortive ovules are needed. In this study, de novo transcriptome sequencing and RNA-seq analysis were conducted using short-read sequencing technology (Illumina HiSeq 2000). The results of the transcriptome assembly analysis revealed genetic information that was associated with the fruit development stage. Two digital gene expression libraries were constructed, one for a full (normally developing) ovule and one for an empty (abortive) ovule. Transcriptome sequencing and assembly results revealed 55,353 unigenes, including 18,751 clusters and 36,602 singletons. These results were annotated using the public databases NR, NT, Swiss-Prot, KEGG, COG, and GO. Using digital gene expression profiling, gene expression differences in developing and abortive ovules were identified. A total of 1,637 and 715 unigenes were significantly upregulated and downregulated, respectively, in abortive ovules, compared with developing ovules. Quantitative real-time polymerase chain reaction analysis was used in order to verify the differential expression of some genes. The transcriptome and digital gene expression profiling data of normally developing and abortive ovules in hazelnut provide exhaustive information that will improve our understanding of the molecular mechanisms of abortive ovule formation in hazelnut.

Identification of Fur, Aconitase, and Other Proteins Expressed by Mycobacterium tuberculosis under Conditions of Low and High Concentrations of Iron by Combined Two-Dimensional Gel Electrophoresis and Mass Spectrometry

PubMed Central

Wong, Diane K.; Lee, Bai-Yu; Horwitz, Marcus A.; Gibson, Bradford W.

1999-01-01

Iron plays a critical role in the pathophysiology of Mycobacterium tuberculosis. To gain a better understanding of iron regulation by this organism, we have used two-dimensional (2-D) gel electrophoresis, mass spectrometry, and database searching to study protein expression in M. tuberculosis under conditions of high and low iron concentration. Proteins in cellular extracts from M. tuberculosis Erdman strain grown under low-iron (1 μM) and high-iron (70 μM) conditions were separated by 2-D polyacrylamide gel electrophoresis, which allowed high-resolution separation of several hundred proteins, as visualized by Coomassie staining. The expression of at least 15 proteins was induced, and the expression of at least 12 proteins was decreased under low-iron conditions. In-gel trypsin digestion was performed on these differentially expressed proteins, and the digestion mixtures were analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry to determine the molecular masses of the resulting tryptic peptides. Partial sequence data on some of the peptides were obtained by using after source decay and/or collision-induced dissociation. The fragmentation data were used to search computerized peptide mass and protein sequence databases for known proteins. Ten iron-regulated proteins were identified, including Fur and aconitase proteins, both of which are known to be regulated by iron in other bacterial systems. Our study shows that, where large protein sequence databases are available from genomic studies, the combined use of 2-D gel electrophoresis, mass spectrometry, and database searching to analyze proteins expressed under defined environmental conditions is a powerful tool for identifying expressed proteins and their physiologic relevance. PMID:9864233

Genomic organization, sequence characterization and expression analysis of Tenebrio molitor apolipophorin-III in response to an intracellular pathogen, Listeria monocytogenes.

PubMed

Noh, Ju Young; Patnaik, Bharat Bhusan; Tindwa, Hamisi; Seo, Gi Won; Kim, Dong Hyun; Patnaik, Hongray Howrelia; Jo, Yong Hun; Lee, Yong Seok; Lee, Bok Luel; Kim, Nam Jung; Han, Yeon Soo

2014-01-25

Apolipophorin III (apoLp-III) is a well-known hemolymph protein having a functional role in lipid transport and immune response of insects. We cloned full-length cDNA encoding putative apoLp-III from larvae of the coleopteran beetle, Tenebrio molitor (TmapoLp-III), by identification of clones corresponding to the partial sequence of TmapoLp-III, subsequently followed with full length sequencing by a clone-by-clone primer walking method. The complete cDNA consists of 890 nucleotides, including an ORF encoding 196 amino acid residues. Excluding a putative signal peptide of the first 20 amino acid residues, the 176-residue mature apoLp-III has a calculated molecular mass of 19,146Da. Genomic sequence analysis with respect to its cDNA showed that TmapoLp-III was organized into four exons interrupted by three introns. Several immune-related transcription factor binding sites were discovered in the putative 5'-flanking region. BLAST and phylogenetic analyses reveal that TmapoLp-III has high sequence identity (88%) with Tribolium castaneum apoLp-III but shares little sequence homologies (<26%) with other apoLp-IIIs. Homology modeling of Tm apoLp-III shows a bundle of five amphipathic alpha helices, including a short helix 3'. The 'helix-short helix-helix' motif was predicted to be implicated in lipid binding interactions, through reversible conformational changes and accommodating the hydrophobic residues to the exterior for stability. Highest level of TmapoLp-III mRNA was detected at late pupal stages, albeit it is expressed in the larval and adult stages at lower levels. The tissue specific expression of the transcripts showed significantly higher numbers in larval fat body and adult integument. In addition, TmapoLp-III mRNA was found to be highly upregulated in late stages of L. monocytogenes or E. coli challenge. These results indicate that TmapoLp-III may play an important role in innate immune responses against bacterial pathogens in T. molitor. Copyright © 2013 Elsevier B.V. All rights reserved.

Terminal oxidase diversity and function in "Metallosphaera yellowstonensis": gene expression and protein modeling suggest mechanisms of Fe(II) oxidation in the sulfolobales.

PubMed

Kozubal, M A; Dlakic, M; Macur, R E; Inskeep, W P

2011-03-01

"Metallosphaera yellowstonensis" is a thermoacidophilic archaeon isolated from Yellowstone National Park that is capable of autotrophic growth using Fe(II), elemental S, or pyrite as electron donors. Analysis of the draft genome sequence from M. yellowstonensis strain MK1 revealed seven different copies of heme copper oxidases (subunit I) in a total of five different terminal oxidase complexes, including doxBCEF, foxABCDEFGHIJ, soxABC, and the soxM supercomplex, as well as a novel hypothetical two-protein doxB-like polyferredoxin complex. Other genes found in M. yellowstonensis with possible roles in S and or Fe cycling include a thiosulfate oxidase (tqoAB), a sulfite oxidase (som), a cbsA cytochrome b(558/566), several small blue copper proteins, and a novel gene sequence coding for a putative multicopper oxidase (Mco). Results from gene expression studies, including reverse transcriptase (RT) quantitative PCR (qPCR) of cultures grown autotrophically on either Fe(II), pyrite, or elemental S showed that the fox gene cluster and mco are highly expressed under conditions where Fe(II) is an electron donor. Metagenome sequence and gene expression studies of Fe-oxide mats confirmed the importance of fox genes (e.g., foxA and foxC) and mco under Fe(II)-oxidizing conditions. Protein modeling of FoxC suggests a novel lysine-lysine or lysine-arginine heme B binding domain, indicating that it is likely the cytochrome component of a heterodimer complex with foxG as a ferredoxin subunit. Analysis of mco shows that it encodes a novel multicopper blue protein with two plastocyanin type I copper domains that may play a role in the transfer of electrons within the Fox protein complex. An understanding of metabolic pathways involved in aerobic iron and sulfur oxidation in Sulfolobales has broad implications for understanding the evolution and niche diversification of these thermophiles as well as practical applications in fields such as bioleaching of trace metals from pyritic ores.

Gene expression profiling of adult female tissues in feeding Rhipicephalus microplus cattle ticks.

PubMed

Stutzer, Christian; van Zyl, Willem A; Olivier, Nicholas A; Richards, Sabine; Maritz-Olivier, Christine

2013-06-01

The southern cattle tick, Rhipicephalus microplus, is an economically important pest, especially for resource-poor countries, both as a highly adaptive invasive species and prominent vector of disease. The increasing prevalence of resistance to chemical acaricides and variable efficacy of current tick vaccine candidates highlight the need for more effective control methods. In the absence of a fully annotated genome, the wealth of available expressed sequence tag sequence data for this species presents a unique opportunity to study the genes that are expressed in tissues involved in blood meal acquisition, digestion and reproduction during feeding. Utilising a custom oligonucleotide microarray designed from available singletons (BmiGI Version 2.1) and expressed sequence tag sequences of R. microplus, the expression profiles in feeding adult female midgut, salivary glands and ovarian tissues were compared. From 13,456 assembled transcripts, 588 genes expressed in all three tissues were identified from fed adult females 20 days post infestation. The greatest complement of genes relate to translation and protein turnover. Additionally, a number of unique transcripts were identified for each tissue that relate well to their respective physiological/biological function/role(s). These transcripts include secreted anti-hemostatics and defense proteins from the salivary glands for acquisition of a blood meal, proteases as well as enzymes and transporters for digestion and nutrient acquisition from ingested blood in the midgut, and finally proteins and associated factors involved in DNA replication and cell-cycle control for oogenesis in the ovaries. Comparative analyses of adult female tissues during feeding enabled the identification of a catalogue of transcripts that may be essential for successful feeding and reproduction in the cattle tick, R. microplus. Future studies will increase our understanding of basic tick biology, allowing the identification of shared proteins/pathways among different tissues that may offer novel targets for the development of new tick control strategies. Copyright © 2013 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Long Term Follow up of the Delayed Effects of Acute Radiation Exposure in Primates

DTIC Science & Technology

2017-10-01

66 of 94 We will then use shRNAs and/or CRISPR constructs targeting the gene of interest to knock down its expression in stem cells prior to...DLBCLs Mutational profiling identifies 150 driver genes Gene expression identifies sub- groups including cell of origin Unbiased CRISPR screen...Exome sequencing in 1,001 DLBCL patients comprehensively identifies 150 driver genes d Unbiased CRISPR screen in DLBCL cell lines identifies essential

Identification and validation of differentially expressed transcripts by RNA-sequencing of formalin-fixed, paraffin-embedded (FFPE) lung tissue from patients with Idiopathic Pulmonary Fibrosis.

PubMed

Vukmirovic, Milica; Herazo-Maya, Jose D; Blackmon, John; Skodric-Trifunovic, Vesna; Jovanovic, Dragana; Pavlovic, Sonja; Stojsic, Jelena; Zeljkovic, Vesna; Yan, Xiting; Homer, Robert; Stefanovic, Branko; Kaminski, Naftali

2017-01-12

Idiopathic Pulmonary Fibrosis (IPF) is a lethal lung disease of unknown etiology. A major limitation in transcriptomic profiling of lung tissue in IPF has been a dependence on snap-frozen fresh tissues (FF). In this project we sought to determine whether genome scale transcript profiling using RNA Sequencing (RNA-Seq) could be applied to archived Formalin-Fixed Paraffin-Embedded (FFPE) IPF tissues. We isolated total RNA from 7 IPF and 5 control FFPE lung tissues and performed 50 base pair paired-end sequencing on Illumina 2000 HiSeq. TopHat2 was used to map sequencing reads to the human genome. On average ~62 million reads (53.4% of ~116 million reads) were mapped per sample. 4,131 genes were differentially expressed between IPF and controls (1,920 increased and 2,211 decreased (FDR < 0.05). We compared our results to differentially expressed genes calculated from a previously published dataset generated from FF tissues analyzed on Agilent microarrays (GSE47460). The overlap of differentially expressed genes was very high (760 increased and 1,413 decreased, FDR < 0.05). Only 92 differentially expressed genes changed in opposite directions. Pathway enrichment analysis performed using MetaCore confirmed numerous IPF relevant genes and pathways including extracellular remodeling, TGF-beta, and WNT. Gene network analysis of MMP7, a highly differentially expressed gene in both datasets, revealed the same canonical pathways and gene network candidates in RNA-Seq and microarray data. For validation by NanoString nCounter® we selected 35 genes that had a fold change of 2 in at least one dataset (10 discordant, 10 significantly differentially expressed in one dataset only and 15 concordant genes). High concordance of fold change and FDR was observed for each type of the samples (FF vs FFPE) with both microarrays (r = 0.92) and RNA-Seq (r = 0.90) and the number of discordant genes was reduced to four. Our results demonstrate that RNA sequencing of RNA obtained from archived FFPE lung tissues is feasible. The results obtained from FFPE tissue are highly comparable to FF tissues. The ability to perform RNA-Seq on archived FFPE IPF tissues should greatly enhance the availability of tissue biopsies for research in IPF.

Analysis of Two Cosmid Clones from Chromosome 4 of Drosophila melanogaster Reveals Two New Genes Amid an Unusual Arrangement of Repeated Sequences

PubMed Central

Locke, John; Podemski, Lynn; Roy, Ken; Pilgrim, David; Hodgetts, Ross

1999-01-01

Chromosome 4 from Drosophila melanogaster has several unusual features that distinguish it from the other chromosomes. These include a diffuse appearance in salivary gland polytene chromosomes, an absence of recombination, and the variegated expression of P-element transgenes. As part of a larger project to understand these properties, we are assembling a physical map of this chromosome. Here we report the sequence of two cosmids representing ∼5% of the polytenized region. Both cosmid clones contain numerous repeated DNA sequences, as identified by cross hybridization with labeled genomic DNA, BLAST searches, and dot matrix analysis, which are positioned between and within the transcribed sequences. The repetitive sequences include three copies of the mobile element Hoppel, one copy of the mobile element HB, and 18 DINE repeats. DINE is a novel, short repeated sequence dispersed throughout both cosmid sequences. One cosmid includes the previously described cubitus interruptus (ci) gene and two new genes: that a gene with a predicted amino acid sequence similar to ribosomal protein S3a which is consistent with the Minute(4)101 locus thought to be in the region, and a novel member of the protein family that includes plexin and met–hepatocyte growth factor receptor. The other cosmid contains only the two short 5′-most exons from the zinc-finger-homolog-2 (zfh-2) gene. This is the first extensive sequence analysis of noncoding DNA from chromosome 4. The distribution of the various repeats suggests its organization is similar to the β-heterochromatic regions near the base of the major chromosome arms. Such a pattern may account for the diffuse banding of the polytene chromosome 4 and the variegation of many P-element transgenes on the chromosome. PMID:10022978

Recombinant protein secretion in Pseudozyma flocculosa and Pseudozyma antarctica with a novel signal peptide.

PubMed

Cheng, Yali; Avis, Tyler J; Bolduc, Sébastien; Zhao, Yingyi; Anguenot, Raphaël; Neveu, Bertrand; Labbé, Caroline; Belzile, François; Bélanger, Richard R

2008-12-01

Secretion of recombinant proteins aims to reproduce the correct posttranslational modifications of the expressed protein while simplifying its recovery. In this study, secretion signal sequences from an abundantly secreted 34-kDa protein (P34) from Pseudozyma flocculosa were cloned. The efficiency of these sequences in the secretion of recombinant green fluorescent protein (GFP) was investigated in two Pseudozyma species and compared with other secretion signal sequences, from S. cerevisiae and Pseudozyma spp. The results indicate that various secretion signal sequences were functional and that the P34 signal peptide was the most effective secretion signal sequence in both P. flocculosa and P. antarctica. The cells correctly processed the secretion signal sequences, including P34 signal peptide, and mature GFP was recovered from the culture medium. This is the first report of functional secretion signal sequences in P. flocculosa. These sequences can be used to test the secretion of other recombinant proteins and for studying the secretion pathway in P. flocculosa and P. antarctica.

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

Overview Article: Identifying transcriptional cis-regulatory modules in animal genomes

PubMed Central

Suryamohan, Kushal; Halfon, Marc S.

2014-01-01

Gene expression is regulated through the activity of transcription factors and chromatin modifying proteins acting on specific DNA sequences, referred to as cis-regulatory elements. These include promoters, located at the transcription initiation sites of genes, and a variety of distal cis-regulatory modules (CRMs), the most common of which are transcriptional enhancers. Because regulated gene expression is fundamental to cell differentiation and acquisition of new cell fates, identifying, characterizing, and understanding the mechanisms of action of CRMs is critical for understanding development. CRM discovery has historically been challenging, as CRMs can be located far from the genes they regulate, have few readily-identifiable sequence characteristics, and for many years were not amenable to high-throughput discovery methods. However, the recent availability of complete genome sequences and the development of next-generation sequencing methods has led to an explosion of both computational and empirical methods for CRM discovery in model and non-model organisms alike. Experimentally, CRMs can be identified through chromatin immunoprecipitation directed against transcription factors or histone post-translational modifications, identification of nucleosome-depleted “open” chromatin regions, or sequencing-based high-throughput functional screening. Computational methods include comparative genomics, clustering of known or predicted transcription factor binding sites, and supervised machine-learning approaches trained on known CRMs. All of these methods have proven effective for CRM discovery, but each has its own considerations and limitations, and each is subject to a greater or lesser number of false-positive identifications. Experimental confirmation of predictions is essential, although shortcomings in current methods suggest that additional means of validation need to be developed. PMID:25704908

Increased complexity of circRNA expression during species evolution.

PubMed

Dong, Rui; Ma, Xu-Kai; Chen, Ling-Ling; Yang, Li

2017-08-03

Circular RNAs (circRNAs) are broadly identified from precursor mRNA (pre-mRNA) back-splicing across various species. Recent studies have suggested a cell-/tissue- specific manner of circRNA expression. However, the distinct expression pattern of circRNAs among species and its underlying mechanism still remain to be explored. Here, we systematically compared circRNA expression from human and mouse, and found that only a small portion of human circRNAs could be determined in parallel mouse samples. The conserved circRNA expression between human and mouse is correlated with the existence of orientation-opposite complementary sequences in introns that flank back-spliced exons in both species, but not the circRNA sequences themselves. Quantification of RNA pairing capacity of orientation-opposite complementary sequences across circRNA-flanking introns by Complementary Sequence Index (CSI) identifies that among all types of complementary sequences, SINEs, especially Alu elements in human, contribute the most for circRNA formation and that their diverse distribution across species leads to the increased complexity of circRNA expression during species evolution. Together, our integrated and comparative reference catalog of circRNAs in different species reveals a species-specific pattern of circRNA expression and suggests a previously under-appreciated impact of fast-evolved SINEs on the regulation of (circRNA) gene expression.

NCBI GEO: archive for functional genomics data sets--10 years on.

PubMed

Barrett, Tanya; Troup, Dennis B; Wilhite, Stephen E; Ledoux, Pierre; Evangelista, Carlos; Kim, Irene F; Tomashevsky, Maxim; Marshall, Kimberly A; Phillippy, Katherine H; Sherman, Patti M; Muertter, Rolf N; Holko, Michelle; Ayanbule, Oluwabukunmi; Yefanov, Andrey; Soboleva, Alexandra

2011-01-01

A decade ago, the Gene Expression Omnibus (GEO) database was established at the National Center for Biotechnology Information (NCBI). The original objective of GEO was to serve as a public repository for high-throughput gene expression data generated mostly by microarray technology. However, the research community quickly applied microarrays to non-gene-expression studies, including examination of genome copy number variation and genome-wide profiling of DNA-binding proteins. Because the GEO database was designed with a flexible structure, it was possible to quickly adapt the repository to store these data types. More recently, as the microarray community switches to next-generation sequencing technologies, GEO has again adapted to host these data sets. Today, GEO stores over 20,000 microarray- and sequence-based functional genomics studies, and continues to handle the majority of direct high-throughput data submissions from the research community. Multiple mechanisms are provided to help users effectively search, browse, download and visualize the data at the level of individual genes or entire studies. This paper describes recent database enhancements, including new search and data representation tools, as well as a brief review of how the community uses GEO data. GEO is freely accessible at http://www.ncbi.nlm.nih.gov/geo/.

ARMOUR - A Rice miRNA: mRNA Interaction Resource.

PubMed

Sanan-Mishra, Neeti; Tripathi, Anita; Goswami, Kavita; Shukla, Rohit N; Vasudevan, Madavan; Goswami, Hitesh

2018-01-01

ARMOUR was developed as A Rice miRNA:mRNA interaction resource. This informative and interactive database includes the experimentally validated expression profiles of miRNAs under different developmental and abiotic stress conditions across seven Indian rice cultivars. This comprehensive database covers 689 known and 1664 predicted novel miRNAs and their expression profiles in more than 38 different tissues or conditions along with their predicted/known target transcripts. The understanding of miRNA:mRNA interactome in regulation of functional cellular machinery is supported by the sequence information of the mature and hairpin structures. ARMOUR provides flexibility to users in querying the database using multiple ways like known gene identifiers, gene ontology identifiers, KEGG identifiers and also allows on the fly fold change analysis and sequence search query with inbuilt BLAST algorithm. ARMOUR database provides a cohesive platform for novel and mature miRNAs and their expression in different experimental conditions and allows searching for their interacting mRNA targets, GO annotation and their involvement in various biological pathways. The ARMOUR database includes a provision for adding more experimental data from users, with an aim to develop it as a platform for sharing and comparing experimental data contributed by research groups working on rice.

MNDA binds NPM/B23 and the NPM-MLF1 chimera generated by the t(3;5) associated with myelodysplastic syndrome and acute myeloid leukemia.

PubMed

Xie, J; Briggs, J A; Morris, S W; Olson, M O; Kinney, M C; Briggs, R C

1997-10-01

The myeloid cell nuclear differentiation antigen (MNDA) is a nuclear protein expressed specifically in developing cells of the human myelomonocytic lineage, including the end-stage monocytes/macrophages and granulocytes. Nuclear localization, lineage- and stage-specific expression, association with chromatin, and regulation by interferon alpha indicate that this protein is involved in regulating gene expression uniquely associated with the differentiation process and/or function of the monocyte/macrophage. MNDA does not bind specific DNA sequences, but rather a set of nuclear proteins that includes nucleolin (C23). Both in vitro binding assays and co-immunoprecipitation were used to demonstrate that MNDA also binds protein B23 (nucleophosmin/NPM). Three reciprocal chromosome translocations found in certain cases of leukemia/lymphoma involve fusions with the NPM/B23 gene, t(5;17) NPM-RARalpha, t(2;5) NPM-ALK, and the t(3;5) NPM-MLF1. In the current study, MNDA was not able to bind the NPM-ALK chimera originating from the t(2;5) and containing residues 1-117 of NPM. However, MNDA did bind the NPM-MLF1 product of the t(3;5) that contains the N-terminal 175 residues of NPM. The additional 58 amino acids (amino acids 117-175) of the NPM sequence that are contained in the product of the NPM-MLF1 fusion gene relative to the product of the NPM-ALK fusion appear responsible for MNDA binding. This additional NPM sequence contains a nuclear localization signal and clusters of acidic residues believed to bind nuclear localization signals of other proteins. Whereas NPM and nucleolin are primarily localized within the nucleolus, MNDA is distributed throughout the nucleus including the nucleolus, suggesting that additional interactions define overall MNDA localization.

Positive Selection Underlies Faster-Z Evolution of Gene Expression in Birds.

PubMed

Dean, Rebecca; Harrison, Peter W; Wright, Alison E; Zimmer, Fabian; Mank, Judith E

2015-10-01

The elevated rate of evolution for genes on sex chromosomes compared with autosomes (Fast-X or Fast-Z evolution) can result either from positive selection in the heterogametic sex or from nonadaptive consequences of reduced relative effective population size. Recent work in birds suggests that Fast-Z of coding sequence is primarily due to relaxed purifying selection resulting from reduced relative effective population size. However, gene sequence and gene expression are often subject to distinct evolutionary pressures; therefore, we tested for Fast-Z in gene expression using next-generation RNA-sequencing data from multiple avian species. Similar to studies of Fast-Z in coding sequence, we recover clear signatures of Fast-Z in gene expression; however, in contrast to coding sequence, our data indicate that Fast-Z in expression is due to positive selection acting primarily in females. In the soma, where gene expression is highly correlated between the sexes, we detected Fast-Z in both sexes, although at a higher rate in females, suggesting that many positively selected expression changes in females are also expressed in males. In the gonad, where intersexual correlations in expression are much lower, we detected Fast-Z for female gene expression, but crucially, not males. This suggests that a large amount of expression variation is sex-specific in its effects within the gonad. Taken together, our results indicate that Fast-Z evolution of gene expression is the product of positive selection acting on recessive beneficial alleles in the heterogametic sex. More broadly, our analysis suggests that the adaptive potential of Z chromosome gene expression may be much greater than that of gene sequence, results which have important implications for the role of sex chromosomes in speciation and sexual selection. © The Author 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

High throughput protein production screening

DOEpatents

Beernink, Peter T [Walnut Creek, CA; Coleman, Matthew A [Oakland, CA; Segelke, Brent W [San Ramon, CA

2009-09-08

Methods, compositions, and kits for the cell-free production and analysis of proteins are provided. The invention allows for the production of proteins from prokaryotic sequences or eukaryotic sequences, including human cDNAs using PCR and IVT methods and detecting the proteins through fluorescence or immunoblot techniques. This invention can be used to identify optimized PCR and WT conditions, codon usages and mutations. The methods are readily automated and can be used for high throughput analysis of protein expression levels, interactions, and functional states.

B-Bolivia, an Allele of the Maize b1 Gene with Variable Expression, Contains a High Copy Retrotransposon-Related Sequence Immediately Upstream1

PubMed Central

Selinger, David A.; Chandler, Vicki L.

2001-01-01

The maize (Zea mays) b1 gene encodes a transcription factor that regulates the anthocyanin pigment pathway. Of the b1 alleles with distinct tissue-specific expression, B-Peru and B-Bolivia are the only alleles that confer seed pigmentation. B-Bolivia produces variable and weaker seed expression but darker, more regular plant expression relative to B-Peru. Our experiments demonstrated that B-Bolivia is not expressed in the seed when transmitted through the male. When transmitted through the female the proportion of kernels pigmented and the intensity of pigment varied. Molecular characterization of B-Bolivia demonstrated that it shares the first 530 bp of the upstream region with B-Peru, a region sufficient for seed expression. Immediately upstream of 530 bp, B-Bolivia is completely divergent from B-Peru. These sequences share sequence similarity to retrotransposons. Transient expression assays of various promoter constructs identified a 33-bp region in B-Bolivia that can account for the reduced aleurone pigment amounts (40%) observed with B-Bolivia relative to B-Peru. Transgenic plants carrying the B-Bolivia promoter proximal region produced pigmented seeds. Similar to native B-Bolivia, some transgene loci are variably expressed in seeds. In contrast to native B-Bolivia, the transgene loci are expressed in seeds when transmitted through both the male and female. Some transgenic lines produced pigment in vegetative tissues, but the tissue-specificity was different from B-Bolivia, suggesting the introduced sequences do not contain the B-Bolivia plant-specific regulatory sequences. We hypothesize that the chromatin context of the B-Bolivia allele controls its epigenetic seed expression properties, which could be influenced by the adjacent highly repeated retrotransposon sequence. PMID:11244116

Generation of expressed sequence tags for discovery of genes responsible for floral traits of Chrysanthemum morifolium by next-generation sequencing technology.

PubMed

Sasaki, Katsutomo; Mitsuda, Nobutaka; Nashima, Kenji; Kishimoto, Kyutaro; Katayose, Yuichi; Kanamori, Hiroyuki; Ohmiya, Akemi

2017-09-04

Chrysanthemum morifolium is one of the most economically valuable ornamental plants worldwide. Chrysanthemum is an allohexaploid plant with a large genome that is commercially propagated by vegetative reproduction. New cultivars with different floral traits, such as color, morphology, and scent, have been generated mainly by classical cross-breeding and mutation breeding. However, only limited genetic resources and their genome information are available for the generation of new floral traits. To obtain useful information about molecular bases for floral traits of chrysanthemums, we read expressed sequence tags (ESTs) of chrysanthemums by high-throughput sequencing using the 454 pyrosequencing technology. We constructed normalized cDNA libraries, consisting of full-length, 3'-UTR, and 5'-UTR cDNAs derived from various tissues of chrysanthemums. These libraries produced a total number of 3,772,677 high-quality reads, which were assembled into 213,204 contigs. By comparing the data obtained with those of full genome-sequenced species, we confirmed that our chrysanthemum contig set contained the majority of all expressed genes, which was sufficient for further molecular analysis in chrysanthemums. We confirmed that our chrysanthemum EST set (contigs) contained a number of contigs that encoded transcription factors and enzymes involved in pigment and aroma compound metabolism that was comparable to that of other species. This information can serve as an informative resource for identifying genes involved in various biological processes in chrysanthemums. Moreover, the findings of our study will contribute to a better understanding of the floral characteristics of chrysanthemums including the myriad cultivars at the molecular level.

Genomic Heat Shock Element Sequences Drive Cooperative Human Heat Shock Factor 1 DNA Binding and Selectivity*

PubMed Central

Jaeger, Alex M.; Makley, Leah N.; Gestwicki, Jason E.; Thiele, Dennis J.

2014-01-01

The heat shock transcription factor 1 (HSF1) activates expression of a variety of genes involved in cell survival, including protein chaperones, the protein degradation machinery, anti-apoptotic proteins, and transcription factors. Although HSF1 activation has been linked to amelioration of neurodegenerative disease, cancer cells exhibit a dependence on HSF1 for survival. Indeed, HSF1 drives a program of gene expression in cancer cells that is distinct from that activated in response to proteotoxic stress, and HSF1 DNA binding activity is elevated in cycling cells as compared with arrested cells. Active HSF1 homotrimerizes and binds to a DNA sequence consisting of inverted repeats of the pentameric sequence nGAAn, known as heat shock elements (HSEs). Recent comprehensive ChIP-seq experiments demonstrated that the architecture of HSEs is very diverse in the human genome, with deviations from the consensus sequence in the spacing, orientation, and extent of HSE repeats that could influence HSF1 DNA binding efficacy and the kinetics and magnitude of target gene expression. To understand the mechanisms that dictate binding specificity, HSF1 was purified as either a monomer or trimer and used to evaluate DNA-binding site preferences in vitro using fluorescence polarization and thermal denaturation profiling. These results were compared with quantitative chromatin immunoprecipitation assays in vivo. We demonstrate a role for specific orientations of extended HSE sequences in driving preferential HSF1 DNA binding to target loci in vivo. These studies provide a biochemical basis for understanding differential HSF1 target gene recognition and transcription in neurodegenerative disease and in cancer. PMID:25204655

[Research progress in neuropsychopharmacology updated for the post-genomic era].

PubMed

Nakanishi, Toru

2009-11-01

Neuropsychopharmacological research in the post genomic (genomic sequence) era has been developing rapidly through the use of novel techniques including DNA chips. We have applied these techniques to investigate the anti-tumor effect of NSAIDs, isolate novel genes specifically expressed in rheumatoid arthritis, and analyze gene expression profiles in mesenchymal stem cells. Recently, we have developed a novel system of quantitative PCR for detection of BDNF mRNA isoforms. By using this system, we identified the exon-specific mode of expression in acute and chronic pain. In addition, we have made gene expression profiles of KO mice of beta2 subunits in acetylcholine receptors.

Generation of a total of 6483 expressed sequence tags from 60 day-old bovine whole fetus and fetal placenta.

PubMed

Oishi, M; Gohma, H; Lejukole, H Y; Taniguchi, Y; Yamada, T; Suzuki, K; Shinkai, H; Uenishi, H; Yasue, H; Sasaki, Y

2004-05-01

Expressed sequence tags (ESTs) generated based on characterization of clones isolated randomly from cDNA libraries are used to study gene expression profiles in specific tissues and to provide useful information for characterizing tissue physiology. In this study, two directionally cloned cDNA libraries were constructed from 60 day-old bovine whole fetus and fetal placenta. We have characterized 5357 and 1126 clones, and then identified 3464 and 795 unique sequences for the fetus and placenta cDNA libraries: 1851 and 504 showed homology to already identified genes, and 1613 and 291 showed no significant matches to any of the sequences in DNA databases, respectively. Further, we found 94 unique sequences overlapping in both the fetus and the placenta, leading to a catalog of 4165 genes expressed in 60 day-old fetus and placenta. The catalog is used to examine expression profile of genes in 60 day-old bovine fetus and placenta.

Whole genome sequencing analysis of the cutaneous pathogenic yeast Malassezia restricta and identification of the major lipase expressed on the scalp of patients with dandruff.

PubMed

Park, Minji; Cho, Yong-Joon; Lee, Yang Won; Jung, Won Hee

2017-03-01

Malassezia species are opportunistic pathogenic fungi that are frequently associated with seborrhoeic dermatitis, including dandruff. Most Malassezia species are lipid dependent, a property that is compensated by breaking down host sebum into fatty acids by lipases. In this study, we aimed to sequence and analyse the whole genome of Malassezia restricta KCTC 27527, a clinical isolate from a Korean patient with severe dandruff, to search for lipase orthologues and identify the lipase that is the most frequently expressed on the scalp of patients with dandruff. The genome of M. restricta KCTC 27527 was sequenced using the Illumina MiSeq and PacBio platforms. Lipase orthologues were identified by comparison with known lipase genes in the genomes of Malassezia globosa and Malassezia sympodialis. The expression of the identified lipase genes was directly evaluated in swab samples from the scalps of 56 patients with dandruff. We found that, among the identified lipase-encoding genes, the gene encoding lipase homolog MRES_03670, named LIP5 in this study, was the most frequently expressed lipase in the swab samples. Our study provides an overview of the genome of a clinical isolate of M. restricta and fundamental information for elucidating the role of lipases during fungus-host interaction. © 2016 Blackwell Verlag GmbH.

Isolation and expression analysis of EcbZIP17 from different finger millet genotypes shows conserved nature of the gene.

PubMed

Chopperla, Ramakrishna; Singh, Sonam; Mohanty, Sasmita; Reddy, Nanja; Padaria, Jasdeep C; Solanke, Amolkumar U

2017-10-01

Basic leucine zipper (bZIP) transcription factors comprise one of the largest gene families in plants. They play a key role in almost every aspect of plant growth and development and also in biotic and abiotic stress tolerance. In this study, we report isolation and characterization of EcbZIP17 , a group B bZIP transcription factor from a climate smart cereal, finger millet ( Eleusine coracana L.). The genomic sequence of EcbZIP17 is 2662 bp long encompassing two exons and one intron with ORF of 1722 bp and peptide length of 573 aa. This gene is homologous to AtbZIP17 ( Arabidopsis ), ZmbZIP17 (maize) and OsbZIP60 (rice) which play a key role in endoplasmic reticulum (ER) stress pathway. In silico analysis confirmed the presence of basic leucine zipper (bZIP) and transmembrane (TM) domains in the EcbZIP17 protein. Allele mining of this gene in 16 different genotypes by Sanger sequencing revealed no variation in nucleotide sequence, including the 618 bp long intron. Expression analysis of EcbZIP17 under heat stress exhibited similar pattern of expression in all the genotypes across time intervals with highest upregulation after 4 h. The present study established the conserved nature of EcbZIP17 at nucleotide and expression level.

Comparison and correlation of Simple Sequence Repeats distribution in genomes of Brucella species

PubMed Central

Kiran, Jangampalli Adi Pradeep; Chakravarthi, Veeraraghavulu Praveen; Kumar, Yellapu Nanda; Rekha, Somesula Swapna; Kruti, Srinivasan Shanthi; Bhaskar, Matcha

2011-01-01

Computational genomics is one of the important tools to understand the distribution of closely related genomes including simple sequence repeats (SSRs) in an organism, which gives valuable information regarding genetic variations. The central objective of the present study was to screen the SSRs distributed in coding and non-coding regions among different human Brucella species which are involved in a range of pathological disorders. Computational analysis of the SSRs in the Brucella indicates few deviations from expected random models. Statistical analysis also reveals that tri-nucleotide SSRs are overrepresented and tetranucleotide SSRs underrepresented in Brucella genomes. From the data, it can be suggested that over expressed tri-nucleotide SSRs in genomic and coding regions might be responsible in the generation of functional variation of proteins expressed which in turn may lead to different pathogenicity, virulence determinants, stress response genes, transcription regulators and host adaptation proteins of Brucella genomes. Abbreviations SSRs - Simple Sequence Repeats, ORFs - Open Reading Frames. PMID:21738309

CAPA-peptides of praying mantids (Mantodea).

PubMed

Koehler, Rene; Predel, Reinhard

2010-03-01

Dictyoptera which consist of cockroaches, termites, and praying mantids are among the oldest pterygote insects known. Whereas the localization and sequences of neuropeptides from a number of cockroaches are very well known, nearly nothing is known about the neuropeptides typical of praying mantids. In this study, the neuroanatomy of the median neuroendocrine system in the abdominal ventral nerve cord and the sequences of the CAPA-peptides which are expressed in the respective neuroendocrine cells were analyzed. Altogether, 40 species belonging to different families of Mantodea were included. In contrast to cockroaches, the mantids mostly express two CAPA-periviscerokinins (PVKs), only in Mantis religiosa a third PVK was identified. These PVKs are sequence-related to the PVKs of basal cockroaches (Polyphagidae). In a group of closely related Mantodea (Paramantinae), extended forms of PVK-2 were observed. As shown, these forms are possibly the result of substitutions in the N-terminal cleavage sites of the respective PVKs. No trace of a CAPA-pyrokinin was found in any of the praying mantids. (c) 2009 Elsevier Inc. All rights reserved.

Disruption of long-distance highly conserved noncoding elements in neurocristopathies.

PubMed

Amiel, Jeanne; Benko, Sabina; Gordon, Christopher T; Lyonnet, Stanislas

2010-12-01

One of the key discoveries of vertebrate genome sequencing projects has been the identification of highly conserved noncoding elements (CNEs). Some characteristics of CNEs include their high frequency in mammalian genomes, their potential regulatory role in gene expression, and their enrichment in gene deserts nearby master developmental genes. The abnormal development of neural crest cells (NCCs) leads to a broad spectrum of congenital malformation(s), termed neurocristopathies, and/or tumor predisposition. Here we review recent findings that disruptions of CNEs, within or at long distance from the coding sequences of key genes involved in NCC development, result in neurocristopathies via the alteration of tissue- or stage-specific long-distance regulation of gene expression. While most studies on human genetic disorders have focused on protein-coding sequences, these examples suggest that investigation of genomic alterations of CNEs will provide a broader understanding of the molecular etiology of both rare and common human congenital malformations. © 2010 New York Academy of Sciences.

Cloning and expression of recombinant adhesive protein MEFP-2 of the blue mussel, Mytilus edulis

DOEpatents

Silverman, Heather G.; Roberto, Francisco F.

2006-02-07

The present invention includes a Mytilus edulis cDNA having a nucleotide sequence that encodes for the Mytilus edulis foot protein-2 (Mefp-2), an example of a mollusk foot protein. Mefp-2 is an integral component of the blue mussels' adhesive protein complex, which allows the mussel to attach to objects underwater. The isolation, purification and sequencing of the Mefp-2 gene will allow researchers to produce Mefp-2 protein using genetic engineering techniques. The discovery of Mefp-2 gene sequences will also allow scientists to better understand how the blue mussel creates its waterproof adhesive protein complex.

Data in support of qPCR primer design and verification in a Pink1 -/- rat model of Parkinson disease.

PubMed

Kelm-Nelson, Cynthia A; Stevenson, Sharon A; Ciucci, Michelle R

2016-09-01

Datasets provided in this article represent the Rattus norvegicus primer design and verification used in Pink1 -/- and wildtype Long Evans brain tissue. Accessible tables include relevant information, accession numbers, sequences, temperatures and product length, describing primer design specific to the transcript amplification use. Additionally, results of Sanger sequencing of qPCR reaction products (FASTA aligned sequences) are presented for genes of interest. Results and further interpretation and discussion can be found in the original research article "Atp13a2 expression in the periaqueductal gray is decreased in the Pink1 -/- rat model of Parkinson disease" [1].

Comparisons between Arabidopsis thaliana and Drosophila melanogaster in relation to Coding and Noncoding Sequence Length and Gene Expression

PubMed Central

Caldwell, Rachel; Lin, Yan-Xia; Zhang, Ren

2015-01-01

There is a continuing interest in the analysis of gene architecture and gene expression to determine the relationship that may exist. Advances in high-quality sequencing technologies and large-scale resource datasets have increased the understanding of relationships and cross-referencing of expression data to the large genome data. Although a negative correlation between expression level and gene (especially transcript) length has been generally accepted, there have been some conflicting results arising from the literature concerning the impacts of different regions of genes, and the underlying reason is not well understood. The research aims to apply quantile regression techniques for statistical analysis of coding and noncoding sequence length and gene expression data in the plant, Arabidopsis thaliana, and fruit fly, Drosophila melanogaster, to determine if a relationship exists and if there is any variation or similarities between these species. The quantile regression analysis found that the coding sequence length and gene expression correlations varied, and similarities emerged for the noncoding sequence length (5′ and 3′ UTRs) between animal and plant species. In conclusion, the information described in this study provides the basis for further exploration into gene regulation with regard to coding and noncoding sequence length. PMID:26114098

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

Characterization and differential expression of microRNAs elicited by sulfur deprivation in Chlamydomonas reinhardtii

PubMed Central

2012-01-01

Background microRNAs (miRNAs) have been found to play an essential role in the modulation of numerous biological processes in eukaryotes. Chlamydomonas reinhardtii is an ideal model organism for the study of many metabolic processes including responses to sulfur-deprivation. We used a deep sequencing platform to extensively profile and identify changes in the miRNAs expression that occurred under sulfur-replete and sulfur-deprived conditions. The aim of our research was to characterize the differential expression of Chlamydomonas miRNAs under sulfur-deprived conditions, and subsequently, the target genes of miRNA involved in sulfur-deprivation were further predicted and analyzed. Results By using high-throughput sequencing, we characterized the microRNA transcriptomes under sulphur-replete and sulfur-deprived conditions in Chlamydomonas reinhardtii. We predicted a total of 310 miRNAs which included 85 known miRNAs and 225 novel miRNAs. 13 miRNAs were the specific to the sulfur-deprived conditions. 47 miRNAs showed significantly differential expressions responding to sulfur-deprivation, and most were up-regulated in the small RNA libraries with sulfur-deprivation. Using a web-based integrated system (Web MicroRNAs Designer 3) and combing the former information from a transcriptome of Chlamydomonas reinhardtii, 22 miRNAs and their targets involved in metabolism regulation with sulfur-deprivation were verified. Conclusions Our results indicate that sulfur-deprivation may have a significant influence on small RNA expression patterns, and the differential expressions of miRNAs and interactions between miRNA and its targets might further reveal the molecular mechanism responding to sulfur-deprivation in Chlamydomonas reinhardtii. PMID:22439676

Deep sequencing of cardiac microRNA-mRNA interactomes in clinical and experimental cardiomyopathy

PubMed Central

Matkovich, Scot J.; Dorn, Gerald W.

2018-01-01

Summary MicroRNAs are a family of short (~21 nucleotide) noncoding RNAs that serve key roles in cellular growth and differentiation and the response of the heart to stress stimuli. As the sequence-specific recognition element of RNA-induced silencing complexes (RISCs), microRNAs bind mRNAs and prevent their translation via mechanisms that may include transcript degradation and/or prevention of ribosome binding. Short microRNA sequences and the ability of microRNAs to bind to mRNA sites having only partial/imperfect sequence complementarity complicates purely computational analyses of microRNA-mRNA interactomes. Furthermore, computational microRNA target prediction programs typically ignore biological context, and therefore the principal determinants of microRNA-mRNA binding: the presence and quantity of each. To address these deficiencies we describe an empirical method, developed via studies of stressed and failing hearts, to determine disease-induced changes in microRNAs, mRNAs, and the mRNAs targeted to the RISC, without cross-linking mRNAs to RISC proteins. Deep sequencing methods are used to determine RNA abundances, delivering unbiased, quantitative RNA data limited only by their annotation in the genome of interest. We describe the laboratory bench steps required to perform these experiments, experimental design strategies to achieve an appropriate number of sequencing reads per biological replicate, and computer-based processing tools and procedures to convert large raw sequencing data files into gene expression measures useful for differential expression analyses. PMID:25836573

Deep sequencing of cardiac microRNA-mRNA interactomes in clinical and experimental cardiomyopathy.

PubMed

Matkovich, Scot J; Dorn, Gerald W

2015-01-01

MicroRNAs are a family of short (~21 nucleotide) noncoding RNAs that serve key roles in cellular growth and differentiation and the response of the heart to stress stimuli. As the sequence-specific recognition element of RNA-induced silencing complexes (RISCs), microRNAs bind mRNAs and prevent their translation via mechanisms that may include transcript degradation and/or prevention of ribosome binding. Short microRNA sequences and the ability of microRNAs to bind to mRNA sites having only partial/imperfect sequence complementarity complicate purely computational analyses of microRNA-mRNA interactomes. Furthermore, computational microRNA target prediction programs typically ignore biological context, and therefore the principal determinants of microRNA-mRNA binding: the presence and quantity of each. To address these deficiencies we describe an empirical method, developed via studies of stressed and failing hearts, to determine disease-induced changes in microRNAs, mRNAs, and the mRNAs targeted to the RISC, without cross-linking mRNAs to RISC proteins. Deep sequencing methods are used to determine RNA abundances, delivering unbiased, quantitative RNA data limited only by their annotation in the genome of interest. We describe the laboratory bench steps required to perform these experiments, experimental design strategies to achieve an appropriate number of sequencing reads per biological replicate, and computer-based processing tools and procedures to convert large raw sequencing data files into gene expression measures useful for differential expression analyses.

Predicting earthquakes by analyzing accelerating precursory seismic activity

USGS Publications Warehouse

Varnes, D.J.

1989-01-01

During 11 sequences of earthquakes that in retrospect can be classed as foreshocks, the accelerating rate at which seismic moment is released follows, at least in part, a simple equation. This equation (1) is {Mathematical expression},where {Mathematical expression} is the cumulative sum until time, t, of the square roots of seismic moments of individual foreshocks computed from reported magnitudes;C and n are constants; and tfis a limiting time at which the rate of seismic moment accumulation becomes infinite. The possible time of a major foreshock or main shock, tf,is found by the best fit of equation (1), or its integral, to step-like plots of {Mathematical expression} versus time using successive estimates of tfin linearized regressions until the maximum coefficient of determination, r2,is obtained. Analyzed examples include sequences preceding earthquakes at Cremasta, Greece, 2/5/66; Haicheng, China 2/4/75; Oaxaca, Mexico, 11/29/78; Petatlan, Mexico, 3/14/79; and Central Chile, 3/3/85. In 29 estimates of main-shock time, made as the sequences developed, the errors in 20 were less than one-half and in 9 less than one tenth the time remaining between the time of the last data used and the main shock. Some precursory sequences, or parts of them, yield no solution. Two sequences appear to include in their first parts the aftershocks of a previous event; plots using the integral of equation (1) show that the sequences are easily separable into aftershock and foreshock segments. Synthetic seismic sequences of shocks at equal time intervals were constructed to follow equation (1), using four values of n. In each series the resulting distributions of magnitudes closely follow the linear Gutenberg-Richter relation log N=a-bM, and the product n times b for each series is the same constant. In various forms and for decades, equation (1) has been used successfully to predict failure times of stressed metals and ceramics, landslides in soil and rock slopes, and volcanic eruptions. Results of more recent experiments and theoretical studies on crack propagation, fault mechanics, and acoustic emission can be closely reproduced by equation (1). Rate-process theory and continuum damage mechanics offer leads toward understanding the physical processes. ?? 1989 Birkha??user Verlag.

Digital transcriptome analysis of putative sex-determination genes in papaya (Carica papaya).

PubMed

Urasaki, Naoya; Tarora, Kazuhiko; Shudo, Ayano; Ueno, Hiroki; Tamaki, Moritoshi; Miyagi, Norimichi; Adaniya, Shinichi; Matsumura, Hideo

2012-01-01

Papaya (Carica papaya) is a trioecious plant species that has male, female and hermaphrodite flowers on different plants. The primitive sex chromosomes genetically determine the sex of the papaya. Although draft sequences of the papaya genome are already available, the genes for sex determination have not been identified, likely due to the complicated structure of its sex-chromosome sequences. To identify the candidate genes for sex determination, we conducted a transcriptome analysis of flower samples from male, female and hermaphrodite plants using high-throughput SuperSAGE for digital gene expression analysis. Among the short sequence tags obtained from the transcripts, 312 unique tags were specifically mapped to the primitive sex chromosome (X or Y(h)) sequences. An annotation analysis revealed that retroelements are the most abundant sequences observed in the genes corresponding to these tags. The majority of tags on the sex chromosomes were located on the X chromosome, and only 30 tags were commonly mapped to both the X and Y(h) chromosome, implying a loss of many genes on the Y(h) chromosome. Nevertheless, candidate Y(h) chromosome-specific female determination genes, including a MADS-box gene, were identified. Information on these sex chromosome-specific expressed genes will help elucidating sex determination in the papaya.

Digital Transcriptome Analysis of Putative Sex-Determination Genes in Papaya (Carica papaya)

PubMed Central

Urasaki, Naoya; Tarora, Kazuhiko; Shudo, Ayano; Ueno, Hiroki; Tamaki, Moritoshi; Miyagi, Norimichi; Adaniya, Shinichi; Matsumura, Hideo

2012-01-01

Papaya (Carica papaya) is a trioecious plant species that has male, female and hermaphrodite flowers on different plants. The primitive sex chromosomes genetically determine the sex of the papaya. Although draft sequences of the papaya genome are already available, the genes for sex determination have not been identified, likely due to the complicated structure of its sex-chromosome sequences. To identify the candidate genes for sex determination, we conducted a transcriptome analysis of flower samples from male, female and hermaphrodite plants using high-throughput SuperSAGE for digital gene expression analysis. Among the short sequence tags obtained from the transcripts, 312 unique tags were specifically mapped to the primitive sex chromosome (X or Yh) sequences. An annotation analysis revealed that retroelements are the most abundant sequences observed in the genes corresponding to these tags. The majority of tags on the sex chromosomes were located on the X chromosome, and only 30 tags were commonly mapped to both the X and Yh chromosome, implying a loss of many genes on the Yh chromosome. Nevertheless, candidate Yh chromosome-specific female determination genes, including a MADS-box gene, were identified. Information on these sex chromosome-specific expressed genes will help elucidating sex determination in the papaya. PMID:22815863

Olfactomedin-like 2 A and B (OLFML2A and OLFML2B) expression profile in primates (human and baboon).

PubMed

Pérez-Ibave, Diana Cristina; González-Alvarez, Rafael; de La Luz Martinez-Fierro, Margarita; Ruiz-Ayma, Gabriel; Luna-Muñoz, Maricela; Martínez-De-Villarreal, Laura Elia; De Lourdes Garza-Rodríguez, María; Reséndez-Pérez, Diana; Mohamed-Noriega, Jibran; Garza-Guajardo, Raquel; Bautista-De-Lucío, Víctor Manuel; Mohamed-Noriega, Karim; Barboza-Quintana, Oralia; Arámburo-De-La-Hoz, Carlos; Barrera-Saldaña, Hugo Alberto; Rodríguez-Sánchez, Irám Pablo

2016-11-08

The olfactomedin-like domain (OLFML) is present in at least four families of proteins, including OLFML2A and OLFML2B, which are expressed in adult rat retina cells. However, no expression of their orthologous has ever been reported in human and baboon. The aim of this study was to investigate the expression of OLFML2A and OLFML2B in ocular tissues of baboons (Papio hamadryas) and humans, as a key to elucidate OLFML function in eye physiology. OLFML2A and OLFML2B cDNA detection in ocular tissues of these species was performed by RT-PCR. The amplicons were cloned and sequenced, phylogenetically analyzed and their proteins products were confirmed by immunofluorescence assays. OLFML2A and OLFML2B transcripts were found in human cornea, lens and retina and in baboon cornea, lens, iris and retina. The baboon OLFML2A and OLFML2B ORF sequences have 96% similarity with their human's orthologous. OLFML2A and OLFML2B evolution fits the hypothesis of purifying selection. Phylogenetic analysis shows clear orthology in OLFML2A genes, while OLFML2B orthology is not clear. Expression of OLFML2A and OLFML2B in human and baboon ocular tissues, including their high similarity, make the baboon a powerful model to deduce the physiological and/or metabolic function of these proteins in the eye.

Identification of differentially expressed lncRNAs involved in transient regeneration of the neonatal C57BL/6J mouse heart by next-generation high-throughput RNA sequencing.

PubMed

Chen, Yu-Mei; Li, Hua; Fan, Yi; Zhang, Qi-Jun; Li, Xing; Wu, Li-Jie; Chen, Zi-Jie; Zhu, Chun; Qian, Ling-Mei

2017-04-25

Previous studies have shown that mammalian cardiac tissue has a regenerative capacity. Remarkably, neonatal mice can regenerate their cardiac tissue for up to 6 days after birth, but this capacity is lost by day 7. In this study, we aimed to explore the expression pattern of long noncoding RNA (lncRNA) during this period and examine the mechanisms underlying this process. We found that 685 lncRNAs and 1833 mRNAs were differentially expressed at P1 and P7 by the next-generation high-throughput RNA sequencing. The coding genes associated with differentially expressed lncRNAs were mainly involved in metabolic processes and cell proliferation, and also were potentially associated with several key regeneration signalling pathways, including PI3K-Akt, MAPK, Hippo and Wnt. In addition, we identified some correlated targets of highly-dysregulated lncRNAs such as Igfbp3, Trnp1, Itgb6, and Pim3 by the coding-noncoding gene co-expression network. These data may offer a reference resource for further investigation about the mechanisms by which lncRNAs regulate cardiac regeneration.

Calcium-Dependent Protein Kinase Genes in Corn Roots

NASA Technical Reports Server (NTRS)

Takezawa, D.; Patil, S.; Bhatia, A.; Poovaiah, B. W.

1996-01-01

Two cDNAs encoding Ca-2(+) - Dependent Protein Kinases (CDPKs), Corn Root Protein Kinase 1 and 2 (CRPK 1, CRPK 2) were isolated from the root tip library of corn (Zea mays L., cv. Merit) and their nucleotide sequences were determined. Deduced amino acid sequences of both the clones have features characteristic of plant CDPKS, including all 11 conserved serine/threonine kinase subdomains, a junction domain and a calmodulin-like domain with four Ca-2(+), -binding sites. Northern analysis revealed that CRPKI mRNA is preferentially expressed in roots, especially in the root tip; whereas, the expression of CRPK2 mRNA was very low in all the tissues tested. In situ hybridization experiments revealed that CRPKI mRNA is highly expressed in the root apex, as compared to other parts of the root. Partially purified CDPK from the root tip phosphorylates syntide-2, a common peptide substrate for plant CDPKs, and the phosphorylation was stimulated 7-fold by the addition of Ca-2(+). Our results show that two CDPK isoforms are expressed in corn roots and they may be involved in the Ca-2(+)-dependent signal transduction process.

Signatures from Tissue-specific MPSS Libraries Identify Transcripts Preferentially Expressed in the Mouse Inner Ear

PubMed Central

Peters, Linda M.; Belyantseva, Inna A.; Lagziel, Ayala; Battey, James F.; Friedman, Thomas B.; Morell, Robert J.

2007-01-01

Specialization in cell function and morphology is influenced by the differential expression of mRNAs, many of which are expressed at low abundance and restricted to certain cell types. Detecting such transcripts in cDNA libraries may require sequencing millions of clones. Massively parallel signature sequencing (MPSS) is well-suited for identifying transcripts that are expressed in discrete cell types and in low abundance. We have made MPSS libraries from microdissections of three inner ear tissues. By comparing these MPSS libraries to those of 87 other tissues included in the Mouse Reference Transcriptome (MRT) online resource, we have identified genes that are highly enriched in, or specific to, the inner ear. We show by RT-PCR and in situ hybridization that signatures unique to the inner ear libraries identify transcripts with highly specific cell-type localizations. These transcripts serve to illustrate the utility of a resource that is available to the research community. Utilization of these resources will increase the number of known transcription units and expand our knowledge of the tissue-specific regulation of the transcriptome. PMID:17049805

Detection of Sirtuin-1 protein expression in peripheral blood leukocytes in dogs.

PubMed

Yoshimura, Kuniko; Matsuu, Aya; Sasaki, Kai; Momoi, Yasuyuki

2018-05-11

Sirtuin-1 (SIRT1) is a nicotinamide adenine dinucleotide (NAD + )-dependent histone deacetylase with a large number of protein substrates. It has attracted a lot of attention in association with extending lifespan. The objective of this study was to enable the evaluation of SIRT1 expression in peripheral blood mononuclear cells (PBMCs) from dogs by flow cytometry. Three transcript variants were amplified from PBMCs by reverse transcription PCR and the nucleotide sequences were analyzed. On the basis deduced amino acid sequence, a monoclonal antibody against human SIRT1, 1F3, was selected to detect canine SIRT1. Canine SIRT1 in peripheral blood mononuclear cells was successfully detected by western blotting using this antibody. Intracellular canine SIRT1 was also detected in permeabilized 293T cells transfected with a canine SIRT1 expression plasmid by flow cytometry using this antibody. SIRT1 was detected in all leukocyte subsets including lymphocytes, granulocytes and monocytes. The expression level was markedly different among individual dogs. These results indicated that the method applied in this study is useful for evaluating canine SIRT1 levels in PBMCs from dogs.

It's DE-licious: A Recipe for Differential Expression Analyses of RNA-seq Experiments Using Quasi-Likelihood Methods in edgeR.

PubMed

Lun, Aaron T L; Chen, Yunshun; Smyth, Gordon K

2016-01-01

RNA sequencing (RNA-seq) is widely used to profile transcriptional activity in biological systems. Here we present an analysis pipeline for differential expression analysis of RNA-seq experiments using the Rsubread and edgeR software packages. The basic pipeline includes read alignment and counting, filtering and normalization, modelling of biological variability and hypothesis testing. For hypothesis testing, we describe particularly the quasi-likelihood features of edgeR. Some more advanced downstream analysis steps are also covered, including complex comparisons, gene ontology enrichment analyses and gene set testing. The code required to run each step is described, along with an outline of the underlying theory. The chapter includes a case study in which the pipeline is used to study the expression profiles of mammary gland cells in virgin, pregnant and lactating mice.

Genome-wide characterization and selection of expressed sequence tag simple sequence repeat primers for optimized marker distribution and reliability in peach

USDA-ARS?s Scientific Manuscript database

Expressed sequence tag (EST) simple sequence repeats (SSRs) in Prunus were mined, and flanking primers designed and used for genome-wide characterization and selection of primers to optimize marker distribution and reliability. A total of 12,618 contigs were assembled from 84,727 ESTs, along with 34...

DNA DAMAGE REPAIR AND CELL CYCLE CONTROL: A NATURAL BIO-DEFENSE MECHANISM

EPA Science Inventory

DNA DAMAGE REPAIR AND CELL CYCLE CONTROL: A natural bio-defense mechanism
Anuradha Mudipalli.

Maintenance of genetic information, including the correct sequence of nucleotides in DNA, is essential for replication, gene expression, and protein synthesis. DNA lesions onto...

DNA copy number, including telomeres and mitochondria, assayed using next-generation sequencing.

PubMed

Castle, John C; Biery, Matthew; Bouzek, Heather; Xie, Tao; Chen, Ronghua; Misura, Kira; Jackson, Stuart; Armour, Christopher D; Johnson, Jason M; Rohl, Carol A; Raymond, Christopher K

2010-04-16

DNA copy number variations occur within populations and aberrations can cause disease. We sought to develop an improved lab-automatable, cost-efficient, accurate platform to profile DNA copy number. We developed a sequencing-based assay of nuclear, mitochondrial, and telomeric DNA copy number that draws on the unbiased nature of next-generation sequencing and incorporates techniques developed for RNA expression profiling. To demonstrate this platform, we assayed UMC-11 cells using 5 million 33 nt reads and found tremendous copy number variation, including regions of single and homogeneous deletions and amplifications to 29 copies; 5 times more mitochondria and 4 times less telomeric sequence than a pool of non-diseased, blood-derived DNA; and that UMC-11 was derived from a male individual. The described assay outputs absolute copy number, outputs an error estimate (p-value), and is more accurate than array-based platforms at high copy number. The platform enables profiling of mitochondrial levels and telomeric length. The assay is lab-automatable and has a genomic resolution and cost that are tunable based on the number of sequence reads.

DNA copy number, including telomeres and mitochondria, assayed using next-generation sequencing

PubMed Central

2010-01-01

Background DNA copy number variations occur within populations and aberrations can cause disease. We sought to develop an improved lab-automatable, cost-efficient, accurate platform to profile DNA copy number. Results We developed a sequencing-based assay of nuclear, mitochondrial, and telomeric DNA copy number that draws on the unbiased nature of next-generation sequencing and incorporates techniques developed for RNA expression profiling. To demonstrate this platform, we assayed UMC-11 cells using 5 million 33 nt reads and found tremendous copy number variation, including regions of single and homogeneous deletions and amplifications to 29 copies; 5 times more mitochondria and 4 times less telomeric sequence than a pool of non-diseased, blood-derived DNA; and that UMC-11 was derived from a male individual. Conclusion The described assay outputs absolute copy number, outputs an error estimate (p-value), and is more accurate than array-based platforms at high copy number. The platform enables profiling of mitochondrial levels and telomeric length. The assay is lab-automatable and has a genomic resolution and cost that are tunable based on the number of sequence reads. PMID:20398377

Generate Optimized Genetic Rhythm for Enzyme Expression in Non-native systems

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

2016-11-03

Most amino acids are represented by more than one codon, resulting in redundancy in the genetic code. Silent codon substitutions that do not alter the amino acid sequence still have an effect on protein expression. We have developed an algorithm, GoGREEN, to enhance the expression of foreign proteins in a host organism. GoGREEN selects codons according to frequency patterns seen in the gene of interest using the codon usage table from the host organism. GoGREEN is also designed to accommodate gaps in the sequence.This software takes for input (1) the aligned protein sequences for genes the user wishes to express,more » (2) the codon usage table for the host organism, (3) and the DNA sequence for the target protein found in the host organism. The program will select codons based on codon usage patterns for the target DNA sequence. The program will also select codons for “gaps” found in the aligned protein sequences using the codon usage table from the host organism.« less

Transcriptome Dynamics of Developing Photoreceptors in Three‐Dimensional Retina Cultures Recapitulates Temporal Sequence of Human Cone and Rod Differentiation Revealing Cell Surface Markers and Gene Networks

PubMed Central

Kaewkhaw, Rossukon; Kaya, Koray Dogan; Brooks, Matthew; Homma, Kohei; Zou, Jizhong; Chaitankar, Vijender; Rao, Mahendra

2015-01-01

Abstract The derivation of three‐dimensional (3D) stratified neural retina from pluripotent stem cells has permitted investigations of human photoreceptors. We have generated a H9 human embryonic stem cell subclone that carries a green fluorescent protein (GFP) reporter under the control of the promoter of cone‐rod homeobox (CRX), an established marker of postmitotic photoreceptor precursors. The CRXp‐GFP reporter replicates endogenous CRX expression in vitro when the H9 subclone is induced to form self‐organizing 3D retina‐like tissue. At day 37, CRX+ photoreceptors appear in the basal or middle part of neural retina and migrate to apical side by day 67. Temporal and spatial patterns of retinal cell type markers recapitulate the predicted sequence of development. Cone gene expression is concomitant with CRX, whereas rod differentiation factor neural retina leucine zipper protein (NRL) is first observed at day 67. At day 90, robust expression of NRL and its target nuclear receptor NR2E3 is evident in many CRX+ cells, while minimal S‐opsin and no rhodopsin or L/M‐opsin is present. The transcriptome profile, by RNA‐seq, of developing human photoreceptors is remarkably concordant with mRNA and immunohistochemistry data available for human fetal retina although many targets of CRX, including phototransduction genes, exhibit a significant delay in expression. We report on temporal changes in gene signatures, including expression of cell surface markers and transcription factors; these expression changes should assist in isolation of photoreceptors at distinct stages of differentiation and in delineating coexpression networks. Our studies establish the first global expression database of developing human photoreceptors, providing a reference map for functional studies in retinal cultures. Stem Cells 2015;33:3504–3518 PMID:26235913

Transcripts and MicroRNAs Responding to Salt Stress in Musa acuminata Colla (AAA Group) cv. Berangan Roots

PubMed Central

Lee, Wan Sin; Gudimella, Ranganath; Wong, Gwo Rong; Tammi, Martti Tapani; Khalid, Norzulaani; Harikrishna, Jennifer Ann

2015-01-01

Physiological responses to stress are controlled by expression of a large number of genes, many of which are regulated by microRNAs. Since most banana cultivars are salt-sensitive, improved understanding of genetic regulation of salt induced stress responses in banana can support future crop management and improvement in the face of increasing soil salinity related to irrigation and climate change. In this study we focused on determining miRNA and their targets that respond to NaCl exposure and used transcriptome sequencing of RNA and small RNA from control and NaCl-treated banana roots to assemble a cultivar-specific reference transcriptome and identify orthologous and Musa-specific miRNA responding to salinity. We observed that, banana roots responded to salinity stress with changes in expression for a large number of genes (9.5% of 31,390 expressed unigenes) and reduction in levels of many miRNA, including several novel miRNA and banana-specific miRNA-target pairs. Banana roots expressed a unique set of orthologous and Musa-specific miRNAs of which 59 respond to salt stress in a dose-dependent manner. Gene expression patterns of miRNA compared with those of their predicted mRNA targets indicated that a majority of the differentially expressed miRNAs were down-regulated in response to increased salinity, allowing increased expression of targets involved in diverse biological processes including stress signaling, stress defence, transport, cellular homeostasis, metabolism and other stress-related functions. This study may contribute to the understanding of gene regulation and abiotic stress response of roots and the high-throughput sequencing data sets generated may serve as important resources related to salt tolerance traits for functional genomic studies and genetic improvement in banana. PMID:25993649

Characterization of Gonadal Transcriptomes from Nile Tilapia (Oreochromis niloticus) Reveals Differentially Expressed Genes

PubMed Central

Sun, Yunlv; Yang, Shijie; Li, Minghui; Zeng, Sheng; Huang, Baofeng; Wang, Deshou

2013-01-01

Four pairs of XX and XY gonads from Nile tilapia were sequenced at four developmental stages, 5, 30, 90, and 180 days after hatching (dah) using Illumina HiseqTM technology. This produced 28 Gb sequences, which were mapped to 21,334 genes. Of these, 259 genes were found to be specifically expressed in XY gonads, and 69 were found to be specific to XX gonads. Totally, 187 XX- and 1,358 XY-enhanced genes were identified, and 2,978 genes were found to be co-expressed in XX and XY gonads. Almost all steroidogenic enzymes, including cyp19a1a, were up-regulated in XX gonads at 5 dah; but in XY gonads these enzymes, including cyp11b2, were significantly up-regulated at 90 dah, indicating that, at a time critical to sex determination, the XX fish produced estrogen and the XY fish did not produce androgens. The most pronounced expression of steroidogenic enzyme genes was observed at 30 and 90 dah for XX and XY gonads, corresponding to the initiation of germ cell meiosis in the female and male gonads, respectively. Both estrogen and androgen receptors were found to be expressed in XX gonads, but only estrogen receptors were expressed in XY gonads at 5 dah. This could explain why exogenous steroid treatment induced XX and XY sex reversal. The XX-enhanced expression of cyp19a1a and cyp19a1b at all stages suggests an important role for estrogen in female sex determination and maintenance of phenotypic sex. This work is the largest collection of gonadal transcriptome data in tilapia and lays the foundation for future studies into the molecular mechanisms of sex determination and maintenance of phenotypic sex in non-model teleosts. PMID:23658843

Transcripts and MicroRNAs Responding to Salt Stress in Musa acuminata Colla (AAA Group) cv. Berangan Roots.

PubMed

Lee, Wan Sin; Gudimella, Ranganath; Wong, Gwo Rong; Tammi, Martti Tapani; Khalid, Norzulaani; Harikrishna, Jennifer Ann

2015-01-01

Physiological responses to stress are controlled by expression of a large number of genes, many of which are regulated by microRNAs. Since most banana cultivars are salt-sensitive, improved understanding of genetic regulation of salt induced stress responses in banana can support future crop management and improvement in the face of increasing soil salinity related to irrigation and climate change. In this study we focused on determining miRNA and their targets that respond to NaCl exposure and used transcriptome sequencing of RNA and small RNA from control and NaCl-treated banana roots to assemble a cultivar-specific reference transcriptome and identify orthologous and Musa-specific miRNA responding to salinity. We observed that, banana roots responded to salinity stress with changes in expression for a large number of genes (9.5% of 31,390 expressed unigenes) and reduction in levels of many miRNA, including several novel miRNA and banana-specific miRNA-target pairs. Banana roots expressed a unique set of orthologous and Musa-specific miRNAs of which 59 respond to salt stress in a dose-dependent manner. Gene expression patterns of miRNA compared with those of their predicted mRNA targets indicated that a majority of the differentially expressed miRNAs were down-regulated in response to increased salinity, allowing increased expression of targets involved in diverse biological processes including stress signaling, stress defence, transport, cellular homeostasis, metabolism and other stress-related functions. This study may contribute to the understanding of gene regulation and abiotic stress response of roots and the high-throughput sequencing data sets generated may serve as important resources related to salt tolerance traits for functional genomic studies and genetic improvement in banana.

Serine proteases in rodent hippocampus.

PubMed

Davies, B J; Pickard, B S; Steel, M; Morris, R G; Lathe, R

1998-09-04

Brain serine proteases are implicated in developmental processes, synaptic plasticity, and in disorders including Alzheimer's disease. The spectrum of the major enzymes expressed in brain has not been established previously. We now present a systematic study of the serine proteases expressed in adult rat and mouse hippocampus. Using a combination of techniques including polymerase chain reaction amplification and Northern blotting we show that tissue-type plasminogen activator (t-PA) is the major species represented. Unexpectedly, the next most abundant species were RNK-Met-1, a lymphocyte protease not reported previously in brain, and two new family members, BSP1 (brain serine protease 1) and BSP2. We report full-length sequences of the two new proteases; homologies indicate that these are of tryptic specificity. Although BSP2 is expressed in several brain regions, BSP1 expression is strikingly restricted to hippocampus. Other enzymes represented, but at lower levels, included elastase IV, proteinase 3, complement C2, chymotrypsin B, chymotrypsin-like protein, and Hageman factor. Although thrombin and urokinase-type plasminogen activator were not detected in the primary screen, low level expression was confirmed using specific polymerase chain reaction primers. In contrast, and despite robust expression of t-PA, the usual t-PA substrate plasminogen was not expressed at detectable levels.

Integrated analysis of whole-exome sequencing and transcriptome profiling in males with autism spectrum disorders.

PubMed

Codina-Solà, Marta; Rodríguez-Santiago, Benjamín; Homs, Aïda; Santoyo, Javier; Rigau, Maria; Aznar-Laín, Gemma; Del Campo, Miguel; Gener, Blanca; Gabau, Elisabeth; Botella, María Pilar; Gutiérrez-Arumí, Armand; Antiñolo, Guillermo; Pérez-Jurado, Luis Alberto; Cuscó, Ivon

2015-01-01

Autism spectrum disorders (ASD) are a group of neurodevelopmental disorders with high heritability. Recent findings support a highly heterogeneous and complex genetic etiology including rare de novo and inherited mutations or chromosomal rearrangements as well as double or multiple hits. We performed whole-exome sequencing (WES) and blood cell transcriptome by RNAseq in a subset of male patients with idiopathic ASD (n = 36) in order to identify causative genes, transcriptomic alterations, and susceptibility variants. We detected likely monogenic causes in seven cases: five de novo (SCN2A, MED13L, KCNV1, CUL3, and PTEN) and two inherited X-linked variants (MAOA and CDKL5). Transcriptomic analyses allowed the identification of intronic causative mutations missed by the usual filtering of WES and revealed functional consequences of some rare mutations. These included aberrant transcripts (PTEN, POLR3C), deregulated expression in 1.7% of mutated genes (that is, SEMA6B, MECP2, ANK3, CREBBP), allele-specific expression (FUS, MTOR, TAF1C), and non-sense-mediated decay (RIT1, ALG9). The analysis of rare inherited variants showed enrichment in relevant pathways such as the PI3K-Akt signaling and the axon guidance. Integrative analysis of WES and blood RNAseq data has proven to be an efficient strategy to identify likely monogenic forms of ASD (19% in our cohort), as well as additional rare inherited mutations that can contribute to ASD risk in a multifactorial manner. Blood transcriptomic data, besides validating 88% of expressed variants, allowed the identification of missed intronic mutations and revealed functional correlations of genetic variants, including changes in splicing, expression levels, and allelic expression.

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

The ura5 gene of the ascomycete Sordaria macrospora: molecular cloning, characterization and expression in Escherichia coli.

PubMed

Le Chevanton, L; Leblon, G

1989-04-15

We cloned the ura5 gene coding for the orotate phosphoribosyl transferase from the ascomycete Sordaria macrospora by heterologous probing of a Sordaria genomic DNA library with the corresponding Podospora anserina sequence. The Sordaria gene was expressed in an Escherichia coli pyrE mutant strain defective for the same enzyme, and expression was shown to be promoted by plasmid sequences. The nucleotide sequence of the 1246-bp DNA fragment encompassing the region of homology with the Podospora gene has been determined. This sequence contains an open reading frame of 699 nucleotides. The deduced amino acid sequence shows 72% similarity with the corresponding Podospora protein.

Hypoexpression and epigenetic regulation of candidate tumor suppressor gene CADM-2 in human prostate cancer.

PubMed

Chang, Guimin; Xu, Shuping; Dhir, Rajiv; Chandran, Uma; O'Keefe, Denise S; Greenberg, Norman M; Gingrich, Jeffrey R

2010-11-15

Cell adhesion molecules (CADM) comprise a newly identified protein family whose functions include cell polarity maintenance and tumor suppression. CADM-1, CADM-3, and CADM-4 have been shown to act as tumor suppressor genes in multiple cancers including prostate cancer. However, CADM-2 expression has not been determined in prostate cancer. The CADM-2 gene was cloned and characterized and its expression in human prostatic cell lines and cancer specimens was analyzed by reverse transcription-PCR and an immunohistochemical tissue array, respectively. The effects of adenovirus-mediated CADM-2 expression on prostate cancer cells were also investigated. CADM-2 promoter methylation was evaluated by bisulfite sequencing and methylation-specific PCR. We report the initial characterization of CADM-2 isoforms: CADM-2a and CADM-2b, each with separate promoters, in human chromosome 3p12.1. Prostate cancer cell lines, LNCaP and DU145, expressed negligible CADM-2a relative to primary prostate tissue and cell lines, RWPE-1 and PPC-1, whereas expression of CADM-2b was maintained. Using immunohistochemistry, tissue array results from clinical specimens showed statistically significant decreased expression in prostate carcinoma compared with normal donor prostate, benign prostatic hyperplasia, prostatic intraepithelial neoplasia, and normal tissue adjacent to tumor (P < 0.001). Adenovirus-mediated CADM-2a expression suppressed DU145 cell proliferation in vitro and colony formation in soft agar. The decrease in CADM-2a mRNA in cancer cell lines correlated with promoter region hypermethylation as determined by bisulfite sequencing and methylation-specific PCR. Accordingly, treatment of cells with the demethylating agent 5-aza-2'-deoxycytidine alone or in combination with the histone deacetylase inhibitor trichostatin A resulted in the reactivation of CADM-2a expression. CADM-2a protein expression is significantly reduced in prostate cancer. Its expression is regulated in part by promoter methylation and implicates CADM-2 as a previously unrecognized tumor suppressor gene in a proportion of human prostate cancers. ©2010 AACR.

Bioinformatics and reanalysis of subtracted expressed sequence tags from the human ciliary body: Identification of novel biological functions.

PubMed

Escribano, Julio; Coca-Prados, Miguel

2002-08-28

The ciliary body is largely known for its major roles in the regulation of aqueous humor secretion, intraocular pressure, and accommodation of the lens. In this review article we applied bioinformatics to re-examine hundreds of expressed sequence tags (ESTs) previously isolated by subtractive hybridization from a human ciliary body library [1]. The DNA sequences of these clones have been recently added to the web site of NEIBank. DNA sequence comparisons of subtracted ESTs were performed against all entries in the last available release of the non-redundant database containing GenBank, EMBL, DDBJ and PDB sequences using the BlastN program accessed through NCBI's BLAST services on the internet (NCBI). Sequences were also compared and mapped using the Blast search program provided through the Internet by the Human Genome Project (UCSC). A total number of 284 independent ESTs were classified in 17 functional groups. Analysis of their relationships allowed to define the expression of five major groups of known genes: (i) protein synthesis, folding, secretion and degradation (20%); (ii) energy supply and biosynthesis (12%); (iii) contractility and cytoskeleton structure (6%); (iv) cellular signaling and cell cycle regulation (7%); and (v) nerve cell related tasks (2%), including neuropeptide processing and putative non-visual phototransduction and circadian rhythm control. The largest group contain unidentified sequences, a total of 105 sequences, accounting for 37% of ESTs. The unidentified sequences show similarity to genomic non-coding regions, or genes of unknown function. The most highly represented EST, correspond to myocilin, a gene involved in glaucoma. The data also confirms the secretory functions of the ciliary epithelium, and its high metabolism; the presence of a neuroendocrine peptidergic system presumably involved in the regulation of the intraocular pressure and/or aqueous humor secretion. Additional genes may be related to a non-visual phototransduction cascade and/or to circadian rhythms. Overall this initial group of subtracted ESTs can lead to uncover novel physiological functions of the ciliary body in normal and in disease, as well as novel candidate genes for ocular diseases.

STAT5A and STAT5B have opposite correlations with drug response gene expression

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

Lamba, V., E-mail: vlamba@ufl.edu; Jia, B.; Liang, F.

Introduction: STAT5A and STAT5B are important transcription factors that play a key role in regulation of several important physiological processes including proliferation, survival, mediation of responses to cytokines and in regulating gender differences in drug response genes such as the hepatic cytochrome P450s (CYPs) that are responsible for a large majority of drug metabolism reactions in the human body. STAT5A and STAT5b have a high degree of sequence homology and have been reported to have largely similar functions. Recent studies have, however, indicated that they can also often have distinct and unique roles in regulating gene expression. Objective: In thismore » study, we evaluated the association of STAT5A and STAT5B mRNA expression levels with those of several key hepatic cytochrome P450s (CYPs) and hepatic transcription factors (TFs) and evaluated the potential roles of STAT5A and 5b in mediating gender differences in these CYPs and TFs. Methods: Expression profiling for major hepatic CYP isoforms and transcription factors was performed using RNA sequencing (RNA-seq) in 102 human liver samples (57 female, 45 male). Real time PCR gene expression data for selected CYPs and TFs was available on a subset of 50 human liver samples (25 female, 25 male) and was used to validate the RNA-seq findings. Results: While STAT5A demonstrated significant negative correlation with expression levels of multiple hepatic transcription factors (including NR1I2 and HNF4A) and DMEs such as CYP3A4 and CYP2C19, STAT5B expression was observed to demonstrate positive associations with several CYPs and TFs analyzed. As STAT5A and STAT5B have been shown to be important in regulation of gender differences in CYPs, we also analyzed STAT5A and 5b associations with CYPs and TFs separately in males and females and observed gender dependent differential associations of STATs with several CYPs and TFs. Results from the real time PCR validation largely supported our RNA-seq findings. Conclusions: Using both RNA sequencing and real time PCR, we examined the association of STAT5A and STAT5B mRNA expression with CYP and TF gene expression. While STAT5A demonstrated significant negative correlations with expression levels of multiple hepatic TFs (including NR1I2 and HNF4α) and CYPs (eg. CYP3A4, CYP2C19), STAT5B expression was observed to demonstrate positive association with most of the CYPs/TFs analyzed suggesting that STAT5A and STAT5b have potentially different and distinct roles in regulating expression of hepatic drug response genes. Further studies are needed to elucidate the potential roles of STAT5A and 5b in regulation of CYPs/TFs and the potential implications of these findings.« less

Metallothionein from Wild Populations of the African Catfish Clarias gariepinus: From Sequence, Protein Expression and Metal Binding Properties to Transcriptional Biomarker of Metal Pollution.

PubMed

M'kandawire, Ethel; Mierek-Adamska, Agnieszka; Stürzenbaum, Stephen R; Choongo, Kennedy; Yabe, John; Mwase, Maxwell; Saasa, Ngonda; Blindauer, Claudia A

2017-07-18

Anthropogenic pollution with heavy metals is an on-going concern throughout the world, and methods to monitor release and impact of heavy metals are of high importance. With a view to probe its suitability as molecular biomarker of metal pollution, this study has determined a coding sequence for metallothionein of the African sharptooth catfish Clarias gariepinus . The gene product was recombinantly expressed in Escherichia coli in presence of Zn(II), Cd(II), or Cu, and characterised by Electrospray Ionisation Mass Spectrometry and elemental analysis. C. gariepinus MT displays typical features of fish MTs, including 20 conserved cysteines, and seven bound divalent cations (Zn(II) or Cd(II)) when saturated. Livers from wild C. gariepinus fish collected in all three seasons from four different sites on the Kafue River of Zambia were analysed for their metal contents and for MT expression levels by quantitative PCR. Significant correlations were found between Zn and Cu levels and MT expression in livers, with MT expression clearly highest at the most polluted site, Chililabombwe, which is situated in the Copperbelt region. Based on our findings, hepatic expression of MT from C. gariepinus may be further developed as a major molecular biomarker of heavy metal pollution resulting from mining activities in this region.

The Suppressor of AAC2 Lethality SAL1 Modulates Sensitivity of Heterologously Expressed Artemia ADP/ATP Carrier to Bongkrekate in Yeast

PubMed Central

Wysocka-Kapcinska, Monika; Torocsik, Beata; Turiak, Lilla; Tsaprailis, George; David, Cynthia L.; Hunt, Andrea M.; Vekey, Karoly; Adam-Vizi, Vera; Kucharczyk, Roza; Chinopoulos, Christos

2013-01-01

The ADP/ATP carrier protein (AAC) expressed in Artemia franciscana is refractory to bongkrekate. We generated two strains of Saccharomyces cerevisiae where AAC1 and AAC3 were inactivated and the AAC2 isoform was replaced with Artemia AAC containing a hemagglutinin tag (ArAAC-HA). In one of the strains the suppressor of ΔAAC2 lethality, SAL1, was also inactivated but a plasmid coding for yeast AAC2 was included, because the ArAACΔsal1Δ strain was lethal. In both strains ArAAC-HA was expressed and correctly localized to the mitochondria. Peptide sequencing of ArAAC expressed in Artemia and that expressed in the modified yeasts revealed identical amino acid sequences. The isolated mitochondria from both modified strains developed 85% of the membrane potential attained by mitochondria of control strains, and addition of ADP yielded bongkrekate-sensitive depolarizations implying acquired sensitivity of ArAAC-mediated adenine nucleotide exchange to this poison, independent from SAL1. However, growth of ArAAC-expressing yeasts in glycerol-containing media was arrested by bongkrekate only in the presence of SAL1. We conclude that the mitochondrial environment of yeasts relying on respiratory growth conferred sensitivity of ArAAC to bongkrekate in a SAL1-dependent manner. PMID:24073201

Expression profiles of mRNA and long noncoding RNA in the ovaries of letrozole-induced polycystic ovary syndrome rat model through deep sequencing.

PubMed

Fu, Lu-Lu; Xu, Ying; Li, Dan-Dan; Dai, Xiao-Wei; Xu, Xin; Zhang, Jing-Shun; Ming, Hao; Zhang, Xue-Ying; Zhang, Guo-Qing; Ma, Ya-Lan; Zheng, Lian-Wen

2018-05-30

Polycystic ovary syndrome (PCOS) is one of the most common endocrine disorders in reproductive-aged women. However, the exact pathophysiology of PCOS remains largely unclear. We performed deep sequencing to investigate the mRNA and long noncoding RNA (lncRNA) expression profiles in the ovarian tissues of letrozole-induced PCOS rat model and control rats. A total of 2147 mRNAs and 158 lncRNAs were differentially expressed between the PCOS models and control. Gene ontology analysis indicated that differentially expressed mRNAs were associated with biological adhesion, reproduction, and metabolic process. Pathway analysis results indicated that these aberrantly expressed mRNAs were related to several specific signaling pathways, including insulin resistance, steroid hormone biosynthesis, PPAR signaling pathway, cell adhesion molecules, autoimmune thyroid disease, and AMPK signaling pathway. The relative expression levels of mRNAs and lncRNAs were validated through qRT-PCR. LncRNA-miRNA-mRNA network was constructed to explore ceRNAs involved in the PCOS model and were also verified by qRTPCR experiment. These findings may provide insight into the pathogenesis of PCOS and clues to find key diagnostic and therapeutic roles of lncRNA in PCOS. Copyright © 2018 Elsevier B.V. All rights reserved.

A Review of Recommendations for Sequencing Receptive and Expressive Language Instruction

ERIC Educational Resources Information Center

Petursdottir, Anna Ingeborg; Carr, James E.

2011-01-01

We review recommendations for sequencing instruction in receptive and expressive language objectives in early and intensive behavioral intervention (EIBI) programs. Several books recommend completing receptive protocols before introducing corresponding expressive protocols. However, this recommendation has little empirical support, and some…

CpG methylation in human papillomavirus (HPV) type 31 long control region (LCR) in cervical infections associated with cytological abnormalities.

PubMed

László, Brigitta; Ferenczi, Annamária; Madar, László; Gyöngyösi, Eszter; Szalmás, Anita; Szakács, Levente; Veress, György; Kónya, József

2016-08-01

The mechanisms that regulate papillomavirus gene expression include DNA methylation. The transcription of papillomavirus oncogenes E6 and E7 is controlled by certain regulatory elements in the LCR, which include binding sites for the E2 protein, a viral regulator of oncogene expression. In HPV-31-infected exfoliated cervical cells, the CpG methylation of the entire LCR was determined by next-generation sequencing after bisulfite modification. Six of the 22 cases had methylated CpG sites in the HPV-31 LCR, including position 7479 and/or 7485, at the promoter distal E2 binding site, thus suggesting a potential regulatory mechanism for papillomavirus transcription.

Differential DNA methylation and transcription profiles in date palm roots exposed to salinity

PubMed Central

Al-Harrasi, Ibtisam; Al-Yahyai, Rashid

2018-01-01

As a salt-adaptive plant, the date palm (Phoenix dactylifera L.) requires a suitable mechanism to adapt to the stress of saline soils. There is growing evidence that DNA methylation plays an important role in regulating gene expression in response to abiotic stresses, including salinity. Thus, the present study sought to examine the differential methylation status that occurs in the date palm genome when plants are exposed to salinity, and to identify salinity responsive genes that are regulated by DNA methylation. To achieve these, whole-genome bisulfite sequencing (WGBS) was employed and mRNA was sequenced from salinity-treated and untreated roots. The WGBS analysis included 324,987,795 and 317,056,091 total reads of the control and the salinity-treated samples, respectively. The analysis covered about 81% of the total genomic DNA with about 40% of mapping efficiency of the sequenced reads and an average read depth of 17-fold coverage per DNA strand, and with a bisulfite conversion rate of around 99%. The level of methylation within the differentially methylated regions (DMRs) was significantly (p < 0.05, FDR ≤ 0.05) increased in response to salinity specifically at the mCHG and mCHH sequence contexts. Consistently, the mass spectrometry and the enzyme-linked immunosorbent assay (ELISA) showed that there was a significant (p < 0.05) increase in the global DNA methylation in response to salinity. mRNA sequencing revealed the presence of 6,405 differentially regulated genes with a significant value (p < 0.001, FDR ≤ 0.05) in response to salinity. Integration of high-resolution methylome and transcriptome analyses revealed a negative correlation between mCG methylation located within the promoters and the gene expression, while a positive correlation was noticed between mCHG/mCHH methylation rations and gene expression specifically when plants grew under control conditions. Therefore, the methylome and transcriptome relationships vary based on the methylated sequence context, the methylated region within the gene, the protein-coding ability of the gene, and the salinity treatment. These results provide insights into interplay among DNA methylation and gene expression, and highlight the effect of salinity on the nature of this relationship, which may involve other genetic and epigenetic players under salt stress conditions. The results obtained from this project provide the first draft map of the differential methylome and transcriptome of date palm when exposed to an abiotic stress. PMID:29352281

Differential DNA methylation and transcription profiles in date palm roots exposed to salinity.

PubMed

Al-Harrasi, Ibtisam; Al-Yahyai, Rashid; Yaish, Mahmoud W

2018-01-01

As a salt-adaptive plant, the date palm (Phoenix dactylifera L.) requires a suitable mechanism to adapt to the stress of saline soils. There is growing evidence that DNA methylation plays an important role in regulating gene expression in response to abiotic stresses, including salinity. Thus, the present study sought to examine the differential methylation status that occurs in the date palm genome when plants are exposed to salinity, and to identify salinity responsive genes that are regulated by DNA methylation. To achieve these, whole-genome bisulfite sequencing (WGBS) was employed and mRNA was sequenced from salinity-treated and untreated roots. The WGBS analysis included 324,987,795 and 317,056,091 total reads of the control and the salinity-treated samples, respectively. The analysis covered about 81% of the total genomic DNA with about 40% of mapping efficiency of the sequenced reads and an average read depth of 17-fold coverage per DNA strand, and with a bisulfite conversion rate of around 99%. The level of methylation within the differentially methylated regions (DMRs) was significantly (p < 0.05, FDR ≤ 0.05) increased in response to salinity specifically at the mCHG and mCHH sequence contexts. Consistently, the mass spectrometry and the enzyme-linked immunosorbent assay (ELISA) showed that there was a significant (p < 0.05) increase in the global DNA methylation in response to salinity. mRNA sequencing revealed the presence of 6,405 differentially regulated genes with a significant value (p < 0.001, FDR ≤ 0.05) in response to salinity. Integration of high-resolution methylome and transcriptome analyses revealed a negative correlation between mCG methylation located within the promoters and the gene expression, while a positive correlation was noticed between mCHG/mCHH methylation rations and gene expression specifically when plants grew under control conditions. Therefore, the methylome and transcriptome relationships vary based on the methylated sequence context, the methylated region within the gene, the protein-coding ability of the gene, and the salinity treatment. These results provide insights into interplay among DNA methylation and gene expression, and highlight the effect of salinity on the nature of this relationship, which may involve other genetic and epigenetic players under salt stress conditions. The results obtained from this project provide the first draft map of the differential methylome and transcriptome of date palm when exposed to an abiotic stress.

Compositions and methods for xylem-specific expression in plant cells

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

Han, Kyung-Hwan; Ko, Jae-Heung

The invention provides promoter sequences that regulate specific expression of operably linked sequences in developing xylem cells and/or in developing xylem tissue. The developing xylem-specific sequences are exemplified by the DX5, DX8, DX11, and DX15 promoters, portions thereof, and homologs thereof. The invention further provides expression vectors, cells, tissues and plants that contain the invention's sequences. The compositions of the invention and methods of using them are useful in, for example, improving the quantity (biomass) and/or the quality (wood density, lignin content, sugar content etc.) of expressed biomass feedstock products that may be used for bioenergy, biorefinary, and generating woodmore » products such as pulp, paper, and solid wood.« less

Mammalian cDNA Library from the NIH Mammalian Gene Collection (MGC) | Office of Cancer Genomics

Cancer.gov

The MGC provides the research community full-length clones for most of the defined (as of 2006) human and mouse genes, along with selected clones of cow and rat genes. Clones were designed to allow easy transfer of the ORF sequences into nearly any type of expression vector. MGC provides protein ‘expression-ready’ clones for each of the included human genes. MGC is part of the ORFeome Collaboration (OC).

Natural selection in avian protein-coding genes expressed in brain.

PubMed

Axelsson, Erik; Hultin-Rosenberg, Lina; Brandström, Mikael; Zwahlén, Martin; Clayton, David F; Ellegren, Hans

2008-06-01

The evolution of birds from theropod dinosaurs took place approximately 150 million years ago, and was associated with a number of specific adaptations that are still evident among extant birds, including feathers, song and extravagant secondary sexual characteristics. Knowledge about the molecular evolutionary background to such adaptations is lacking. Here, we analyse the evolution of > 5000 protein-coding gene sequences expressed in zebra finch brain by comparison to orthologous sequences in chicken. Mean d(N)/d(S) is 0.085 and genes with their maximal expression in the eye and central nervous system have the lowest mean d(N)/d(S) value, while those expressed in digestive and reproductive tissues exhibit the highest. We find that fast-evolving genes (those which have higher than expected rate of nonsynonymous substitution, indicative of adaptive evolution) are enriched for biological functions such as fertilization, muscle contraction, defence response, response to stress, wounding and endogenous stimulus, and cell death. After alignment to mammalian orthologues, we identify a catalogue of 228 genes that show a significantly higher rate of protein evolution in the two bird lineages than in mammals. These accelerated bird genes, representing candidates for avian-specific adaptations, include genes implicated in vocal learning and other cognitive processes. Moreover, colouration genes evolve faster in birds than in mammals, which may have been driven by sexual selection for extravagant plumage characteristics.

PSI:Biology-Materials Repository: A Biologist’s Resource for Protein Expression Plasmids

PubMed Central

Cormier, Catherine Y.; Park, Jin G.; Fiacco, Michael; Steel, Jason; Hunter, Preston; Kramer, Jason; Singla, Rajeev; LaBaer, Joshua

2011-01-01

The Protein Structure Initiative:Biology-Materials Repository (PSI:Biology-MR; MR; http://psimr.asu.edu) sequence-verifies, annotates, stores, and distributes the protein expression plasmids and vectors created by the Protein Structure Initiative (PSI). The MR has developed an informatics and sample processing pipeline that manages this process for thousands of samples per month from nearly a dozen PSI centers. DNASU (http://dnasu.asu.edu), a freely searchable database, stores the plasmid annotations, which include the full-length sequence, vector information, and associated publications for over 130,000 plasmids created by our laboratory, by the PSI and other consortia, and by individual laboratories for distribution to researchers worldwide. Each plasmid links to external resources, including the PSI Structural Biology Knowledgebase (http://sbkb.org), which facilitates cross-referencing of a particular plasmid to additional protein annotations and experimental data. To expedite and simplify plasmid requests, the MR uses an expedited material transfer agreement (EP-MTA) network, where researchers from network institutions can order and receive PSI plasmids without institutional delays. Currently over 39,000 protein expression plasmids and 78 empty vectors from the PSI are available upon request from DNASU. Overall, the MR’s repository of expression-ready plasmids, its automated pipeline, and the rapid process for receiving and distributing these plasmids more effectively allows the research community to dissect the biological function of proteins whose structures have been studied by the PSI. PMID:21360289

The use of a viral 2A sequence for the simultaneous over-expression of both the vgf gene and enhanced green fluorescent protein (eGFP) in vitro and in vivo.

PubMed

Lewis, Jo E; Brameld, John M; Hill, Phil; Barrett, Perry; Ebling, Francis J P; Jethwa, Preeti H

2015-12-30

The viral 2A sequence has become an attractive alternative to the traditional internal ribosomal entry site (IRES) for simultaneous over-expression of two genes and in combination with recombinant adeno-associated viruses (rAAV) has been used to manipulate gene expression in vitro. To develop a rAAV construct in combination with the viral 2A sequence to allow long-term over-expression of the vgf gene and fluorescent marker gene for tracking of the transfected neurones in vivo. Transient transfection of the AAV plasmid containing the vgf gene, viral 2A sequence and eGFP into SH-SY5Y cells resulted in eGFP fluorescence comparable to a commercially available reporter construct. This increase in fluorescent cells was accompanied by an increase in VGF mRNA expression. Infusion of the rAAV vector containing the vgf gene, viral 2A sequence and eGFP resulted in eGFP fluorescence in the hypothalamus of both mice and Siberian hamsters, 32 weeks post infusion. In situ hybridisation confirmed that the location of VGF mRNA expression in the hypothalamus corresponded to the eGFP pattern of fluorescence. The viral 2A sequence is much smaller than the traditional IRES and therefore allowed over-expression of the vgf gene with fluorescent tracking without compromising viral capacity. The use of the viral 2A sequence in the AAV plasmid allowed the simultaneous expression of both genes in vitro. When used in combination with rAAV it resulted in long-term over-expression of both genes at equivalent locations in the hypothalamus of both Siberian hamsters and mice, without any adverse effects. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

The Medicago sativa gene index 1.2: a web-accessible gene expression atlas for investigating expression differences between Medicago sativa subspecies.

PubMed

O'Rourke, Jamie A; Fu, Fengli; Bucciarelli, Bruna; Yang, S Sam; Samac, Deborah A; Lamb, JoAnn F S; Monteros, Maria J; Graham, Michelle A; Gronwald, John W; Krom, Nick; Li, Jun; Dai, Xinbin; Zhao, Patrick X; Vance, Carroll P

2015-07-07

Alfalfa (Medicago sativa L.) is the primary forage legume crop species in the United States and plays essential economic and ecological roles in agricultural systems across the country. Modern alfalfa is the result of hybridization between tetraploid M. sativa ssp. sativa and M. sativa ssp. falcata. Due to its large and complex genome, there are few genomic resources available for alfalfa improvement. A de novo transcriptome assembly from two alfalfa subspecies, M. sativa ssp. sativa (B47) and M. sativa ssp. falcata (F56) was developed using Illumina RNA-seq technology. Transcripts from roots, nitrogen-fixing root nodules, leaves, flowers, elongating stem internodes, and post-elongation stem internodes were assembled into the Medicago sativa Gene Index 1.2 (MSGI 1.2) representing 112,626 unique transcript sequences. Nodule-specific and transcripts involved in cell wall biosynthesis were identified. Statistical analyses identified 20,447 transcripts differentially expressed between the two subspecies. Pair-wise comparisons of each tissue combination identified 58,932 sequences differentially expressed in B47 and 69,143 sequences differentially expressed in F56. Comparing transcript abundance in floral tissues of B47 and F56 identified expression differences in sequences involved in anthocyanin and carotenoid synthesis, which determine flower pigmentation. Single nucleotide polymorphisms (SNPs) unique to each M. sativa subspecies (110,241) were identified. The Medicago sativa Gene Index 1.2 increases the expressed sequence data available for alfalfa by ninefold and can be expanded as additional experiments are performed. The MSGI 1.2 transcriptome sequences, annotations, expression profiles, and SNPs were assembled into the Alfalfa Gene Index and Expression Database (AGED) at http://plantgrn.noble.org/AGED/ , a publicly available genomic resource for alfalfa improvement and legume research.

Low-coverage single-cell mRNA sequencing reveals cellular heterogeneity and activated signaling pathways in developing cerebral cortex.

PubMed

Pollen, Alex A; Nowakowski, Tomasz J; Shuga, Joe; Wang, Xiaohui; Leyrat, Anne A; Lui, Jan H; Li, Nianzhen; Szpankowski, Lukasz; Fowler, Brian; Chen, Peilin; Ramalingam, Naveen; Sun, Gang; Thu, Myo; Norris, Michael; Lebofsky, Ronald; Toppani, Dominique; Kemp, Darnell W; Wong, Michael; Clerkson, Barry; Jones, Brittnee N; Wu, Shiquan; Knutsson, Lawrence; Alvarado, Beatriz; Wang, Jing; Weaver, Lesley S; May, Andrew P; Jones, Robert C; Unger, Marc A; Kriegstein, Arnold R; West, Jay A A

2014-10-01

Large-scale surveys of single-cell gene expression have the potential to reveal rare cell populations and lineage relationships but require efficient methods for cell capture and mRNA sequencing. Although cellular barcoding strategies allow parallel sequencing of single cells at ultra-low depths, the limitations of shallow sequencing have not been investigated directly. By capturing 301 single cells from 11 populations using microfluidics and analyzing single-cell transcriptomes across downsampled sequencing depths, we demonstrate that shallow single-cell mRNA sequencing (~50,000 reads per cell) is sufficient for unbiased cell-type classification and biomarker identification. In the developing cortex, we identify diverse cell types, including multiple progenitor and neuronal subtypes, and we identify EGR1 and FOS as previously unreported candidate targets of Notch signaling in human but not mouse radial glia. Our strategy establishes an efficient method for unbiased analysis and comparison of cell populations from heterogeneous tissue by microfluidic single-cell capture and low-coverage sequencing of many cells.

Sequence Requirements of the 5-Enolpyruvylshikimate-3-phosphate Synthase 5[prime]-Upstream Region for Tissue-Specific Expression in Flowers and Seedlings.

PubMed Central

Benfey, PN; Takatsuji, H; Ren, L; Shah, DM; Chua, NH

1990-01-01

We have analyzed expression from deletion derivatives of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) 5[prime]-upstream region in transgenic petunia flowers and seedlings. In seedlings, expression was strongest in root cortex cells and in trichomes. High-level expression in petals and in seedling roots was conferred by large (>500 base-pair) stretches of sequence, but was lost when smaller fragments were analyzed individually. This apparent requirement for extensive sequence suggests that combinations of cis-elements that are widely separated control tissue-specific expression from the EPSPS promoter. We have also used the high-level, petal-specific expression of the EPSPS promoter to change petal color in two mutant petunia lines. PMID:12354968

Characterisation of microRNAs from apple (Malus domestica 'Royal Gala') vascular tissue and phloem sap.

PubMed

Varkonyi-Gasic, Erika; Gould, Nick; Sandanayaka, Manoharie; Sutherland, Paul; MacDiarmid, Robin M

2010-08-04

Plant microRNAs (miRNAs) are a class of small, non-coding RNAs that play an important role in development and environmental responses. Hundreds of plant miRNAs have been identified to date, mainly from the model species for which there are available genome sequences. The current challenge is to characterise miRNAs from plant species with agricultural and horticultural importance, to aid our understanding of important regulatory mechanisms in crop species and enable improvement of crops and rootstocks. Based on the knowledge that many miRNAs occur in large gene families and are highly conserved among distantly related species, we analysed expression of twenty-one miRNA sequences in different tissues of apple (Malus x domestica 'Royal Gala'). We identified eighteen sequences that are expressed in at least one of the tissues tested. Some, but not all, miRNAs expressed in apple tissues including the phloem tissue were also detected in the phloem sap sample derived from the stylets of woolly apple aphids. Most of the miRNAs detected in apple phloem sap were also abundant in the phloem sap of herbaceous species. Potential targets for apple miRNAs were identified that encode putative proteins shown to be targets of corresponding miRNAs in a number of plant species. Expression patterns of potential targets were analysed and correlated with expression of corresponding miRNAs. This study validated tissue-specific expression of apple miRNAs that target genes responsible for plant growth, development, and stress response. A subset of characterised miRNAs was also present in the apple phloem translocation stream. A comparative analysis of phloem miRNAs in herbaceous species and woody perennials will aid our understanding of non-cell autonomous roles of miRNAs in plants.

Characterisation of microRNAs from apple (Malus domestica 'Royal Gala') vascular tissue and phloem sap

PubMed Central

2010-01-01

Background Plant microRNAs (miRNAs) are a class of small, non-coding RNAs that play an important role in development and environmental responses. Hundreds of plant miRNAs have been identified to date, mainly from the model species for which there are available genome sequences. The current challenge is to characterise miRNAs from plant species with agricultural and horticultural importance, to aid our understanding of important regulatory mechanisms in crop species and enable improvement of crops and rootstocks. Results Based on the knowledge that many miRNAs occur in large gene families and are highly conserved among distantly related species, we analysed expression of twenty-one miRNA sequences in different tissues of apple (Malus x domestica 'Royal Gala'). We identified eighteen sequences that are expressed in at least one of the tissues tested. Some, but not all, miRNAs expressed in apple tissues including the phloem tissue were also detected in the phloem sap sample derived from the stylets of woolly apple aphids. Most of the miRNAs detected in apple phloem sap were also abundant in the phloem sap of herbaceous species. Potential targets for apple miRNAs were identified that encode putative proteins shown to be targets of corresponding miRNAs in a number of plant species. Expression patterns of potential targets were analysed and correlated with expression of corresponding miRNAs. Conclusions This study validated tissue-specific expression of apple miRNAs that target genes responsible for plant growth, development, and stress response. A subset of characterised miRNAs was also present in the apple phloem translocation stream. A comparative analysis of phloem miRNAs in herbaceous species and woody perennials will aid our understanding of non-cell autonomous roles of miRNAs in plants. PMID:20682080

Curcumin activates human glutathione S-transferase P1 expression through antioxidant response element.

PubMed

Nishinaka, Toru; Ichijo, Yusuke; Ito, Maki; Kimura, Masayoshi; Katsuyama, Masato; Iwata, Kazumi; Miura, Takeshi; Terada, Tomoyuki; Yabe-Nishimura, Chihiro

2007-05-15

Curcumin is a plant-derived diferuloylmethane compound extracted from Curcuma longa, possessing antioxidative and anticarcinogenic properties. Antioxidants and oxidative stress are known to induce the expression of certain classes of detoxification enzymes. Since the upregulation of detoxifying enzymes affects the drug metabolism and cell defense system, it is important to understand the gene regulation by such agents. In this study, we demonstrated that curcumin could induce the expression of human glutathione S-transferase P1 (GSTP1). In HepG2 cells treated with 20muM curcumin, the level of GSTP1 mRNA was significantly increased. In luciferase reporter assays, curcumin augmented the promoter activity of a reporter construct carrying 336bp upstream of the 5'-flanking region of the GSTP1 gene. Mutation analyses revealed that the region including antioxidant response element (ARE), which overlaps AP1 in sequence, was essential to the response to curcumin. While the introduction of a wild-type Nrf2 expression construct augmented the promoter activity of the GSTP1 gene, co-expression of a dominant-negative Nrf2 abolished the responsiveness to curcumin. In addition, curcumin activated the expression of the luciferase gene from a reporter construct carrying multiple ARE consensus sequences but not one with multiple AP1 sites. In a gel mobility shift assay with an oligonucleotide with GSTP1 ARE, an increase in the amount of the binding complex was observed in the nuclear extracts of curcumin-treated HepG2 cells. These results suggested that ARE is the primary sequence for the curcumin-induced transactivation of the GSTP1 gene. The induction of GSTP1 may be one of the mechanisms underlying the multiple actions of curcumin.

NHE3 in an ancestral vertebrate: primary sequence, distribution, localization, and function in gills.

PubMed

Choe, Keith P; Kato, Akira; Hirose, Shigehisa; Plata, Consuelo; Sindic, Aleksandra; Romero, Michael F; Claiborne, J B; Evans, David H

2005-11-01

In mammals, the Na+/H+ exchanger 3 (NHE3) is expressed with Na+/K+-ATPase in renal proximal tubules, where it secretes H+ and absorbs Na+ to maintain blood pH and volume. In elasmobranchs (sharks, skates, and stingrays), the gills are the dominant site of pH and osmoregulation. This study was conducted to determine whether epithelial NHE homologs exist in elasmobranchs and, if so, to localize their expression in gills and determine whether their expression is altered by environmental salinity or hypercapnia. Degenerate primers and RT-PCR were used to deduce partial sequences of mammalian NHE2 and NHE3 homologs from the gills of the euryhaline Atlantic stingray (Dasyatis sabina). Real-time PCR was then used to demonstrate that mRNA expression of the NHE3 homolog increased when stingrays were transferred to low salinities but not during hypercapnia. Expression of the NHE2 homolog did not change with either treatment. Rapid amplification of cDNA was then used to deduce the complete sequence of a putative NHE3. The 2,744-base pair cDNA includes a coding region for a 2,511-amino acid protein that is 70% identical to human NHE3 (SLC9A3). Antisera generated against the carboxyl tail of the putative stingray NHE3 labeled the apical membranes of Na+/K+-ATPase-rich epithelial cells, and acclimation to freshwater caused a redistribution of labeling in the gills. This study provides the first NHE3 cloned from an elasmobranch and is the first to demonstrate an increase in gill NHE3 expression during acclimation to low salinities, suggesting that NHE3 can absorb Na+ from ion-poor environments.

Subgenome-specific assembly of vitamin E biosynthesis genes and expression patterns during seed development provide insight into the evolution of oat genome.

PubMed

Gutierrez-Gonzalez, Juan J; Garvin, David F

2016-11-01

Vitamin E is essential for humans and thus must be a component of a healthy diet. Among the cereal grains, hexaploid oats (Avena sativa L.) have high vitamin E content. To date, no gene sequences in the vitamin E biosynthesis pathway have been reported for oats. Using deep sequencing and orthology-guided assembly, coding sequences of genes for each step in vitamin E synthesis in oats were reconstructed, including resolution of the sequences of homeologs. Three homeologs, presumably representing each of the three oat subgenomes, were identified for the main steps of the pathway. Partial sequences, likely representing pseudogenes, were recovered in some instances as well. Pairwise comparisons among homeologs revealed that two of the three putative subgenome-specific homeologs are almost identical for each gene. Synonymous substitution rates indicate the time of divergence of the two more similar subgenomes from the distinct one at 7.9-8.7 MYA, and a divergence between the similar subgenomes from a common ancestor 1.1 MYA. A new proposed evolutionary model for hexaploid oat formation is discussed. Homeolog-specific gene expression was quantified during oat seed development and compared with vitamin E accumulation. Homeolog expression largely appears to be similar for most of genes; however, for some genes, homoeolog-specific transcriptional bias was observed. The expression of HPPD, as well as certain homoeologs of VTE2 and VTE4, is highly correlated with seed vitamin E accumulation. Our findings expand our understanding of oat genome evolution and will assist efforts to modify vitamin E content and composition in oats. Published 2016. This article is a U.S. Government work and is in the public domain in the USA. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

miRNAs involved in the development and differentiation of fertile and sterile flowers in Viburnum macrocephalum f. keteleeri.

PubMed

Li, Weixing; He, Zhichong; Zhang, Li; Lu, Zhaogeng; Xu, Jing; Cui, Jiawen; Wang, Li; Jin, Biao

2017-10-13

Sterile and fertile flowers are important evolutionary developmental phenotypes in angiosperm flowers. The development of floral organs, critical in angiosperm reproduction, is regulated by microRNAs (miRNAs). However, the mechanisms underpinning the miRNA regulation of the differentiation and development of sterile and fertile flowers remain unclear. Here, based on investigations of the morphological differences between fertile and sterile flowers, we used high-throughput sequencing to characterize the miRNAs in the differentiated floral organs of Viburnum macrocephalum f. keteleeri. We identified 49 known miRNAs and 67 novel miRNAs by small RNA (sRNA) sequencing and bioinformatics analysis, and 17 of these known and novel miRNA precursors were validated by polymerase chain reaction (PCR) and Sanger sequencing. Furthermore, by comparing the sequencing results of two sRNA libraries, we found that 30 known and 39 novel miRNA sequences were differentially expressed, and 35 were upregulated and 34 downregulated in sterile compared with fertile flowers. Combined with their predicted targets, the potential roles of miRNAs in V. macrocephalum f. keteleeri flowers include involvement in floral organogenesis, cell proliferation, hormonal pathways, and stress responses. miRNA precursors and targets were further validated by quantitative real-time PCR (qRT-PCR). Specifically, miR156a-5p, miR156g, and miR156j expression levels were significantly higher in fertile flowers than in sterile flowers, while SPL genes displayed the opposite expression pattern. Considering that the targets of miR156 are predicted to be SPL genes, we propose that miR156 may be involved in the regulation of stamen development in V. macrocephalum f. keteleeri. We identified miRNAs differentially expressed between fertile and sterile flowers in V. macrocephalum f. keteleeri and provided new insights into the important regulatory roles of miRNAs in the differentiation and development of fertile and sterile flowers.

Gut transcriptome of replete adult female cattle ticks, Rhipicephalus (Boophilus) microplus, feeding upon a Babesia bovis-infected bovine host.

PubMed

Heekin, Andrew M; Guerrero, Felix D; Bendele, Kylie G; Saldivar, Leo; Scoles, Glen A; Dowd, Scot E; Gondro, Cedric; Nene, Vishvanath; Djikeng, Appolinaire; Brayton, Kelly A

2013-09-01

As it feeds upon cattle, Rhipicephalus (Boophilus) microplus is capable of transmitting a number of pathogenic organisms, including the apicomplexan hemoparasite Babesia bovis, a causative agent of bovine babesiosis. The R. microplus female gut transcriptome was studied for two cohorts: adult females feeding on a bovine host infected with B. bovis and adult females feeding on an uninfected bovine. RNA was purified and used to generate a subtracted cDNA library from B. bovis-infected female gut, and 4,077 expressed sequence tags (ESTs) were sequenced. Gene expression was also measured by a microarray designed from the publicly available R. microplus gene index: BmiGI Version 2. We compared gene expression in the tick gut from females feeding upon an uninfected bovine to gene expression in tick gut from females feeding upon a splenectomized bovine infected with B. bovis. Thirty-three ESTs represented on the microarray were expressed at a higher level in female gut samples from the ticks feeding upon a B. bovis-infected calf compared to expression levels in female gut samples from ticks feeding on an uninfected calf. Forty-three transcripts were expressed at a lower level in the ticks feeding upon B. bovis-infected female guts compared with expression in female gut samples from ticks feeding on the uninfected calf. These array data were used as initial characterization of gene expression associated with the infection of R. microplus by B. bovis.

Tandem alternative polyadenylation events of genes in non-eosinophilic nasal polyp tissue identified by high-throughput sequencing analysis

PubMed Central

TIAN, PENG; LI, JIE; LIU, XIANG; LI, YUXI; CHEN, MEIHENG; MA, YUN; ZHENG, YI QING; FU, YONGGUI; ZOU, HUA

2014-01-01

Nasal polyps (NP) is highly associated with the disorder of immune cells. Alternative polyadenylation (APA) produces mRNA isoforms with different length of 3′-untranslated region (UTR) and regulates gene expression. It has been proven that this APA-mediated regulation of 3′UTR length is an immune-associated phenomenon. The aim of this study was to investigate the genome-wide alternative tandem 3′UTR length switching events in non-eosinophilic nasal polyp tissue. Thirteen patients diagnosed as having non-eosinophilic nasal polyps were included in this study. Nasal polyp tissue and control mucosa were collected during surgery. The 3′ end library of cDNA was constructed. The recovered libraries were sequenced with second sequencing technology, and the sequencing data were analyzed by an in-house bioinformatics pipeline. Tandem 3′UTR length switching between samples was detected by a test of linear trend alternative to independence. We found a significant alteration in the tandem 3′UTR length in 1,920 genes in nasal polyp samples. Functional annotation results showed that several gene ontology (GO) terms were enriched in the list of genes with switched APA sites, including regulation of transcription, macromolecule catabolic localization and mRNA processing. The results suggested that APA-mediated alternative 3′UTR regulation plays an important role in the post-transcriptional regulation of gene expression in non-eosinophilic nasal polyps. PMID:24715051

Cloning, sequencing, and expression of interferon-γ from elk in North America

USGS Publications Warehouse

Sweeney, Steven J.; Emerson, Carlene; Eriks, Inge S.

2001-01-01

Eradication of Mycobacterium bovis relies on accurate detection of infected animals, including potential domestic and wildlife reservoirs. Available diagnostic tests lack the sensitivity and specificity necessary for accurate detection, particularly in infected wildlife populations. Recently, an in vitro diagnostic test for cattle which measures plasma interferon-gamma (IFN-γ) levels in blood following in vitro incubation with M. bovis purified protein derivative has been enveloped. This test appears to have increased sensitivity over traditional testing. Unfortunately, it does not detect IFN-γ from Cervidae. To begin to address this problem, the IFN-γ gene from elk (Cervus elaphus) was cloned, sequenced, expressed, and characterized. cDNA was cloned from mitogen stimulated peripheral blood mononuclear cells. The predicted amino acid (aa) sequence was compared to known sequences from cattle, sheep, goats, red deer (Cervus elaphus), humans, and mice. Biological activity of the recombinant elk IFN-γ (rElkIFN-γ) was confirmed in a vesicular stomatitis virus cytopathic effect reduction assay. Production of monoclonal antibodies to IFN-γ epitopes conserved between ruminant species could provide an important tool for the development of reliable, practical diagnostic assays for detection of a delayed type hypersensitivity response to a variety of persistent infectious agents in ruminants, including M. bovis and Brucella abortus. Moreover, development of these reagents will aid investigators in studies to explore immunological responses of elk that are associated with resistance to infectious diseases.

Rose spring dwarf-associated virus has RNA structural and gene-expression features like those of Barley yellow dwarf virus

PubMed Central

Salem, Nida’ M.; Miller, W. Allen; Rowhani, Adib; Golino, Deborah A.; Moyne, Anne-Laure; Falk, Bryce W.

2015-01-01

We determined the complete nucleotide sequence of the Rose spring dwarf-associated virus (RSDaV) genomic RNA (GenBank accession no. EU024678) and compared its predicted RNA structural characteristics affecting gene expression. A cDNA library was derived from RSDaV double-stranded RNAs (dsRNAs) purified from infected tissue. Nucleotide sequence analysis of the cloned cDNAs, plus for clones generated by 5′- and 3′-RACE showed the RSDaV genomic RNA to be 5,808 nucleotides. The genomic RNA contains five major open reading frames (ORFs), and three small ORFs in the 3′-terminal 800 nucleotides, typical for viruses of genus Luteovirus in the family Luteoviridae. Northern blot hybridization analysis revealed the genomic RNA and two prominent subgenomic RNAs of approximately 3 kb and 1 kb. Putative 5′ ends of the sgRNAs were predicted by identification of conserved sequences and secondary structures which resembled the Barley yellow dwarf virus (BYDV) genomic RNA 5′ end and subgenomic RNA promoter sequences. Secondary structures of the BYDV-like ribosomal frameshift elements and cap-independent translation elements, including long-distance base pairing spanning four kb were identified. These contain similarities but also informative differences with the BYDV structures, including a strikingly different structure predicted for the 3′ cap-independent translation element. These analyses of the RSDaV genomic RNA show more complexity for the RNA structural elements for members of the Luteoviridae. PMID:18329064

Rose spring dwarf-associated virus has RNA structural and gene-expression features like those of Barley yellow dwarf virus.

PubMed

Salem, Nida' M; Miller, W Allen; Rowhani, Adib; Golino, Deborah A; Moyne, Anne-Laure; Falk, Bryce W

2008-06-05

We determined the complete nucleotide sequence of the Rose spring dwarf-associated virus (RSDaV) genomic RNA (GenBank accession no. EU024678) and compared its predicted RNA structural characteristics affecting gene expression. A cDNA library was derived from RSDaV double-stranded RNAs (dsRNAs) purified from infected tissue. Nucleotide sequence analysis of the cloned cDNAs, plus for clones generated by 5'- and 3'-RACE showed the RSDaV genomic RNA to be 5808 nucleotides. The genomic RNA contains five major open reading frames (ORFs), and three small ORFs in the 3'-terminal 800 nucleotides, typical for viruses of genus Luteovirus in the family Luteoviridae. Northern blot hybridization analysis revealed the genomic RNA and two prominent subgenomic RNAs of approximately 3 kb and 1 kb. Putative 5' ends of the sgRNAs were predicted by identification of conserved sequences and secondary structures which resembled the Barley yellow dwarf virus (BYDV) genomic RNA 5' end and subgenomic RNA promoter sequences. Secondary structures of the BYDV-like ribosomal frameshift elements and cap-independent translation elements, including long-distance base pairing spanning four kb were identified. These contain similarities but also informative differences with the BYDV structures, including a strikingly different structure predicted for the 3' cap-independent translation element. These analyses of the RSDaV genomic RNA show more complexity for the RNA structural elements for members of the Luteoviridae.

Transfection and heat-inducible expression of molluscan promoter-luciferase reporter gene constructs in the Biomphalaria glabrata embryonic snail cell line.

PubMed

Yoshino, T P; Wu, X J; Liu, H D

1998-09-01

Studies were initiated to begin developing a genetic transformation system for cells derived from the freshwater gastropod, Biomphalaria glabrata, an intermediate host of the human blood fluke Schistosoma mansoni. Using a 70-kD heat-shock protein (HSP70) cDNA probe obtained from the B. glabrata embryonic (Bge) cell line, we cloned from Bge cells a complete HSP70 gene including a 1-kb genomic DNA fragment in its 5'-flanking region containing sequences indicative of a HSP promoter. Identified in the 5'-half (416 nucleotides) of this genomic fragment were TATA and CAAT boxes, two putative transcription initiation sites, and a series of palindromic DNA repeats with shared homology to the heat-shock element consensus sequence (Bge HSP70(0.5k) promoter). The 3'-half of this upstream flanking region was comprised of a 508-base intron located immediately 5' of the ATG start codon. To determine the functionality of the putative snail promoter sequence, Bge HSP promoter/luciferase (Luc) reporter gene constructs were introduced into Bge cells by N-(1-(2,3-dioleoyloxy) propyl)-N,N,N-trimethylammonium methylsulfate (DOTAP)-mediated transfection methods, and assayed for Luc activity 48 hr following a 1.5-hr heat-shock treatment (40 degrees C). Compared with control vectors or the Bge HSP70(0.5k/1.0k) promoter constructs at 26 degrees C, a 10- to 300-fold increase in Luc expression was obtained only in the Bge HSP70 promoter/Luc-transfected cells following heat-shock. Results of transfection experiments demonstrate that the Bge HSP70(0.5k) DNA segment contains appropriate promoter sequences for driving temperature-inducible gene expression in the Bge snail cell line. This report represents the first isolation and functional characterization of an inducible promoter from a freshwater gastropod mollusc. Successful transient expression of a foreign reporter gene in Bge cells using a homologous, inducible promoter sequence now paves the way for development of methods for stable integration and expression of snail genes of interest into the Bge cell line.

Gene therapy for prostate cancer: where are we now?

PubMed

Steiner, M S; Gingrich, J R

2000-10-01

The ability to recombine specifically and alter DNA sequences followed by techniques to transfer these sequences or even whole genes into normal and diseased cells has revolutionized medical research and ushered the clinicians of today into the age of gene therapy. We provide urologists a review of relevant background information, outline current treatment strategies and clinical trials, and delineate current challenges facing the field of gene therapy for advanced prostate cancer. We comprehensively reviewed the literature, including PubMed and recent abstract proceedings from national meetings, relevant to gene therapy and advanced prostate cancer. We selected for review literature representative of the principal scientific background for current gene therapy strategies and National Institutes of Health Recombinant DNA Advisory Committee approved clinical trials. Current prostate cancer gene therapy strategies include correcting aberrant gene expression, exploiting programmed cell death pathways, targeting critical cell biological functions, introducing toxic or cell lytic suicide genes, enhancing the immune system antitumor response and combining treatment with conventional cytotoxic chemotherapy or radiation therapy. Many challenges lie ahead for gene therapy, including improving DNA transfer efficiency to cells locally and at distant sites, enhancing levels of gene expression and overcoming immune responses that limit the time that genes are expressed. Nevertheless, despite these current challenges it is almost certain that gene therapy will be part of the urological armamentarium against prostate cancer in this century.

Viruses and miRNAs: More Friends than Foes.

PubMed

Bruscella, Patrice; Bottini, Silvia; Baudesson, Camille; Pawlotsky, Jean-Michel; Feray, Cyrille; Trabucchi, Michele

2017-01-01

There is evidence that eukaryotic miRNAs (hereafter called host miRNAs) play a role in the replication and propagation of viruses. Expression or targeting of host miRNAs can be involved in cellular antiviral responses. Most times host miRNAs play a role in viral life-cycles and promote infection through complex regulatory pathways. miRNAs can also be encoded by a viral genome and be expressed in the host cell. Viral miRNAs can share common sequences with host miRNAs or have totally different sequences. They can regulate a variety of biological processes involved in viral infection, including apoptosis, evasion of the immune response, or modulation of viral life-cycle phases. Overall, virus/miRNA pathway interaction is defined by a plethora of complex mechanisms, though not yet fully understood. This article review summarizes recent advances and novel biological concepts related to the understanding of miRNA expression, control and function during viral infections. The article also discusses potential therapeutic applications of this particular host-pathogen interaction.

Viruses and miRNAs: More Friends than Foes

PubMed Central

Bruscella, Patrice; Bottini, Silvia; Baudesson, Camille; Pawlotsky, Jean-Michel; Feray, Cyrille; Trabucchi, Michele

2017-01-01

There is evidence that eukaryotic miRNAs (hereafter called host miRNAs) play a role in the replication and propagation of viruses. Expression or targeting of host miRNAs can be involved in cellular antiviral responses. Most times host miRNAs play a role in viral life-cycles and promote infection through complex regulatory pathways. miRNAs can also be encoded by a viral genome and be expressed in the host cell. Viral miRNAs can share common sequences with host miRNAs or have totally different sequences. They can regulate a variety of biological processes involved in viral infection, including apoptosis, evasion of the immune response, or modulation of viral life-cycle phases. Overall, virus/miRNA pathway interaction is defined by a plethora of complex mechanisms, though not yet fully understood. This article review summarizes recent advances and novel biological concepts related to the understanding of miRNA expression, control and function during viral infections. The article also discusses potential therapeutic applications of this particular host–pathogen interaction. PMID:28555130

MicroRNA-21 promotes proliferation of rat hepatocyte BRL-3A by targeting FASLG.

PubMed

Li, J J; Chan, W H; Leung, W Y; Wang, Y; Xu, C S

2015-04-27

Rat liver regeneration (RLR) induced by partial hepatectomy involves cell proliferation regulated by numerous factors, including microRNAs (miRNAs). miRNA high-throughput sequencing has been established and used to analyze miRNA expression profiles. This study showed that 39 miRNAs were related to RLR through the analysis of miRNA high-throughput sequencing. Their role toward rat normal hepatocyte line BRL-3A was studied by gain- and loss-of-function analyses, and one of them, microRNA-21 (miR-21), obviously upregulated and promoted BRL-3A cell proliferation. Using bioinformatics to search for miR-21 targets revealed that Fas ligand (FASLG) is one of miR-21's target genes. A dual-luciferase report assay and Western blot assay showed that miR-21 directly targeted the 3'-untranslated region of FASLG and inhibited the expression of FASLG, which suggests that miR-21 promoted BRL-3A cell proliferation by reducing FASLG expression.

Lipopolysaccharide-induced innate immune factors in the bottlenose dolphin (Tursiops truncatus) detected in expression sequence tag analysis.

PubMed

Ohishi, Kazue; Shishido, Reiko; Iwata, Yasunao; Saitoh, Masafumi; Takenaka, Ryota; Ohtsu, Dai; Okutsu, Kenji; Maruyama, Tadashi

2011-11-01

EST analysis based on the megaclone-megasorting method was performed using leukocytes from the bottlenose dolphin (Tursiops truncatus) with or without LPS stimulation. A total of 849 upregulated and 384 downregulated EST clones were sequenced, annotated, and functionally classified. Ferritin heavy peptide I was the most abundant upregulated transcript, suggesting that LPS stimulation induced high production of reactive oxygen species, which were sequestered in ferritin. Among the immune factors, the transcripts coding for an IL-1Ra, homologs to bovine serum amyloid A3, and canine intercellular adhesion molecule-1 were highly expressed. Markedly downregulated transcripts of immune factors were those for homologs of calcium-binding proteins belonging to the S100 family, S100A12, S100A8, and S100A6. Time-course experiments on the expression of some immune factors including IL-1Ra suggested that these factors interact and control cetacean innate immunity. © 2011 The Societies and Blackwell Publishing Asia Pty Ltd.

Growth of surface and corner cracks in beta-processed and mill-annealed Ti-6Al-4V

NASA Technical Reports Server (NTRS)

Bell, P. D.

1975-01-01

Empirical stress-intensity expressions were developed to relate the growth of cracks from corner flaws to the growth of cracks from surface flaws. An experimental program using beta-processed Ti-6Al-4V verified these expressions for stress ratios, R greater than or equal to 0. An empirical crack growth-rate expression which included stress-ratio and stress-level effects was also developed. Cracks grew approximately 10 percent faster in transverse-grain material than in longitudinal-grain material and at approximately the same rate in longitudinal-grain mill-annealed Ti-6Al-4V. Specimens having surface and corner cracks and made of longitudinal-grain, beta-processed material were tested with block loads, and increasing the stresses in a block did not significantly change the crack growth rates. Truncation of the basic ascending stress sequence within a block caused more rapid crack growth, whereas both the descending and low-to-high stress sequences slowed crack growth.

Neuropeptide Y (NPY), cocaine- and amphetamine-regulated transcript (CART) and cholecystokinin (CCK) in winter skate (Raja ocellata): cDNA cloning, tissue distribution and mRNA expression responses to fasting.

PubMed

MacDonald, Erin; Volkoff, Hélène

2009-04-01

cDNAs encoding for neuropeptide Y (NPY), cocaine- and amphetamine-regulated transcript (CART) and cholecystokinin (CCK) were cloned in an elasmobranch fish, the winter skate. mRNA tissue distribution was examined for the three peptides as well as the effects of two weeks of fasting on their expression. Skate NPY, CART and CCK sequences display similarities with sequences for teleost fish but in general the degree of identity is relatively low (50%). All three peptides are present in brain and in several peripheral tissues, including gut and gonads. Within the brain, the three peptides are expressed in the hypothalamus, telencephalon, optic tectum and cerebellum. Two weeks of fasting induced an increase in telencephalon NPY and an increase in CCK in the gut but had no effects on hypothalamic NPY, CART and CCK, or on telencephalon CART. Our results provide basis for further investigation into the regulation of feeding in winter skate.

Gene expression divergence and nucleotide differentiation between males of different color morphs and mating strategies in the ruff

PubMed Central

Ekblom, Robert; Farrell, Lindsay L; Lank, David B; Burke, Terry

2012-01-01

By next generation transcriptome sequencing, it is possible to obtain data on both nucleotide sequence variation and gene expression. We have used this approach (RNA-Seq) to investigate the genetic basis for differences in plumage coloration and mating strategies in a non-model bird species, the ruff (Philomachus pugnax). Ruff males show enormous variation in the coloration of ornamental feathers, used for individual recognition. This polymorphism is linked to reproductive strategies, with dark males (Independents) defending territories on leks against other Independents, whereas white morphs (Satellites) co-occupy Independent's courts without agonistic interactions. Previous work found a strong genetic component for mating strategy, but the genes involved were not identified. We present feather transcriptome data of more than 6,000 de-novo sequenced ruff genes (although with limited coverage for many of them). None of the identified genes showed significant expression divergence between males, but many genetic markers showed nucleotide differentiation between different color morphs and mating strategies. These include several feather keratin genes, splicing factors, and the Xg blood-group gene. Many of the genes with significant genetic structure between mating strategies have not yet been annotated and their functions remain to be elucidated. We also conducted in-depth investigations of 28 pre-identified coloration candidate genes. Two of these (EDNRB and TYR) were specifically expressed in black- and rust-colored males, respectively. We have demonstrated the utility of next generation transcriptome sequencing for identifying and genotyping large number of genetic markers in a non-model species without previous genomic resources, and highlight the potential of this approach for addressing the genetic basis of ecologically important variation. PMID:23145334

Expression of human electron transfer flavoprotein-ubiquinone oxidoreductase from a baculovirus vector: kinetic and spectral characterization of the human protein.

PubMed

Simkovic, Martin; Degala, Gregory D; Eaton, Sandra S; Frerman, Frank E

2002-06-15

Electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO) is an iron-sulphur flavoprotein and a component of an electron-transfer system that links 10 different mitochondrial flavoprotein dehydrogenases to the mitochondrial bc1 complex via electron transfer flavoprotein (ETF) and ubiquinone. ETF-QO is an integral membrane protein, and the primary sequences of human and porcine ETF-QO were deduced from the sequences of the cloned cDNAs. We have expressed human ETF-QO in Sf9 insect cells using a baculovirus vector. The cDNA encoding the entire protein, including the mitochondrial targeting sequence, was present in the vector. We isolated a membrane-bound form of the enzyme that has a molecular mass identical with that of the mature porcine protein as determined by SDS/PAGE and has an N-terminal sequence that is identical with that predicted for the mature holoenzyme. These data suggest that the heterologously expressed ETF-QO is targeted to mitochondria and processed to the mature, catalytically active form. The detergent-solubilized protein was purified by ion-exchange and hydroxyapatite chromatography. Absorption and EPR spectroscopy and redox titrations are consistent with the presence of flavin and iron-sulphur centres that are very similar to those in the equivalent porcine and bovine proteins. Additionally, the redox potentials of the two prosthetic groups appear similar to those of the other eukaryotic ETF-QO proteins. The steady-state kinetic constants of human ETF-QO were determined with ubiquinone homologues, a ubiquinone analogue, and with human wild-type ETF and a Paracoccus-human chimaeric ETF as varied substrates. The results demonstrate that this expression system provides sufficient amounts of human ETF-QO to enable crystallization and mechanistic investigations of the iron-sulphur flavoprotein.

cDNA cloning, functional expression and cellular localization of rat liver mitochondrial electron-transfer flavoprotein-ubiquinone oxidoreductase protein.

PubMed

Huang, Shengbing; Song, Wei; Lin, Qishui

2005-08-01

A membrane-bound protein was purified from rat liver mitochondria. After being digested with V8 protease, two peptides containing identical 14 amino acid residue sequences were obtained. Using the 14 amino acid peptide derived DNA sequence as gene specific primer, the cDNA of correspondent gene 5'-terminal and 3'-terminal were obtained by RACE technique. The full-length cDNA that encoded a protein of 616 amino acids was thus cloned, which included the above mentioned peptide sequence. The full length cDNA was highly homologous to that of human ETF-QO, indicating that it may be the cDNA of rat ETF-QO. ETF-QO is an iron sulfur protein located in mitochondria inner membrane containing two kinds of redox center: FAD and [4Fe-4S] center. After comparing the sequence from the cDNA of the 616 amino acids protein with that of the mature protein of rat liver mitochondria, it was found that the N terminal 32 amino acid residues did not exist in the mature protein, indicating that the cDNA was that of ETF-QOp. When the cDNA was expressed in Saccharomyces cerevisiae with inducible vectors, the protein product was enriched in mitochondrial fraction and exhibited electron transfer activity (NBT reductase activity) of ETF-QO. Results demonstrated that the 32 amino acid peptide was a mitochondrial targeting peptide, and both FAD and iron-sulfur cluster were inserted properly into the expressed ETF-QO. ETF-QO had a high level expression in rat heart, liver and kidney. The fusion protein of GFP-ETF-QO co-localized with mitochondria in COS-7 cells.

Tissue-specific transcriptomics of the exotic invasive insect pest emerald ash borer (Agrilus planipennis).

PubMed

Mittapalli, Omprakash; Bai, Xiaodong; Mamidala, Praveen; Rajarapu, Swapna Priya; Bonello, Pierluigi; Herms, Daniel A

2010-10-28

The insect midgut and fat body represent major tissue interfaces that deal with several important physiological functions including digestion, detoxification and immune response. The emerald ash borer (Agrilus planipennis), is an exotic invasive insect pest that has killed millions of ash trees (Fraxinus spp.) primarily in the Midwestern United States and Ontario, Canada. However, despite its high impact status little knowledge exists for A. planipennis at the molecular level. Newer-generation Roche-454 pyrosequencing was used to obtain 126,185 reads for the midgut and 240,848 reads for the fat body, which were assembled into 25,173 and 37,661 high quality expressed sequence tags (ESTs) for the midgut and the fat body of A. planipennis larvae, respectively. Among these ESTs, 36% of the midgut and 38% of the fat body sequences showed similarity to proteins in the GenBank nr database. A high number of the midgut sequences contained chitin-binding peritrophin (248)and trypsin (98) domains; while the fat body sequences showed high occurrence of cytochrome P450s (85) and protein kinase (123) domains. Further, the midgut transcriptome of A. planipennis revealed putative microbial transcripts encoding for cell-wall degrading enzymes such as polygalacturonases and endoglucanases. A significant number of SNPs (137 in midgut and 347 in fat body) and microsatellite loci (317 in midgut and 571 in fat body) were predicted in the A. planipennis transcripts. An initial assessment of cytochrome P450s belonging to various CYP clades revealed distinct expression patterns at the tissue level. To our knowledge this study is one of the first to illuminate tissue-specific gene expression in an invasive insect of high ecological and economic consequence. These findings will lay the foundation for future gene expression and functional studies in A. planipennis.

An RNA-Seq Transcriptome Analysis of Orthophosphate-Deficient White Lupin Reveals Novel Insights into Phosphorus Acclimation in Plants1[W][OA

PubMed Central

O’Rourke, Jamie A.; Yang, S. Samuel; Miller, Susan S.; Bucciarelli, Bruna; Liu, Junqi; Rydeen, Ariel; Bozsoki, Zoltan; Uhde-Stone, Claudia; Tu, Zheng Jin; Allan, Deborah; Gronwald, John W.; Vance, Carroll P.

2013-01-01

Phosphorus, in its orthophosphate form (Pi), is one of the most limiting macronutrients in soils for plant growth and development. However, the whole-genome molecular mechanisms contributing to plant acclimation to Pi deficiency remain largely unknown. White lupin (Lupinus albus) has evolved unique adaptations for growth in Pi-deficient soils, including the development of cluster roots to increase root surface area. In this study, we utilized RNA-Seq technology to assess global gene expression in white lupin cluster roots, normal roots, and leaves in response to Pi supply. We de novo assembled 277,224,180 Illumina reads from 12 complementary DNA libraries to build what is to our knowledge the first white lupin gene index (LAGI 1.0). This index contains 125,821 unique sequences with an average length of 1,155 bp. Of these sequences, 50,734 were transcriptionally active (reads per kilobase per million reads ≥ 3), representing approximately 7.8% of the white lupin genome, using the predicted genome size of Lupinus angustifolius as a reference. We identified a total of 2,128 sequences differentially expressed in response to Pi deficiency with a 2-fold or greater change and P ≤ 0.05. Twelve sequences were consistently differentially expressed due to Pi deficiency stress in three species, Arabidopsis (Arabidopsis thaliana), potato (Solanum tuberosum), and white lupin, making them ideal candidates to monitor the Pi status of plants. Additionally, classic physiological experiments were coupled with RNA-Seq data to examine the role of cytokinin and gibberellic acid in Pi deficiency-induced cluster root development. This global gene expression analysis provides new insights into the biochemical and molecular mechanisms involved in the acclimation to Pi deficiency. PMID:23197803

An RNA-Seq transcriptome analysis of orthophosphate-deficient white lupin reveals novel insights into phosphorus acclimation in plants.

PubMed

O'Rourke, Jamie A; Yang, S Samuel; Miller, Susan S; Bucciarelli, Bruna; Liu, Junqi; Rydeen, Ariel; Bozsoki, Zoltan; Uhde-Stone, Claudia; Tu, Zheng Jin; Allan, Deborah; Gronwald, John W; Vance, Carroll P

2013-02-01

Phosphorus, in its orthophosphate form (P(i)), is one of the most limiting macronutrients in soils for plant growth and development. However, the whole-genome molecular mechanisms contributing to plant acclimation to P(i) deficiency remain largely unknown. White lupin (Lupinus albus) has evolved unique adaptations for growth in P(i)-deficient soils, including the development of cluster roots to increase root surface area. In this study, we utilized RNA-Seq technology to assess global gene expression in white lupin cluster roots, normal roots, and leaves in response to P(i) supply. We de novo assembled 277,224,180 Illumina reads from 12 complementary DNA libraries to build what is to our knowledge the first white lupin gene index (LAGI 1.0). This index contains 125,821 unique sequences with an average length of 1,155 bp. Of these sequences, 50,734 were transcriptionally active (reads per kilobase per million reads ≥ 3), representing approximately 7.8% of the white lupin genome, using the predicted genome size of Lupinus angustifolius as a reference. We identified a total of 2,128 sequences differentially expressed in response to P(i) deficiency with a 2-fold or greater change and P ≤ 0.05. Twelve sequences were consistently differentially expressed due to P(i) deficiency stress in three species, Arabidopsis (Arabidopsis thaliana), potato (Solanum tuberosum), and white lupin, making them ideal candidates to monitor the P(i) status of plants. Additionally, classic physiological experiments were coupled with RNA-Seq data to examine the role of cytokinin and gibberellic acid in P(i) deficiency-induced cluster root development. This global gene expression analysis provides new insights into the biochemical and molecular mechanisms involved in the acclimation to P(i) deficiency.

Differential expression and molecular characterisation of Lmo7, Myo1e, Sash1, and Mcoln2 genes in Btk-defective B-cells.

PubMed

Lindvall, Jessica M; Blomberg, K Emelie M; Wennborg, Anders; Smith, C I Edvard

2005-05-01

Bruton's tyrosine kinase is crucial for B-lymphocyte development. By the use of gene expression profiling, we have identified four expressed sequence tags among 38 potential Btk target genes, which have now been characterised. Bioinformatics tools including data mining of additional unpublished gene expression profiles, sequence verification of PCR products and qualitative RT-PCR were used. Stimulations targeting the B-cell receptor and the protein kinase C were used to activate whole B-cell splenocytes. Target genes were characterised as Lim domain only 7 (Lmo7); Myosin1e (Myo1e); SAM and SH3 domain containing 1 (Sash1); and Mucolipin2 (Mcoln2). Expression was found in cell lines of different origin and developmental stages as well as in whole B-cell splenocytes and Transitional type 1 (T1) splenic B-cells from wild type and Btk-defective mice, respectively. By the use of semi-quantitative RT-PCR we found Sash1 not to be expressed in the investigated haematopoietic cell lines, while transcripts were found in whole splenic B-cells from both wild type and Btk-defective mice, whereas Lmo7, Myo1e, and Mcoln2 were expressed in both B-cell lines and primary B-lymphocytes. Except for Lmo7, the transcript level was similarly affected by stimulation in control and Btk-defective cells.

Cloning, sequencing, and expression of cDNA for human. beta. -glucuronidase

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

Oshima, A.; Kyle, J.W.; Miller, R.D.

1987-02-01

The authors report here the cDNA sequence for human placental ..beta..-glucuronidase (..beta..-D-glucuronoside glucuronosohydrolase, EC 3.2.1.31) and demonstrate expression of the human enzyme in transfected COS cells. They also sequenced a partial cDNA clone from human fibroblasts that contained a 153-base-pair deletion within the coding sequence and found a second type of cDNA clone from placenta that contained the same deletion. Nuclease S1 mapping studies demonstrated two types of mRNAs in human placenta that corresponded to the two types of cDNA clones isolated. The NH/sub 2/-terminal amino acid sequence determined for human spleen ..beta..-glucuronidase agreed with that inferred from the DNAmore » sequence of the two placental clones, beginning at amino acid 23, suggesting a cleaved signal sequence of 22 amino acids. When transfected into COS cells, plasmids containing either placental clone expressed an immunoprecipitable protein that contained N-linked oligosaccharides as evidenced by sensitivity to endoglycosidase F. However, only transfection with the clone containing the 153-base-pair segment led to expression of human ..beta..-glucuronidase activity. These studies provide the sequence for the full-length cDNA for human ..beta..-glucuronidase, demonstrate the existence of two populations of mRNA for ..beta..-glucuronidase in human placenta, only one of which specifies a catalytically active enzyme, and illustrate the importance of expression studies in verifying that a cDNA is functionally full-length.« less

Transcriptome and Gene Expression Analysis of the Rice Leaf Folder, Cnaphalocrosis medinalis

PubMed Central

Li, Shang-Wei; Yang, Hong; Liu, Yue-Feng; Liao, Qi-Rong; Du, Juan; Jin, Dao-Chao

2012-01-01

Background The rice leaf folder (RLF), Cnaphalocrocis medinalis (Guenee) (Lepidoptera: Pyralidae), is one of the most destructive pests affecting rice in Asia. Although several studies have been performed on the ecological and physiological aspects of this species, the molecular mechanisms underlying its developmental regulation, behavior, and insecticide resistance remain largely unknown. Presently, there is a lack of genomic information for RLF; therefore, studies aimed at profiling the RLF transcriptome expression would provide a better understanding of its biological function at the molecular level. Principal Findings De novo assembly of the RLF transcriptome was performed via the short read sequencing technology (Illumina). In a single run, we produced more than 23 million sequencing reads that were assembled into 44,941 unigenes (mean size = 474 bp) by Trinity. Through a similarity search, 25,281 (56.82%) unigenes matched known proteins in the NCBI Nr protein database. The transcriptome sequences were annotated with gene ontology (GO), cluster of orthologous groups of proteins (COG), and KEGG orthology (KO). Additionally, we profiled gene expression during RLF development using a tag-based digital gene expression (DGE) system. Five DGE libraries were constructed, and variations in gene expression were compared between collected samples: eggs vs. 3rd instar larvae, 3rd instar larvae vs. pupae, pupae vs. adults. The results demonstrated that thousands of genes were significantly differentially expressed during various developmental stages. A number of the differentially expressed genes were confirmed by quantitative real-time PCR (qRT-PCR). Conclusions The RLF transcriptome and DGE data provide a comprehensive and global gene expression profile that would further promote our understanding of the molecular mechanisms underlying various biological characteristics, including development, elevated fecundity, flight, sex differentiation, olfactory behavior, and insecticide resistance in RLF. Therefore, these findings could help elucidate the intrinsic factors involved in the RLF-mediated destruction of rice and offer sustainable insect pest management. PMID:23185238

Modelling multimodal expression of emotion in a virtual agent.

PubMed

Pelachaud, Catherine

2009-12-12

Over the past few years we have been developing an expressive embodied conversational agent system. In particular, we have developed a model of multimodal behaviours that includes dynamism and complex facial expressions. The first feature refers to the qualitative execution of behaviours. Our model is based on perceptual studies and encompasses several parameters that modulate multimodal behaviours. The second feature, the model of complex expressions, follows a componential approach where a new expression is obtained by combining facial areas of other expressions. Lately we have been working on adding temporal dynamism to expressions. So far they have been designed statically, typically at their apex. Only full-blown expressions could be modelled. To overcome this limitation, we have defined a representation scheme that describes the temporal evolution of the expression of an emotion. It is no longer represented by a static definition but by a temporally ordered sequence of multimodal signals.

Regulation of the Human Endogenous Retrovirus K (HML-2) Transcriptome by the HIV-1 Tat Protein

PubMed Central

Gonzalez-Hernandez, Marta J.; Cavalcoli, James D.; Sartor, Maureen A.; Contreras-Galindo, Rafael; Meng, Fan; Dai, Manhong; Dube, Derek; Saha, Anjan K.; Gitlin, Scott D.; Omenn, Gilbert S.; Kaplan, Mark H.

2014-01-01

ABSTRACT Approximately 8% of the human genome is made up of endogenous retroviral sequences. As the HIV-1 Tat protein activates the overall expression of the human endogenous retrovirus type K (HERV-K) (HML-2), we used next-generation sequencing to determine which of the 91 currently annotated HERV-K (HML-2) proviruses are regulated by Tat. Transcriptome sequencing of total RNA isolated from Tat- and vehicle-treated peripheral blood lymphocytes from a healthy donor showed that Tat significantly activates expression of 26 unique HERV-K (HML-2) proviruses, silences 12, and does not significantly alter the expression of the remaining proviruses. Quantitative reverse transcription-PCR validation of the sequencing data was performed on Tat-treated PBLs of seven donors using provirus-specific primers and corroborated the results with a substantial degree of quantitative similarity. IMPORTANCE The expression of HERV-K (HML-2) is tightly regulated but becomes markedly increased following infection with HIV-1, in part due to the HIV-1 Tat protein. The findings reported here demonstrate the complexity of the genome-wide regulation of HERV-K (HML-2) expression by Tat. This work also demonstrates that although HERV-K (HML-2) proviruses in the human genome are highly similar in terms of DNA sequence, modulation of the expression of specific proviruses in a given biological situation can be ascertained using next-generation sequencing and bioinformatics analysis. PMID:24872592

First Transcriptome and Digital Gene Expression Analysis in Neuroptera with an Emphasis on Chemoreception Genes in Chrysopa pallens (Rambur).

PubMed

Li, Zhao-Qun; Zhang, Shuai; Ma, Yan; Luo, Jun-Yu; Wang, Chun-Yi; Lv, Li-Min; Dong, Shuang-Lin; Cui, Jin-Jie

2013-01-01

Chrysopa pallens (Rambur) are the most important natural enemies and predators of various agricultural pests. Understanding the sophisticated olfactory system in insect antennae is crucial for studying the physiological bases of olfaction and also could lead to effective applications of C. pallens in integrated pest management. However no transcriptome information is available for Neuroptera, and sequence data for C. pallens are scarce, so obtaining more sequence data is a priority for researchers on this species. To facilitate identifying sets of genes involved in olfaction, a normalized transcriptome of C. pallens was sequenced. A total of 104,603 contigs were obtained and assembled into 10,662 clusters and 39,734 singletons; 20,524 were annotated based on BLASTX analyses. A large number of candidate chemosensory genes were identified, including 14 odorant-binding proteins (OBPs), 22 chemosensory proteins (CSPs), 16 ionotropic receptors, 14 odorant receptors, and genes potentially involved in olfactory modulation. To better understand the OBPs, CSPs and cytochrome P450s, phylogenetic trees were constructed. In addition, 10 digital gene expression libraries of different tissues were constructed and gene expression profiles were compared among different tissues in males and females. Our results provide a basis for exploring the mechanisms of chemoreception in C. pallens, as well as other insects. The evolutionary analyses in our study provide new insights into the differentiation and evolution of insect OBPs and CSPs. Our study provided large-scale sequence information for further studies in C. pallens.

Cloning and analysis of DnaJ family members in the silkworm, Bombyx mori.

PubMed

Li, Yinü; Bu, Cuiyu; Li, Tiantian; Wang, Shibao; Jiang, Feng; Yi, Yongzhu; Yang, Huipeng; Zhang, Zhifang

2016-01-15

Heat shock proteins (Hsps) are involved in a variety of critical biological functions, including protein folding, degradation, and translocation and macromolecule assembly, act as molecular chaperones during periods of stress by binding to other proteins. Using expressed sequence tag (EST) and silkworm (Bombyx mori) transcriptome databases, we identified 27 cDNA sequences encoding the conserved J domain, which is found in DnaJ-type Hsps. Of the 27 J domain-containing sequences, 25 were complete cDNA sequences. We divided them into three types according to the number and presence of conserved domains. By analyzing the gene structures, intron numbers, and conserved domains and constructing a phylogenetic tree, we found that the DnaJ family had undergone convergent evolution, obtaining new domains to expand the diversity of its family members. The acquisition of the new DnaJ domains most likely occurred prior to the evolutionary divergence of prokaryotes and eukaryotes. The expression of DnaJ genes in the silkworm was generally higher in the fat body. The tissue distribution of DnaJ1 proteins was detected by western blotting, demonstrating that in the fifth-instar larvae, the DnaJ1 proteins were expressed at their highest levels in hemocytes, followed by the fat body and head. We also found that the DnaJ1 transcripts were likely differentially translated in different tissues. Using immunofluorescence cytochemistry, we revealed that in the blood cells, DnaJ1 was mainly localized in the cytoplasm. Copyright © 2015 Elsevier B.V. All rights reserved.

Molecular cloning and characterization of RGA1 encoding a G protein alpha subunit from rice (Oryza sativa L. IR-36).

PubMed

Seo, H S; Kim, H Y; Jeong, J Y; Lee, S Y; Cho, M J; Bahk, J D

1995-03-01

A cDNA clone, RGA1, was isolated by using a GPA1 cDNA clone of Arabidopsis thaliana G protein alpha subunit as a probe from a rice (Oryza sativa L. IR-36) seedling cDNA library from roots and leaves. Sequence analysis of genomic clone reveals that the RGA1 gene has 14 exons and 13 introns, and encodes a polypeptide of 380 amino acid residues with a calculated molecular weight of 44.5 kDa. The encoded protein exhibits a considerable degree of amino acid sequence similarity to all the other known G protein alpha subunits. A putative TATA sequence (ATATGA), a potential CAAT box sequence (AGCAATAC), and a cis-acting element, CCACGTGG (ABRE), known to be involved in ABA induction are found in the promoter region. The RGA1 protein contains all the consensus regions of G protein alpha subunits except the cysteine residue near the C-terminus for ADP-ribosylation by pertussis toxin. The RGA1 polypeptide expressed in Escherichia coli was, however, ADP-ribosylated by 10 microM [adenylate-32P] NAD and activated cholera toxin. Southern analysis indicates that there are no other genes similar to the RGA1 gene in the rice genome. Northern analysis reveals that the RGA1 mRNA is 1.85 kb long and expressed in vegetative tissues, including leaves and roots, and that its expression is regulated by light.

Elucidating the Small Regulatory RNA Repertoire of the Sea Anemone Anemonia viridis Based on Whole Genome and Small RNA Sequencing

PubMed Central

Patel, Hardip; Forêt, Sylvain; Karlsen, Bård Ove; Jørgensen, Tor Erik; Hall-Spencer, Jason M

2018-01-01

Abstract Cnidarians harbor a variety of small regulatory RNAs that include microRNAs (miRNAs) and PIWI-interacting RNAs (piRNAs), but detailed information is limited. Here, we report the identification and expression of novel miRNAs and putative piRNAs, as well as their genomic loci, in the symbiotic sea anemone Anemonia viridis. We generated a draft assembly of the A. viridis genome with putative size of 313 Mb that appeared to be composed of about 36% repeats, including known transposable elements. We detected approximately equal fractions of DNA transposons and retrotransposons. Deep sequencing of small RNA libraries constructed from A. viridis adults sampled at a natural CO2 gradient off Vulcano Island, Italy, identified 70 distinct miRNAs. Eight were homologous to previously reported miRNAs in cnidarians, whereas 62 appeared novel. Nine miRNAs were recognized as differentially expressed along the natural seawater pH gradient. We found a highly abundant and diverse population of piRNAs, with a substantial fraction showing ping–pong signatures. We identified nearly 22% putative piRNAs potentially targeting transposable elements within the A. viridis genome. The A. viridis genome appeared similar in size to that of other hexacorals with a very high divergence of transposable elements resembling that of the sea anemone genus Exaiptasia. The genome encodes and expresses a high number of small regulatory RNAs, which include novel miRNAs and piRNAs. Differentially expressed small RNAs along the seawater pH gradient indicated regulatory gene responses to environmental stressors. PMID:29385567

GarlicESTdb: an online database and mining tool for garlic EST sequences.

PubMed

Kim, Dae-Won; Jung, Tae-Sung; Nam, Seong-Hyeuk; Kwon, Hyuk-Ryul; Kim, Aeri; Chae, Sung-Hwa; Choi, Sang-Haeng; Kim, Dong-Wook; Kim, Ryong Nam; Park, Hong-Seog

2009-05-18

Allium sativum., commonly known as garlic, is a species in the onion genus (Allium), which is a large and diverse one containing over 1,250 species. Its close relatives include chives, onion, leek and shallot. Garlic has been used throughout recorded history for culinary, medicinal use and health benefits. Currently, the interest in garlic is highly increasing due to nutritional and pharmaceutical value including high blood pressure and cholesterol, atherosclerosis and cancer. For all that, there are no comprehensive databases available for Expressed Sequence Tags(EST) of garlic for gene discovery and future efforts of genome annotation. That is why we developed a new garlic database and applications to enable comprehensive analysis of garlic gene expression. GarlicESTdb is an integrated database and mining tool for large-scale garlic (Allium sativum) EST sequencing. A total of 21,595 ESTs collected from an in-house cDNA library were used to construct the database. The analysis pipeline is an automated system written in JAVA and consists of the following components: automatic preprocessing of EST reads, assembly of raw sequences, annotation of the assembled sequences, storage of the analyzed information into MySQL databases, and graphic display of all processed data. A web application was implemented with the latest J2EE (Java 2 Platform Enterprise Edition) software technology (JSP/EJB/JavaServlet) for browsing and querying the database, for creation of dynamic web pages on the client side, and for mapping annotated enzymes to KEGG pathways, the AJAX framework was also used partially. The online resources, such as putative annotation, single nucleotide polymorphisms (SNP) and tandem repeat data sets, can be searched by text, explored on the website, searched using BLAST, and downloaded. To archive more significant BLAST results, a curation system was introduced with which biologists can easily edit best-hit annotation information for others to view. The GarlicESTdb web application is freely available at http://garlicdb.kribb.re.kr. GarlicESTdb is the first incorporated online information database of EST sequences isolated from garlic that can be freely accessed and downloaded. It has many useful features for interactive mining of EST contigs and datasets from each library, including curation of annotated information, expression profiling, information retrieval, and summary of statistics of functional annotation. Consequently, the development of GarlicESTdb will provide a crucial contribution to biologists for data-mining and more efficient experimental studies.

Accelerated Evolution of PAK3- and PIM1-like Kinase Gene Families in the Zebra Finch, Taeniopygia guttata

PubMed Central

Kong, Lesheng; Lovell, Peter V.; Heger, Andreas; Mello, Claudio V.; Ponting, Chris P.

2010-01-01

Genes encoding protein kinases tend to evolve slowly over evolutionary time, and only rarely do they appear as recent duplications in sequenced vertebrate genomes. Consequently, it was a surprise to find two families of kinase genes that have greatly and recently expanded in the zebra finch (Taeniopygia guttata) lineage. In contrast to other amniotic genomes (including chicken) that harbor only single copies of p21-activated serine/threonine kinase 3 (PAK3) and proviral integration site 1 (PIM1) genes, the zebra finch genome appeared at first to additionally contain 67 PAK3-like (PAK3L) and 51 PIM1-like (PIM1L) protein kinase genes. An exhaustive analysis of these gene models, however, revealed most to be incomplete, owing to the absence of terminal exons. After reprediction, 31 PAK3L genes and 10 PIM1L genes remain, and all but three are predicted, from the retention of functional sites and open reading frames, to be enzymatically active. PAK3L, but not PIM1L, gene sequences show evidence of recurrent episodes of positive selection, concentrated within structures spatially adjacent to N- and C-terminal protein regions that have been discarded from zebra finch PAK3L genes. At least seven zebra finch PAK3L genes were observed to be expressed in testis, whereas two sequences were found transcribed in the brain, one broadly including the song nuclei and the other in the ventricular zone and in cells resembling Bergmann's glia in the cerebellar Purkinje cell layer. Two PIM1L sequences were also observed to be expressed with broad distributions in the zebra finch brain, one in both the ventricular zone and the cerebellum and apparently associated with glial cells and the other showing neuronal cell expression and marked enrichment in midbrain/thalamic nuclei. These expression patterns do not correlate with zebra finch-specific features such as vocal learning. Nevertheless, our results show how ancient and conserved intracellular signaling molecules can be co-opted, following duplication, thereby resulting in lineage-specific functions, presumably affecting the zebra finch testis and brain. PMID:20237222

The spectrum of low molecular weight alpha-amylase/protease inhibitor genes expressed in the US bread wheat cultivar Butte 86

PubMed Central

2011-01-01

Background Wheat grains accumulate a variety of low molecular weight proteins that are inhibitors of alpha-amylases and proteases and play an important protective role in the grain. These proteins have more balanced amino acid compositions than the major wheat gluten proteins and contribute important reserves for both seedling growth and human nutrition. The alpha-amylase/protease inhibitors also are of interest because they cause IgE-mediated occupational and food allergies and thereby impact human health. Results The complement of genes encoding alpha-amylase/protease inhibitors expressed in the US bread wheat Butte 86 was characterized by analysis of expressed sequence tags (ESTs). Coding sequences for 19 distinct proteins were identified. These included two monomeric (WMAI), four dimeric (WDAI), and six tetrameric (WTAI) inhibitors of exogenous alpha-amylases, two inhibitors of endogenous alpha-amylases (WASI), four putative trypsin inhibitors (CMx and WTI), and one putative chymotrypsin inhibitor (WCI). A number of the encoded proteins were identical or very similar to proteins in the NCBI database. Sequences not reported previously included variants of WTAI-CM3, three CMx inhibitors and WTI. Within the WDAI group, two different genes encoded the same mature protein. Based on numbers of ESTs, transcripts for WTAI-CM3 Bu-1, WMAI Bu-1 and WTAI-CM16 Bu-1 were most abundant in Butte 86 developing grain. Coding sequences for 16 of the inhibitors were unequivocally associated with specific proteins identified by tandem mass spectrometry (MS/MS) in a previous proteomic analysis of milled white flour from Butte 86. Proteins corresponding to WDAI Bu-1/Bu-2, WMAI Bu-1 and the WTAI subunits CM2 Bu-1, CM3 Bu-1 and CM16 Bu-1 were accumulated to the highest levels in flour. Conclusions Information on the spectrum of alpha-amylase/protease inhibitor genes and proteins expressed in a single wheat cultivar is central to understanding the importance of these proteins in both plant defense mechanisms and human allergies and facilitates both breeding and biotechnology approaches for manipulating the composition of these proteins in plants. PMID:21774824

Analysis and Functional Annotation of an Expressed Sequence Tag Collection for Tropical Crop Sugarcane

PubMed Central

Vettore, André L.; da Silva, Felipe R.; Kemper, Edson L.; Souza, Glaucia M.; da Silva, Aline M.; Ferro, Maria Inês T.; Henrique-Silva, Flavio; Giglioti, Éder A.; Lemos, Manoel V.F.; Coutinho, Luiz L.; Nobrega, Marina P.; Carrer, Helaine; França, Suzelei C.; Bacci, Maurício; Goldman, Maria Helena S.; Gomes, Suely L.; Nunes, Luiz R.; Camargo, Luis E.A.; Siqueira, Walter J.; Van Sluys, Marie-Anne; Thiemann, Otavio H.; Kuramae, Eiko E.; Santelli, Roberto V.; Marino, Celso L.; Targon, Maria L.P.N.; Ferro, Jesus A.; Silveira, Henrique C.S.; Marini, Danyelle C.; Lemos, Eliana G.M.; Monteiro-Vitorello, Claudia B.; Tambor, José H.M.; Carraro, Dirce M.; Roberto, Patrícia G.; Martins, Vanderlei G.; Goldman, Gustavo H.; de Oliveira, Regina C.; Truffi, Daniela; Colombo, Carlos A.; Rossi, Magdalena; de Araujo, Paula G.; Sculaccio, Susana A.; Angella, Aline; Lima, Marleide M.A.; de Rosa, Vicente E.; Siviero, Fábio; Coscrato, Virginia E.; Machado, Marcos A.; Grivet, Laurent; Di Mauro, Sonia M.Z.; Nobrega, Francisco G.; Menck, Carlos F.M.; Braga, Marilia D.V.; Telles, Guilherme P.; Cara, Frank A.A.; Pedrosa, Guilherme; Meidanis, João; Arruda, Paulo

2003-01-01

To contribute to our understanding of the genome complexity of sugarcane, we undertook a large-scale expressed sequence tag (EST) program. More than 260,000 cDNA clones were partially sequenced from 26 standard cDNA libraries generated from different sugarcane tissues. After the processing of the sequences, 237,954 high-quality ESTs were identified. These ESTs were assembled into 43,141 putative transcripts. Of the assembled sequences, 35.6% presented no matches with existing sequences in public databases. A global analysis of the whole SUCEST data set indicated that 14,409 assembled sequences (33% of the total) contained at least one cDNA clone with a full-length insert. Annotation of the 43,141 assembled sequences associated almost 50% of the putative identified sugarcane genes with protein metabolism, cellular communication/signal transduction, bioenergetics, and stress responses. Inspection of the translated assembled sequences for conserved protein domains revealed 40,821 amino acid sequences with 1415 Pfam domains. Reassembling the consensus sequences of the 43,141 transcripts revealed a 22% redundancy in the first assembling. This indicated that possibly 33,620 unique genes had been identified and indicated that >90% of the sugarcane expressed genes were tagged. PMID:14613979

Mutations That Stimulate flhDC Expression in Escherichia coli K-12.

PubMed

Fahrner, Karen A; Berg, Howard C

2015-10-01

Motility is a beneficial attribute that enables cells to access and explore new environments and to escape detrimental ones. The organelle of motility in Escherichia coli is the flagellum, and its production is initiated by the activating transcription factors FlhD and FlhC. The expression of these factors by the flhDC operon is highly regulated and influenced by environmental conditions. The flhDC promoter is recognized by σ(70) and is dependent on the transcriptional activator cyclic AMP (cAMP)-cAMP receptor protein complex (cAMP-CRP). A number of K-12 strains exhibit limited motility due to low expression levels of flhDC. We report here a large number of mutations that stimulate flhDC expression in such strains. They include single nucleotide changes in the -10 element of the promoter, in the promoter spacer, and in the cAMP-CRP binding region. In addition, we show that insertion sequence (IS) elements or a kanamycin gene located hundreds of base pairs upstream of the promoter can effectively enhance transcription, suggesting that the topology of a large upstream region plays a significant role in the regulation of flhDC expression. None of the mutations eliminated the requirement for cAMP-CRP for activation. However, several mutations allowed expression in the absence of the nucleoid organizing protein, H-NS, which is normally required for flhDC expression. The flhDC operon of Escherichia coli encodes transcription factors that initiate flagellar synthesis, an energetically costly process that is highly regulated. Few deregulating mutations have been reported thus far. This paper describes new single nucleotide mutations that stimulate flhDC expression, including a number that map to the promoter spacer region. In addition, this work shows that insertion sequence elements or a kanamycin gene located far upstream from the promoter or repressor binding sites also stimulate transcription, indicating a role of regional topology in the regulation of flhDC expression. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

The contribution of 700,000 ORF sequence tags to the definition of the human transcriptome

PubMed Central

Camargo, Anamaria A.; Samaia, Helena P. B.; Dias-Neto, Emmanuel; Simão, Daniel F.; Migotto, Italo A.; Briones, Marcelo R. S.; Costa, Fernando F.; Aparecida Nagai, Maria; Verjovski-Almeida, Sergio; Zago, Marco A.; Andrade, Luis Eduardo C.; Carrer, Helaine; El-Dorry, Hamza F. A.; Espreafico, Enilza M.; Habr-Gama, Angelita; Giannella-Neto, Daniel; Goldman, Gustavo H.; Gruber, Arthur; Hackel, Christine; Kimura, Edna T.; Maciel, Rui M. B.; Marie, Suely K. N.; Martins, Elizabeth A. L.; Nóbrega, Marina P.; Paçó-Larson, Maria Luisa; Pardini, Maria Inês M. C.; Pereira, Gonçalo G.; Pesquero, João Bosco; Rodrigues, Vanderlei; Rogatto, Silvia R.; da Silva, Ismael D. C. G.; Sogayar, Mari C.; Sonati, Maria de Fátima; Tajara, Eloiza H.; Valentini, Sandro R.; Alberto, Fernando L.; Amaral, Maria Elisabete J.; Aneas, Ivy; Arnaldi, Liliane A. T.; de Assis, Angela M.; Bengtson, Mário Henrique; Bergamo, Nadia Aparecida; Bombonato, Vanessa; de Camargo, Maria E. R.; Canevari, Renata A.; Carraro, Dirce M.; Cerutti, Janete M.; Corrêa, Maria Lucia C.; Corrêa, Rosana F. R.; Costa, Maria Cristina R.; Curcio, Cyntia; Hokama, Paula O. M.; Ferreira, Ari J. S.; Furuzawa, Gilberto K.; Gushiken, Tsieko; Ho, Paulo L.; Kimura, Elza; Krieger, José E.; Leite, Luciana C. C.; Majumder, Paromita; Marins, Mozart; Marques, Everaldo R.; Melo, Analy S. A.; Melo, Monica; Mestriner, Carlos Alberto; Miracca, Elisabete C.; Miranda, Daniela C.; Nascimento, Ana Lucia T. O.; Nóbrega, Francisco G.; Ojopi, Élida P. B.; Pandolfi, José Rodrigo C.; Pessoa, Luciana G.; Prevedel, Aline C.; Rahal, Paula; Rainho, Claudia A.; Reis, Eduardo M. R.; Ribeiro, Marcelo L.; da Rós, Nancy; de Sá, Renata G.; Sales, Magaly M.; Sant'anna, Simone Cristina; dos Santos, Mariana L.; da Silva, Aline M.; da Silva, Neusa P.; Silva, Wilson A.; da Silveira, Rosana A.; Sousa, Josane F.; Stecconi, Daniella; Tsukumo, Fernando; Valente, Valéria; Soares, Fernando; Moreira, Eloisa S.; Nunes, Diana N.; Correa, Ricardo G.; Zalcberg, Heloisa; Carvalho, Alex F.; Reis, Luis F. L.; Brentani, Ricardo R.; Simpson, Andrew J. G.; de Souza, Sandro J.

2001-01-01

Open reading frame expressed sequences tags (ORESTES) differ from conventional ESTs by providing sequence data from the central protein coding portion of transcripts. We generated a total of 696,745 ORESTES sequences from 24 human tissues and used a subset of the data that correspond to a set of 15,095 full-length mRNAs as a means of assessing the efficiency of the strategy and its potential contribution to the definition of the human transcriptome. We estimate that ORESTES sampled over 80% of all highly and moderately expressed, and between 40% and 50% of rarely expressed, human genes. In our most thoroughly sequenced tissue, the breast, the 130,000 ORESTES generated are derived from transcripts from an estimated 70% of all genes expressed in that tissue, with an equally efficient representation of both highly and poorly expressed genes. In this respect, we find that the capacity of the ORESTES strategy both for gene discovery and shotgun transcript sequence generation significantly exceeds that of conventional ESTs. The distribution of ORESTES is such that many human transcripts are now represented by a scaffold of partial sequences distributed along the length of each gene product. The experimental joining of the scaffold components, by reverse transcription–PCR, represents a direct route to transcript finishing that may represent a useful alternative to full-length cDNA cloning. PMID:11593022

The contribution of 700,000 ORF sequence tags to the definition of the human transcriptome.

PubMed

Camargo, A A; Samaia, H P; Dias-Neto, E; Simão, D F; Migotto, I A; Briones, M R; Costa, F F; Nagai, M A; Verjovski-Almeida, S; Zago, M A; Andrade, L E; Carrer, H; El-Dorry, H F; Espreafico, E M; Habr-Gama, A; Giannella-Neto, D; Goldman, G H; Gruber, A; Hackel, C; Kimura, E T; Maciel, R M; Marie, S K; Martins, E A; Nobrega, M P; Paco-Larson, M L; Pardini, M I; Pereira, G G; Pesquero, J B; Rodrigues, V; Rogatto, S R; da Silva, I D; Sogayar, M C; Sonati, M F; Tajara, E H; Valentini, S R; Alberto, F L; Amaral, M E; Aneas, I; Arnaldi, L A; de Assis, A M; Bengtson, M H; Bergamo, N A; Bombonato, V; de Camargo, M E; Canevari, R A; Carraro, D M; Cerutti, J M; Correa, M L; Correa, R F; Costa, M C; Curcio, C; Hokama, P O; Ferreira, A J; Furuzawa, G K; Gushiken, T; Ho, P L; Kimura, E; Krieger, J E; Leite, L C; Majumder, P; Marins, M; Marques, E R; Melo, A S; Melo, M B; Mestriner, C A; Miracca, E C; Miranda, D C; Nascimento, A L; Nobrega, F G; Ojopi, E P; Pandolfi, J R; Pessoa, L G; Prevedel, A C; Rahal, P; Rainho, C A; Reis, E M; Ribeiro, M L; da Ros, N; de Sa, R G; Sales, M M; Sant'anna, S C; dos Santos, M L; da Silva, A M; da Silva, N P; Silva, W A; da Silveira, R A; Sousa, J F; Stecconi, D; Tsukumo, F; Valente, V; Soares, F; Moreira, E S; Nunes, D N; Correa, R G; Zalcberg, H; Carvalho, A F; Reis, L F; Brentani, R R; Simpson, A J; de Souza, S J; Melo, M

2001-10-09

Open reading frame expressed sequences tags (ORESTES) differ from conventional ESTs by providing sequence data from the central protein coding portion of transcripts. We generated a total of 696,745 ORESTES sequences from 24 human tissues and used a subset of the data that correspond to a set of 15,095 full-length mRNAs as a means of assessing the efficiency of the strategy and its potential contribution to the definition of the human transcriptome. We estimate that ORESTES sampled over 80% of all highly and moderately expressed, and between 40% and 50% of rarely expressed, human genes. In our most thoroughly sequenced tissue, the breast, the 130,000 ORESTES generated are derived from transcripts from an estimated 70% of all genes expressed in that tissue, with an equally efficient representation of both highly and poorly expressed genes. In this respect, we find that the capacity of the ORESTES strategy both for gene discovery and shotgun transcript sequence generation significantly exceeds that of conventional ESTs. The distribution of ORESTES is such that many human transcripts are now represented by a scaffold of partial sequences distributed along the length of each gene product. The experimental joining of the scaffold components, by reverse transcription-PCR, represents a direct route to transcript finishing that may represent a useful alternative to full-length cDNA cloning.

Identification and functional analysis of flowering related microRNAs in common wild rice (Oryza rufipogon Griff.).

PubMed

Chen, Zongxiang; Li, Fuli; Yang, Songnan; Dong, Yibo; Yuan, Qianhua; Wang, Feng; Li, Weimin; Jiang, Ying; Jia, Shirong; Pei, Xinwu

2013-01-01

MicroRNAs (miRNAs) is a class of non-coding RNAs involved in post- transcriptional control of gene expression, via degradation and/or translational inhibition. Six-hundred sixty-one rice miRNAs are known that are important in plant development. However, flowering-related miRNAs have not been characterized in Oryza rufipogon Griff. It was approved by supervision department of Guangdong wild rice protection. We analyzed flowering-related miRNAs in O. rufipogon using high-throughput sequencing (deep sequencing) to understand the changes that occurred during rice domestication, and to elucidate their functions in flowering. Three O. rufipogon sRNA libraries, two vegetative stage (CWR-V1 and CWR-V2) and one flowering stage (CWR-F2) were sequenced using Illumina deep sequencing. A total of 20,156,098, 21,531,511 and 20,995,942 high quality sRNA reads were obtained from CWR-V1, CWR-V2 and CWR-F2, respectively, of which 3,448,185, 4,265,048 and 2,833,527 reads matched known miRNAs. We identified 512 known rice miRNAs in 214 miRNA families and predicted 290 new miRNAs. Targeted functional annotation, GO and KEGG pathway analyses predicted that 187 miRNAs regulate expression of flowering-related genes. Differential expression analysis of flowering-related miRNAs showed that: expression of 95 miRNAs varied significantly between the libraries, 66 are flowering-related miRNAs, such as oru-miR97, oru-miR117, oru-miR135, oru-miR137, et al. 17 are early-flowering -related miRNAs, including osa-miR160f, osa-miR164d, osa-miR167d, osa-miR169a, osa-miR172b, oru-miR4, et al., induced during the floral transition. Real-time PCR revealed the same expression patterns as deep sequencing. miRNAs targets were confirmed for cleavage by 5'-RACE in vivo, and were negatively regulated by miRNAs. This is the first investigation of flowering miRNAs in wild rice. The result indicates that variation in miRNAs occurred during rice domestication and lays a foundation for further study of phase change and flowering in O. rufipogon. Complicated regulatory networks mediated by multiple miRNAs regulate the expression of flowering genes that control the induction of flowering.

Immune response of the Caribbean sea fan, Gorgonia ventalina, exposed to an Aplanochytrium parasite as revealed by transcriptome sequencing

PubMed Central

Burge, Colleen A.; Mouchka, Morgan E.; Harvell, C. Drew; Roberts, Steven

2013-01-01

Coral reef communities are undergoing marked declines due to a variety of stressors including disease. The sea fan coral, Gorgonia ventalina, is a tractable study system to investigate mechanisms of immunity to a naturally occurring pathogen. Functional studies in Gorgonia ventalina immunity indicate that several key pathways and cellular components are involved in response to natural microbial invaders, although to date the functional and regulatory pathways remain largely un-described. This study used short-read sequencing (Illumina GAIIx) to identify genes involved in the response of G. ventalina to a naturally occurring Aplanochytrium spp. parasite. De novo assembly of the G. ventalina transcriptome yielded 90,230 contigs of which 40,142 were annotated. RNA-Seq analysis revealed 210 differentially expressed genes in sea fans exposed to the Aplanochytrium parasite. Differentially expressed genes involved in immunity include pattern recognition molecules, anti-microbial peptides, and genes involved in wound repair and reactive oxygen species formation. Gene enrichment analysis indicated eight biological processes were enriched representing 36 genes, largely involved with protein translation and energy production. This is the first report using high-throughput sequencing to characterize the host response of a coral to a natural pathogen. Furthermore, we have generated the first transcriptome for a soft (octocoral or non-scleractinian) coral species. Expression analysis revealed genes important in invertebrate innate immune pathways, as well as those whose role is previously un-described in cnidarians. This resource will be valuable in characterizing G. ventalina immune response to infection and co-infection of pathogens in the context of environmental change. PMID:23898300

Transcription analysis of peloric mutants of Phalaenopsis orchids derived from tissue culture.

PubMed

Chen, Ya Huei; Tsai, Yi Jung; Huang, Jian Zhi; Chen, Fure Chyi

2005-08-01

Tissue culture has been widely used for mass propagation of Phalaenopsis. However, somaclonal variation occurred during micropropagation process posed a severe problem by affecting product quality. In this study, wild type and peloric flower buds of Phalaenopsis hybrids derived from flower stalk nodal culture were used for cDNA-RAPD and cDNA suppression subtractive hybridization analyses in order to study their genetic difference in terms of expressed sequence tags. A total of 209 ESTs from normal flower buds and 230 from mutants were sequenced. These ESTs sequences can be grouped into several functional categories involved in different cellular processes including metabolism, signal transduction, transcription, cell growth and division, protein synthesis, and protein localization, and into a subcategory of proteins with unknown function. Cymbidium mosaic virus transcript was surprisingly found expressed frequently in the peloric mutant of P. Little Mary. Real-time RT-PCR analysis on selected ESTs showed that in mutant flower buds, a bZIP transcription factor (TGA1a-like protein) was down-regulated, while up-regulated genes include auxin-regulated protein kinase, cyclophilin, and TCP-like genes. A retroelement clone was also preferentially expressed in the peloric mutant flowers. On the other hand, ESTs involved in DNA methylation, chromatin remodeling and post-transcriptional regulation, such as DNA methyltransferase, histone acetyltransferase, ERECTA, and DEAD/DEAH RNA helicase, were enriched in normal flower buds than the mutants. The enriched transcripts in the wild type indicate the down regulation of these transcripts in the mutants, and vice versa. The potential roles of the analyzed transcripts in the development of Phalaenopsis flowers are discussed.

Improving RNA-Seq expression estimation by modeling isoform- and exon-specific read sequencing rate.

PubMed

Liu, Xuejun; Shi, Xinxin; Chen, Chunlin; Zhang, Li

2015-10-16

The high-throughput sequencing technology, RNA-Seq, has been widely used to quantify gene and isoform expression in the study of transcriptome in recent years. Accurate expression measurement from the millions or billions of short generated reads is obstructed by difficulties. One is ambiguous mapping of reads to reference transcriptome caused by alternative splicing. This increases the uncertainty in estimating isoform expression. The other is non-uniformity of read distribution along the reference transcriptome due to positional, sequencing, mappability and other undiscovered sources of biases. This violates the uniform assumption of read distribution for many expression calculation approaches, such as the direct RPKM calculation and Poisson-based models. Many methods have been proposed to address these difficulties. Some approaches employ latent variable models to discover the underlying pattern of read sequencing. However, most of these methods make bias correction based on surrounding sequence contents and share the bias models by all genes. They therefore cannot estimate gene- and isoform-specific biases as revealed by recent studies. We propose a latent variable model, NLDMseq, to estimate gene and isoform expression. Our method adopts latent variables to model the unknown isoforms, from which reads originate, and the underlying percentage of multiple spliced variants. The isoform- and exon-specific read sequencing biases are modeled to account for the non-uniformity of read distribution, and are identified by utilizing the replicate information of multiple lanes of a single library run. We employ simulation and real data to verify the performance of our method in terms of accuracy in the calculation of gene and isoform expression. Results show that NLDMseq obtains competitive gene and isoform expression compared to popular alternatives. Finally, the proposed method is applied to the detection of differential expression (DE) to show its usefulness in the downstream analysis. The proposed NLDMseq method provides an approach to accurately estimate gene and isoform expression from RNA-Seq data by modeling the isoform- and exon-specific read sequencing biases. It makes use of a latent variable model to discover the hidden pattern of read sequencing. We have shown that it works well in both simulations and real datasets, and has competitive performance compared to popular methods. The method has been implemented as a freely available software which can be found at https://github.com/PUGEA/NLDMseq.

ISOL@: an Italian SOLAnaceae genomics resource.

PubMed

Chiusano, Maria Luisa; D'Agostino, Nunzio; Traini, Alessandra; Licciardello, Concetta; Raimondo, Enrico; Aversano, Mario; Frusciante, Luigi; Monti, Luigi

2008-03-26

Present-day '-omics' technologies produce overwhelming amounts of data which include genome sequences, information on gene expression (transcripts and proteins) and on cell metabolic status. These data represent multiple aspects of a biological system and need to be investigated as a whole to shed light on the mechanisms which underpin the system functionality. The gathering and convergence of data generated by high-throughput technologies, the effective integration of different data-sources and the analysis of the information content based on comparative approaches are key methods for meaningful biological interpretations. In the frame of the International Solanaceae Genome Project, we propose here ISOLA, an Italian SOLAnaceae genomics resource. ISOLA (available at http://biosrv.cab.unina.it/isola) represents a trial platform and it is conceived as a multi-level computational environment.ISOLA currently consists of two main levels: the genome and the expression level. The cornerstone of the genome level is represented by the Solanum lycopersicum genome draft sequences generated by the International Tomato Genome Sequencing Consortium. Instead, the basic element of the expression level is the transcriptome information from different Solanaceae species, mainly in the form of species-specific comprehensive collections of Expressed Sequence Tags (ESTs). The cross-talk between the genome and the expression levels is based on data source sharing and on tools that enhance data quality, that extract information content from the levels' under parts and produce value-added biological knowledge. ISOLA is the result of a bioinformatics effort that addresses the challenges of the post-genomics era. It is designed to exploit '-omics' data based on effective integration to acquire biological knowledge and to approach a systems biology view. Beyond providing experimental biologists with a preliminary annotation of the tomato genome, this effort aims to produce a trial computational environment where different aspects and details are maintained as they are relevant for the analysis of the organization, the functionality and the evolution of the Solanaceae family.

Pseudomonas aeruginosa AES-1 exhibits increased virulence gene expression during chronic infection of cystic fibrosis lung.

PubMed

Naughton, Sharna; Parker, Dane; Seemann, Torsten; Thomas, Torsten; Turnbull, Lynne; Rose, Barbara; Bye, Peter; Cordwell, Stuart; Whitchurch, Cynthia; Manos, Jim

2011-01-01

Pseudomonas aeruginosa, the leading cause of morbidity and mortality in people with cystic fibrosis (CF), adapts for survival in the CF lung through both mutation and gene expression changes. Frequent clonal strains such as the Australian Epidemic Strain-1 (AES-1), have increased ability to establish infection in the CF lung and to superimpose and replace infrequent clonal strains. Little is known about the factors underpinning these properties. Analysis has been hampered by lack of expression array templates containing CF-strain specific genes. We sequenced the genome of an acute infection AES-1 isolate from a CF infant (AES-1R) and constructed a non-redundant micro-array (PANarray) comprising AES-1R and seven other sequenced P. aeruginosa genomes. The unclosed AES-1R genome comprised 6.254Mbp and contained 6957 putative genes, including 338 not found in the other seven genomes. The PANarray contained 12,543 gene probe spots; comprising 12,147 P. aeruginosa gene probes, 326 quality-control probes and 70 probes for non-P. aeruginosa genes, including phage and plant genes. We grew AES-1R and its isogenic pair AES-1M, taken from the same patient 10.5 years later and not eradicated in the intervening period, in our validated artificial sputum medium (ASMDM) and used the PANarray to compare gene expression of both in duplicate. 675 genes were differentially expressed between the isogenic pairs, including upregulation of alginate, biofilm, persistence genes and virulence-related genes such as dihydroorotase, uridylate kinase and cardiolipin synthase, in AES-1M. Non-PAO1 genes upregulated in AES-1M included pathogenesis-related (PAGI-5) genes present in strains PACS2 and PA7, and numerous phage genes. Elucidation of these genes' roles could lead to targeted treatment strategies for chronically infected CF patients.