Gene pools in wild Lima bean (Phaseolus lunatus L.) from the Americas: evidences for an Andean origin and past migrations.

PubMed

Serrano-Serrano, Martha L; Hernández-Torres, Jorge; Castillo-Villamizar, Genis; Debouck, Daniel G; Sánchez, María I Chacón

2010-01-01

The aims of this research were to assess the genetic structure of wild Phaseolus lunatus L. in the Americas and the hypothesis of a relatively recent Andean origin of the species. For this purpose, nuclear and non-coding chloroplast DNA markers were analyzed in a collection of 59 wild Lima bean accessions and six allied species. Twenty-three chloroplast and 28 nuclear DNA haplotypes were identified and shown to be geographically structured. Three highly divergent wild Lima bean gene pools, AI, MI, and MII, with mostly non-overlapping geographic ranges, are proposed. The results support an Andean origin of wild Lima beans during Pleistocene times and an early divergence of the three gene pools at an age that is posterior to completion of the Isthmus of Panama and major Andean orogeny. Gene pools would have evolved and reached their current geographic distribution mainly in isolation and therefore are of high priority for conservation and breeding programs.

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

Marchisio, Mario Andrea, E-mail: marchisio@hit.edu.cn

Published in 2008, Parts & Pools represents one of the first attempts to conceptualize the modular design of bacterial synthetic gene circuits with Standard Biological Parts (DNA segments) and Pools of molecules referred to as common signal carriers (e.g., RNA polymerases and ribosomes). The original framework for modeling bacterial components and designing prokaryotic circuits evolved over the last years and brought, first, to the development of an algorithm for the automatic design of Boolean gene circuits. This is a remarkable achievement since gene digital circuits have a broad range of applications that goes from biosensors for health and environment caremore » to computational devices. More recently, Parts & Pools was enabled to give a proper formal description of eukaryotic biological circuit components. This was possible by employing a rule-based modeling approach, a technique that permits a faithful calculation of all the species and reactions involved in complex systems such as eukaryotic cells and compartments. In this way, Parts & Pools is currently suitable for the visual and modular design of synthetic gene circuits in yeast and mammalian cells too.« less

Introduced Scotch broom (Cytisus scoparius) invades the genome of native populations in vulnerable heathland habitats.

PubMed

Rostgaard Nielsen, Lene; Brandes, Ursula; Dahl Kjaer, Erik; Fjellheim, Siri

2016-06-01

Cytisus scoparius is a global invasive species that affects local flora and fauna at the intercontinental level. Its natural distribution spans across Europe, but seeds have also been moved among countries, mixing plants of native and non-native genetic origins. Hybridization between the introduced and native gene pool is likely to threaten both the native gene pool and the local flora. In this study, we address the potential threat of invasive C. scoparius to local gene pools in vulnerable heathlands. We used nuclear single nucleotide polymorphic (SNP) and simple sequence repeat (SSR) markers together with plastid SSR and indel markers to investigate the level and direction of gene flow between invasive and native heathland C. scoparius. Analyses of population structures confirmed the presence of two gene pools: one native and the other invasive. The nuclear genome of the native types was highly introgressed with the invasive genome, and we observed advanced-generation hybrids, suggesting that hybridization has been occurring for several generations. There is asymmetrical gene flow from the invasive to the native gene pool, which can be attributed to higher fecundity in the invasive individuals, measured by the number of flowers and seed pods. Strong spatial genetic structure in plastid markers and weaker structure in nuclear markers suggest that seeds spread over relatively short distances and that gene flow over longer distances is mainly facilitated by pollen dispersal. We further show that the growth habits of heathland plants become more vigorous with increased introgression from the invaders. Implications of the findings are discussed in relation to future management of invading C. scoparius. © 2016 John Wiley & Sons Ltd.

[Book review] Fish Gene Pools: preservation of genetic resources in relation to wild fish stocks, edited by N. Ryman

USGS Publications Warehouse

Simon, R.C.

1984-01-01

Review of: Fish Gene Pools: Preservation of Genetic Resources in Relation to Wild Fish Stocks. Edited by N. Ryman. The Editorial Service/FRN, Box 6710, S-11385, Stockholm, Sweden. 1981. 111 pages. $16.00 (paper).

Sampling and Pooling Methods for Capturing Herd Level Antibiotic Resistance in Swine Feces using qPCR and CFU Approaches

PubMed Central

Mellerup, Anders; Ståhl, Marie

2015-01-01

The aim of this article was to define the sampling level and method combination that captures antibiotic resistance at pig herd level utilizing qPCR antibiotic resistance gene quantification and culture-based quantification of antibiotic resistant coliform indicator bacteria. Fourteen qPCR assays for commonly detected antibiotic resistance genes were developed, and used to quantify antibiotic resistance genes in total DNA from swine fecal samples that were obtained using different sampling and pooling methods. In parallel, the number of antibiotic resistant coliform indicator bacteria was determined in the same swine fecal samples. The results showed that the qPCR assays were capable of detecting differences in antibiotic resistance levels in individual animals that the coliform bacteria colony forming units (CFU) could not. Also, the qPCR assays more accurately quantified antibiotic resistance genes when comparing individual sampling and pooling methods. qPCR on pooled samples was found to be a good representative for the general resistance level in a pig herd compared to the coliform CFU counts. It had significantly reduced relative standard deviations compared to coliform CFU counts in the same samples, and therefore differences in antibiotic resistance levels between samples were more readily detected. To our knowledge, this is the first study to describe sampling and pooling methods for qPCR quantification of antibiotic resistance genes in total DNA extracted from swine feces. PMID:26114765

Questioning the utility of pooling samples in microarray experiments with cell lines.

PubMed

Lusa, L; Cappelletti, V; Gariboldi, M; Ferrario, C; De Cecco, L; Reid, J F; Toffanin, S; Gallus, G; McShane, L M; Daidone, M G; Pierotti, M A

2006-01-01

We describe a microarray experiment using the MCF-7 breast cancer cell line in two different experimental conditions for which the same number of independent pools as the number of individual samples was hybridized on Affymetrix GeneChips. Unexpectedly, when using individual samples, the number of probe sets found to be differentially expressed between treated and untreated cells was about three times greater than that found using pools. These findings indicate that pooling samples in microarray experiments where the biological variability is expected to be small might not be helpful and could even decrease one's ability to identify differentially expressed genes.

Use of a pooled clone method to isolate a novel Bacillus thuringiensis Cry2A toxin with activity against Ostrinia furnacalis.

PubMed

Shu, Changlong; Zhang, Jingtao; Chen, Guihua; Liang, Gemei; He, Kanglai; Crickmore, Neil; Huang, Dafang; Zhang, Jie; Song, Fuping

2013-09-01

A pooled clone method was developed to screen for cry2A genes. This metagenomic method avoids the need to analyse isolated Bacillus thuringiensis strains by performing gene specific PCR on plasmid-enriched DNA prepared from a pooled soil sample. Using this approach the novel holotype gene cry2Ah1 was cloned and characterized. The toxin gene was over-expressed in Escherichia coli Rosetta (DE3) and the expressed toxin accumulated in both the soluble and insoluble fractions. The soluble Cry2Ah1 was found to have a weight loss activity against Ostrinia furnacalis, and a growth inhibitory activity to both Cry1Ac-susceptible and resistant Helicoverpa armigera populations. Copyright © 2013 Elsevier Inc. All rights reserved.

Analyses of Thinopyrum bessarabicum, Th. elongation and Th. junceum chromosomes using EST-SSR markers

USDA-ARS?s Scientific Manuscript database

Wild Thinopyrum grasses serve as important gene pools for forage and cereal crops. Knowledge of their chromosome organizations is pivotal for efficient utilization of this important gene pool in germplasm enhancement programs. EST-SSR markers for Th. bessarabicum, Th. elongatum and Th. junceum chr...

GPFrontend and GPGraphics: graphical analysis tools for genetic association studies.

PubMed

Uebe, Steffen; Pasutto, Francesca; Krumbiegel, Mandy; Schanze, Denny; Ekici, Arif B; Reis, André

2010-09-21

Most software packages for whole genome association studies are non-graphical, purely text based programs originally designed to run with UNIX-like operating systems. Graphical output is often not intended or supposed to be performed with other command line tools, e.g. gnuplot. Using the Microsoft .NET 2.0 platform and Visual Studio 2005, we have created a graphical software package to analyze data from microarray whole genome association studies, both for a DNA-pooling based approach as well as regular single sample data. Part of this package was made to integrate with GenePool 0.8.2, a previously existing software suite for GNU/Linux systems, which we have modified to run in a Microsoft Windows environment. Further modifications cause it to generate some additional data. This enables GenePool to interact with the .NET parts created by us. The programs we developed are GPFrontend, a graphical user interface and frontend to use GenePool and create metadata files for it, and GPGraphics, a program to further analyze and graphically evaluate output of different WGA analysis programs, among them also GenePool. Our programs enable regular MS Windows users without much experience in bioinformatics to easily visualize whole genome data from a variety of sources.

Functional genomics platform for pooled screening and mammalian genetic interaction maps

PubMed Central

Kampmann, Martin; Bassik, Michael C.; Weissman, Jonathan S.

2014-01-01

Systematic genetic interaction maps in microorganisms are powerful tools for identifying functional relationships between genes and defining the function of uncharacterized genes. We have recently implemented this strategy in mammalian cells as a two-stage approach. First, genes of interest are robustly identified in a pooled genome-wide screen using complex shRNA libraries. Second, phenotypes for all pairwise combinations of hit genes are measured in a double-shRNA screen and used to construct a genetic interaction map. Our protocol allows for rapid pooled screening under various conditions without a requirement for robotics, in contrast to arrayed approaches. Each stage of the protocol can be implemented in ~2 weeks, with additional time for analysis and generation of reagents. We discuss considerations for screen design, and present complete experimental procedures as well as a full computational analysis suite for identification of hits in pooled screens and generation of genetic interaction maps. While the protocols outlined here were developed for our original shRNA-based approach, they can be applied more generally, including to CRISPR-based approaches. PMID:24992097

Evidence for cryptic northern refugia in the last glacial period in Cryptomeria japonica

PubMed Central

Kimura, Megumi K.; Uchiyama, Kentaro; Nakao, Katsuhiro; Moriguchi, Yoshinari; San Jose-Maldia, Lerma; Tsumura, Yoshihiko

2014-01-01

Background and Aims Distribution shifts and natural selection during past climatic changes are important factors in determining the genetic structure of forest species. In particular, climatic fluctuations during the Quaternary appear to have caused changes in the distribution ranges of plants, and thus strongly affected their genetic structure. This study was undertaken to identify the responses of the conifer Cryptomeria japonica, endemic to the Japanese Archipelago, to past climatic changes using a combination of phylogeography and species distribution modelling (SDM) methods. Specifically, this study focused on the locations of refugia during the last glacial maximum (LGM). Methods Genetic diversity and structure were examined using 20 microsatellite markers in 37 populations of C. japonica. The locations of glacial refugia were assessed using STRUCTURE analysis, and potential habitats under current and past climate conditions were predicted using SDM. The process of genetic divergence was also examined using the approximate Bayesian computation procedure (ABC) in DIY ABC to test the divergence time between the gene pools detected by the STRUCTURE analysis. Key Results STRUCTURE analysis identified four gene pools: northern Tohoku district; from Chubu to Chugoku district; from Tohoku to Shikoku district on the Pacific Ocean side of the Archipelago; and Yakushima Island. DIY ABC analysis indicated that the four gene pools diverged at the same time before the LGM. SDM also indicated potential northern cryptic refugia. Conclusions The combined evidence from microsatellites and SDM clearly indicates that climatic changes have shaped the genetic structure of C. japonica. The gene pool detected in northern Tohoku district is likely to have been established by cryptic northern refugia on the coast of the Japan Sea to the west of the Archipelago. The gene pool in Yakushima Island can probably be explained simply by long-term isolation from the other gene pools since the LGM. These results are supported by those of SDM and the predicted divergence time determined using ABC analysis. PMID:25355521

A comprehensive study of the genomic differentiation between temperate Dent and Flint maize.

PubMed

Unterseer, Sandra; Pophaly, Saurabh D; Peis, Regina; Westermeier, Peter; Mayer, Manfred; Seidel, Michael A; Haberer, Georg; Mayer, Klaus F X; Ordas, Bernardo; Pausch, Hubert; Tellier, Aurélien; Bauer, Eva; Schön, Chris-Carolin

2016-07-08

Dent and Flint represent two major germplasm pools exploited in maize breeding. Several traits differentiate the two pools, like cold tolerance, early vigor, and flowering time. A comparative investigation of their genomic architecture relevant for quantitative trait expression has not been reported so far. Understanding the genomic differences between germplasm pools may contribute to a better understanding of the complementarity in heterotic patterns exploited in hybrid breeding and of mechanisms involved in adaptation to different environments. We perform whole-genome screens for signatures of selection specific to temperate Dent and Flint maize by comparing high-density genotyping data of 70 American and European Dent and 66 European Flint inbred lines. We find 2.2 % and 1.4 % of the genes are under selective pressure, respectively, and identify candidate genes associated with agronomic traits known to differ between the two pools. Taking flowering time as an example for the differentiation between Dent and Flint, we investigate candidate genes involved in the flowering network by phenotypic analyses in a Dent-Flint introgression library and find that the Flint haplotypes of the candidates promote earlier flowering. Within the flowering network, the majority of Flint candidates are associated with endogenous pathways in contrast to Dent candidate genes, which are mainly involved in response to environmental factors like light and photoperiod. The diversity patterns of the candidates in a unique panel of more than 900 individuals from 38 European landraces indicate a major contribution of landraces from France, Germany, and Spain to the candidate gene diversity of the Flint elite lines. In this study, we report the investigation of pool-specific differences between temperate Dent and Flint on a genome-wide scale. The identified candidate genes represent a promising source for the functional investigation of pool-specific haplotypes in different genetic backgrounds and for the evaluation of their potential for future crop improvement like the adaptation to specific environments.

Emergence and Influence of the Zapatista Social Netwar

DTIC Science & Technology

2001-01-01

Dawkins , Richard, The Selfish Gene , New York: Oxford University Press, 1989. Diaz del Castillo, Bernal, The Conquest of New Spain [1568], Balti- more...as biological bodies are based on units of “self-replicating patterns of information” (p. 329). In his view (p. 192), Just as genes propagate...themselves in the gene pool by leaping from body to body via sperm or eggs, so memes propagate themselves in the meme pool by leaping from brain to brain via

The care and handling of the forest gene pool

Treesearch

Roy R. Silen; Ivan Doig

1976-01-01

What must be the world's most magnificent pool of forest genes has timbered our Pacific slopes.Why else do the tallest firs, pines, spruces, hemlocks, redwoods, and larches all rise along the Pacific Coast of North America? Does their hugeness simply thrust up from our deep soils and mild, rainy climate? From a vantage point of three...

A restoration practitioner's guide to the restoration gene pool concept

Treesearch

Thomas A. Jones; Thomas A. Monaco

2007-01-01

Choosing plant materials for each desired species is often one of the most difficult steps in developing a restoration plan. The Restoration Gene Pool concept was developed to clarify the options available to the ecological restoration practitioner in terms of plant materials. We present a decision-making flowchart incorporating the issues delineated in the Restoration...

A two-generation analysis of pollen pool genetic structure in flowering dogwood Cornus florida (Cornaceae), in the Missouri Ozarks

Treesearch

Victoria L. Sork; Peter E. Smouse; Victoria J. Apsit; Rodney J. Dyer; Robert D. Westfall

2005-01-01

Anthropogenic landscape change can disrupt gene flow. As part of the Missouri Ozark Forest Ecosystem Project, this study examined whether silvicultural practices influence pollen-mediated gene movement in the insect-pollinated species, Cornus florida L., by comparing pollen pool structure (ΦST) among clear-cutting,...

Identifying Genetic Signatures of Natural Selection Using Pooled Population Sequencing in Picea abies

PubMed Central

Chen, Jun; Källman, Thomas; Ma, Xiao-Fei; Zaina, Giusi; Morgante, Michele; Lascoux, Martin

2016-01-01

The joint inference of selection and past demography remain a costly and demanding task. We used next generation sequencing of two pools of 48 Norway spruce mother trees, one corresponding to the Fennoscandian domain, and the other to the Alpine domain, to assess nucleotide polymorphism at 88 nuclear genes. These genes are candidate genes for phenological traits, and most belong to the photoperiod pathway. Estimates of population genetic summary statistics from the pooled data are similar to previous estimates, suggesting that pooled sequencing is reliable. The nonsynonymous SNPs tended to have both lower frequency differences and lower FST values between the two domains than silent ones. These results suggest the presence of purifying selection. The divergence between the two domains based on synonymous changes was around 5 million yr, a time similar to a recent phylogenetic estimate of 6 million yr, but much larger than earlier estimates based on isozymes. Two approaches, one of them novel and that considers both FST and difference in allele frequencies between the two domains, were used to identify SNPs potentially under diversifying selection. SNPs from around 20 genes were detected, including genes previously identified as main target for selection, such as PaPRR3 and PaGI. PMID:27172202

Identifying Genetic Signatures of Natural Selection Using Pooled Population Sequencing in Picea abies.

PubMed

Chen, Jun; Källman, Thomas; Ma, Xiao-Fei; Zaina, Giusi; Morgante, Michele; Lascoux, Martin

2016-07-07

The joint inference of selection and past demography remain a costly and demanding task. We used next generation sequencing of two pools of 48 Norway spruce mother trees, one corresponding to the Fennoscandian domain, and the other to the Alpine domain, to assess nucleotide polymorphism at 88 nuclear genes. These genes are candidate genes for phenological traits, and most belong to the photoperiod pathway. Estimates of population genetic summary statistics from the pooled data are similar to previous estimates, suggesting that pooled sequencing is reliable. The nonsynonymous SNPs tended to have both lower frequency differences and lower FST values between the two domains than silent ones. These results suggest the presence of purifying selection. The divergence between the two domains based on synonymous changes was around 5 million yr, a time similar to a recent phylogenetic estimate of 6 million yr, but much larger than earlier estimates based on isozymes. Two approaches, one of them novel and that considers both FST and difference in allele frequencies between the two domains, were used to identify SNPs potentially under diversifying selection. SNPs from around 20 genes were detected, including genes previously identified as main target for selection, such as PaPRR3 and PaGI. Copyright © 2016 Chen et al.

Diagnostic value of secreted frizzled-related protein 2 gene promoter hypermethylation in stool for colorectal cancer: A meta-analysis.

PubMed

Zhou, Zhiran; Zhang, Huitian; Lei, Yunxia

2016-10-01

To evaluate the diagnostic value of secreted frizzled-related protein 2 (SFRP2) gene promoter hypermethylation in stool for colorectal cancer (CRC). Open published diagnostic study of SFRP2 gene promoter hypermethylation in stool for CRC detection was electronic searched in the databases of PubMed, EMBASE, Cochrane Library, Web of Science, and China National Knowledge Infrastructure. The data of true positive, false positive false negative, and true negative identified by stool SFRP2 gene hypermethylation was extracted and pooled for diagnostic sensitivity, specificity, and summary receiver operating characteristic (SROC) curve. According to the inclusion and exclusion criteria, we finally included nine publications with 792 cases in the meta-analysis. Thus, the diagnostic sensitivity was aggregated through random effect model. The pooled sensitivity was 0.82 with the corresponding 95% confidence interval (95% CI) of 0.79-0.85; the pooled specificity and its corresponding 95% CI were 0.47 and 0.40-0.53 by the random effect model; we pooled the SROC curve by sensitivity versus specificity according to data published in the nine studies. The area under the SROC curve was 0.70 (95% CI: 0.65-0.73). SFRP2 gene promoter hypermethylation in stool can was a potential biomarker for CRC diagnosis with relative high sensitivity.

Comprehensive analysis of correlation coefficients estimated from pooling heterogeneous microarray data

PubMed Central

2013-01-01

Background The synthesis of information across microarray studies has been performed by combining statistical results of individual studies (as in a mosaic), or by combining data from multiple studies into a large pool to be analyzed as a single data set (as in a melting pot of data). Specific issues relating to data heterogeneity across microarray studies, such as differences within and between labs or differences among experimental conditions, could lead to equivocal results in a melting pot approach. Results We applied statistical theory to determine the specific effect of different means and heteroskedasticity across 19 groups of microarray data on the sign and magnitude of gene-to-gene Pearson correlation coefficients obtained from the pool of 19 groups. We quantified the biases of the pooled coefficients and compared them to the biases of correlations estimated by an effect-size model. Mean differences across the 19 groups were the main factor determining the magnitude and sign of the pooled coefficients, which showed largest values of bias as they approached ±1. Only heteroskedasticity across the pool of 19 groups resulted in less efficient estimations of correlations than did a classical meta-analysis approach of combining correlation coefficients. These results were corroborated by simulation studies involving either mean differences or heteroskedasticity across a pool of N > 2 groups. Conclusions The combination of statistical results is best suited for synthesizing the correlation between expression profiles of a gene pair across several microarray studies. PMID:23822712

Multiplexed resequencing analysis to identify rare variants in pooled DNA with barcode indexing using next-generation sequencer.

PubMed

Mitsui, Jun; Fukuda, Yoko; Azuma, Kyo; Tozaki, Hirokazu; Ishiura, Hiroyuki; Takahashi, Yuji; Goto, Jun; Tsuji, Shoji

2010-07-01

We have recently found that multiple rare variants of the glucocerebrosidase gene (GBA) confer a robust risk for Parkinson disease, supporting the 'common disease-multiple rare variants' hypothesis. To develop an efficient method of identifying rare variants in a large number of samples, we applied multiplexed resequencing using a next-generation sequencer to identification of rare variants of GBA. Sixteen sets of pooled DNAs from six pooled DNA samples were prepared. Each set of pooled DNAs was subjected to polymerase chain reaction to amplify the target gene (GBA) covering 6.5 kb, pooled into one tube with barcode indexing, and then subjected to extensive sequence analysis using the SOLiD System. Individual samples were also subjected to direct nucleotide sequence analysis. With the optimization of data processing, we were able to extract all the variants from 96 samples with acceptable rates of false-positive single-nucleotide variants.

Mobile genes in the human microbiome are structured from global to individual scales

PubMed Central

Brito, IL; Jupiter, SD; Jenkins, AP; Naisilisili, W; Tamminen, M; Smillie, CS; Wortman, JR; Birren, BW; Xavier, RJ; Blainey, PC; Singh, AK; Gevers, D; Alm, EJ

2016-01-01

Recent work has underscored the importance of the microbiome in human health, largely attributing differences in phenotype to differences in the species present across individuals1,2,3,4,5. But mobile genes can confer profoundly different phenotypes on different strains of the same species. Little is known about the function and distribution of mobile genes in the human microbiome, and in particular whether the gene pool is globally homogenous or constrained by human population structure. Here, we investigate this question by comparing the mobile genes found in the microbiomes of 81 metropolitan North Americans with that of 172 agrarian Fiji islanders using a combination of single-cell genomics and metagenomics. We find large differences in mobile gene content between the Fijian and North American microbiomes, with functional variation that mirrors known dietary differences such as the excess of plant-based starch degradation genes. Remarkably, differences are also observed between the mobile gene pools of proximal Fijian villages, even though microbiome composition across villages is similar. Finally, we observe high rates of recombination leading to individual-specific mobile elements, suggesting that the abundance of some genes may reflect environmental selection rather than dispersal limitation. Together, these data support the hypothesis that human activities and behaviors provide selective pressures that shape mobile gene pools, and that acquisition of mobile genes is important to colonizing specific human populations. PMID:27409808

How voluntary prenatal diagnosis and selective abortion increase the abnormal human gene pool.

PubMed

Boss, J A

1990-06-01

It is often assumed that prenatal diagnosis followed by the selective abortion of "defective" fetuses has a positive eugenic effect. Although mandatory selective abortion of "defective" fetuses and, more important, carriers would tend to reduce the number of deleterious genes in the gene pool, the present program of voluntary prenatal diagnosis and selective abortion actually increases the number of deleterious genes. This raises the issue of freedom of choice regarding selective abortion and societal pressure on parents to undergo prenatal testing and to abort their fetus should it have a genetic disorder or be a carrier of one.

[Estimation of Genetic Diversity of Romanov Sheep by the Coefficient of Genetic Originality Based on ISSR-Fingerprinting Data].

PubMed

Nesteruk, L V; Makarova, N N; Svishcheva, G R; Stolpovsky, Yu A

2015-07-01

Estimation of the state of the genetic diversity and the originality of the breed structure is required for the conservation and management of domestic breeds of agricultural animals. The Romanov breed of sheep from the leading breeding and gene pool farms in Yaroslavl oblast (Russia) is the object of our study. ISS R fingerprinting was used as a molecular method of the study of sheep gene pools. Forty-three DNA fragments were detected (25 and 18, respectively) by two primers ((AG)9C and (GA)9C). Of the discovered ISSR markers, 81% were polymorphic. The coefficient of genetic originality was for the first time used for the study of the specificity and originality of the Romanov-breed gene pool. Based on its values, the studied individuals were divided into five classes depending on the frequency of the ISSR fragment. The most original or the rarest, as well as typical genotypes, were singled out in the Romanov sheep gene pool. Use the obtained data on genetic originality was proposed as a means to increase the efficiency of selection and breeding during the breeding of autochthonous breeds of domesticated animal species.

Pooled Genome-Wide Analysis to Identify Novel Risk Loci for Pediatric Allergic Asthma

PubMed Central

Ricci, Giampaolo; Astolfi, Annalisa; Remondini, Daniel; Cipriani, Francesca; Formica, Serena; Dondi, Arianna; Pession, Andrea

2011-01-01

Background Genome-wide association studies of pooled DNA samples were shown to be a valuable tool to identify candidate SNPs associated to a phenotype. No such study was up to now applied to childhood allergic asthma, even if the very high complexity of asthma genetics is an appropriate field to explore the potential of pooled GWAS approach. Methodology/Principal Findings We performed a pooled GWAS and individual genotyping in 269 children with allergic respiratory diseases comparing allergic children with and without asthma. We used a modular approach to identify the most significant loci associated with asthma by combining silhouette statistics and physical distance method with cluster-adapted thresholding. We found 97% concordance between pooled GWAS and individual genotyping, with 36 out of 37 top-scoring SNPs significant at individual genotyping level. The most significant SNP is located inside the coding sequence of C5, an already identified asthma susceptibility gene, while the other loci regulate functions that are relevant to bronchial physiopathology, as immune- or inflammation-mediated mechanisms and airway smooth muscle contraction. Integration with gene expression data showed that almost half of the putative susceptibility genes are differentially expressed in experimental asthma mouse models. Conclusion/Significance Combined silhouette statistics and cluster-adapted physical distance threshold analysis of pooled GWAS data is an efficient method to identify candidate SNP associated to asthma development in an allergic pediatric population. PMID:21359210

Single cell transcriptomic analysis of prostate cancer cells.

PubMed

Welty, Christopher J; Coleman, Ilsa; Coleman, Roger; Lakely, Bryce; Xia, Jing; Chen, Shu; Gulati, Roman; Larson, Sandy R; Lange, Paul H; Montgomery, Bruce; Nelson, Peter S; Vessella, Robert L; Morrissey, Colm

2013-02-16

The ability to interrogate circulating tumor cells (CTC) and disseminated tumor cells (DTC) is restricted by the small number detected and isolated (typically <10). To determine if a commercially available technology could provide a transcriptomic profile of a single prostate cancer (PCa) cell, we clonally selected and cultured a single passage of cell cycle synchronized C4-2B PCa cells. Ten sets of single, 5-, or 10-cells were isolated using a micromanipulator under direct visualization with an inverted microscope. Additionally, two groups of 10 individual DTC, each isolated from bone marrow of 2 patients with metastatic PCa were obtained. RNA was amplified using the WT-Ovation™ One-Direct Amplification System. The amplified material was hybridized on a 44K Whole Human Gene Expression Microarray. A high stringency threshold, a mean Alexa Fluor® 3 signal intensity above 300, was used for gene detection. Relative expression levels were validated for select genes using real-time PCR (RT-qPCR). Using this approach, 22,410, 20,423, and 17,009 probes were positive on the arrays from 10-cell pools, 5-cell pools, and single-cells, respectively. The sensitivity and specificity of gene detection on the single-cell analyses were 0.739 and 0.972 respectively when compared to 10-cell pools, and 0.814 and 0.979 respectively when compared to 5-cell pools, demonstrating a low false positive rate. Among 10,000 randomly selected pairs of genes, the Pearson correlation coefficient was 0.875 between the single-cell and 5-cell pools and 0.783 between the single-cell and 10-cell pools. As expected, abundant transcripts in the 5- and 10-cell samples were detected by RT-qPCR in the single-cell isolates, while lower abundance messages were not. Using the same stringency, 16,039 probes were positive on the patient single-cell arrays. Cluster analysis showed that all 10 DTC grouped together within each patient. A transcriptomic profile can be reliably obtained from a single cell using commercially available technology. As expected, fewer amplified genes are detected from a single-cell sample than from pooled-cell samples, however this method can be used to reliably obtain a transcriptomic profile from DTC isolated from the bone marrow of patients with PCa.

Construction of the BAC Library of Small Abalone (Haliotis diversicolor) for Gene Screening and Genome Characterization.

PubMed

Jiang, Likun; You, Weiwei; Zhang, Xiaojun; Xu, Jian; Jiang, Yanliang; Wang, Kai; Zhao, Zixia; Chen, Baohua; Zhao, Yunfeng; Mahboob, Shahid; Al-Ghanim, Khalid A; Ke, Caihuan; Xu, Peng

2016-02-01

The small abalone (Haliotis diversicolor) is one of the most important aquaculture species in East Asia. To facilitate gene cloning and characterization, genome analysis, and genetic breeding of it, we constructed a large-insert bacterial artificial chromosome (BAC) library, which is an important genetic tool for advanced genetics and genomics research. The small abalone BAC library includes 92,610 clones with an average insert size of 120 Kb, equivalent to approximately 7.6× of the small abalone genome. We set up three-dimensional pools and super pools of 18,432 BAC clones for target gene screening using PCR method. To assess the approach, we screened 12 target genes in these 18,432 BAC clones and identified 16 positive BAC clones. Eight positive BAC clones were then sequenced and assembled with the next generation sequencing platform. The assembled contigs representing these 8 BAC clones spanned 928 Kb of the small abalone genome, providing the first batch of genome sequences for genome evaluation and characterization. The average GC content of small abalone genome was estimated as 40.33%. A total of 21 protein-coding genes, including 7 target genes, were annotated into the 8 BACs, which proved the feasibility of PCR screening approach with three-dimensional pools in small abalone BAC library. One hundred fifty microsatellite loci were also identified from the sequences for marker development in the future. The BAC library and clone pools provided valuable resources and tools for genetic breeding and conservation of H. diversicolor.

From single-cell to cell-pool transcriptomes: stochasticity in gene expression and RNA splicing.

PubMed

Marinov, Georgi K; Williams, Brian A; McCue, Ken; Schroth, Gary P; Gertz, Jason; Myers, Richard M; Wold, Barbara J

2014-03-01

Single-cell RNA-seq mammalian transcriptome studies are at an early stage in uncovering cell-to-cell variation in gene expression, transcript processing and editing, and regulatory module activity. Despite great progress recently, substantial challenges remain, including discriminating biological variation from technical noise. Here we apply the SMART-seq single-cell RNA-seq protocol to study the reference lymphoblastoid cell line GM12878. By using spike-in quantification standards, we estimate the absolute number of RNA molecules per cell for each gene and find significant variation in total mRNA content: between 50,000 and 300,000 transcripts per cell. We directly measure technical stochasticity by a pool/split design and find that there are significant differences in expression between individual cells, over and above technical variation. Specific gene coexpression modules were preferentially expressed in subsets of individual cells, including one enriched for mRNA processing and splicing factors. We assess cell-to-cell variation in alternative splicing and allelic bias and report evidence of significant differences in splice site usage that exceed splice variation in the pool/split comparison. Finally, we show that transcriptomes from small pools of 30-100 cells approach the information content and reproducibility of contemporary RNA-seq from large amounts of input material. Together, our results define an experimental and computational path forward for analyzing gene expression in rare cell types and cell states.

Systematic investigation of the relationship between high myopia and polymorphisms of the MMP2, TIMP2, and TIMP3 genes by a DNA pooling approach.

PubMed

Leung, Kim Hung; Yiu, Wai Chi; Yap, Maurice K H; Ng, Po Wah; Fung, Wai Yan; Sham, Pak Chung; Yip, Shea Ping

2011-06-01

This study examined the relationship between high myopia and three myopia candidate genes--matrix metalloproteinase 2 (MMP2) and tissue inhibitor of metalloproteinase-2 and -3 (TIMP2 and TIMP3)--involved in scleral remodeling. Recruited for the study were unrelated adult Han Chinese who were high myopes (spherical equivalent, ≤ -6.0 D in both eyes; cases) and emmetropes (within ±1.0 D in both eyes; controls). Sample set 1 had 300 cases and 300 controls, and sample set 2 had 356 cases and 354 controls. Forty-nine tag single-nucleotide polymorphisms (SNPs) were selected from these candidate genes. The first stage was an initial screen of six case pools and six control pools constructed from sample set 1, each pool consisting of 50 distinct subjects of the same affection status. In the second stage, positive SNPs from the first stage were confirmed by genotyping individual samples forming the DNA pools. In the third stage, positive SNPs from stage 2 were replicated, with sample set 2 genotyped individually. Of the 49 SNPs screened by DNA pooling, three passed the lenient threshold of P < 0.10 (nested ANOVA) and were followed up by individual genotyping. Of the three SNPs genotyped, two TIMP3 SNPs were found to be significantly associated with high myopia by single-marker or haplotype analysis. However, the initial positive results could not be replicated by sample set 2. MMP2, TIPM2, and TIMP3 genes were not associated with high myopia in this Chinese sample and hence are unlikely to play a major role in the genetic susceptibility to high myopia.

Rapid gene identification in sugar beet using deep sequencing of DNA from phenotypic pools selected from breeding panels.

PubMed

Ries, David; Holtgräwe, Daniela; Viehöver, Prisca; Weisshaar, Bernd

2016-03-15

The combination of bulk segregant analysis (BSA) and next generation sequencing (NGS), also known as mapping by sequencing (MBS), has been shown to significantly accelerate the identification of causal mutations for species with a reference genome sequence. The usual approach is to cross homozygous parents that differ for the monogenic trait to address, to perform deep sequencing of DNA from F2 plants pooled according to their phenotype, and subsequently to analyze the allele frequency distribution based on a marker table for the parents studied. The method has been successfully applied for EMS induced mutations as well as natural variation. Here, we show that pooling genetically diverse breeding lines according to a contrasting phenotype also allows high resolution mapping of the causal gene in a crop species. The test case was the monogenic locus causing red vs. green hypocotyl color in Beta vulgaris (R locus). We determined the allele frequencies of polymorphic sequences using sequence data from two diverging phenotypic pools of 180 B. vulgaris accessions each. A single interval of about 31 kbp among the nine chromosomes was identified which indeed contained the causative mutation. By applying a variation of the mapping by sequencing approach, we demonstrated that phenotype-based pooling of diverse accessions from breeding panels and subsequent direct determination of the allele frequency distribution can be successfully applied for gene identification in a crop species. Our approach made it possible to identify a small interval around the causative gene. Sequencing of parents or individual lines was not necessary. Whenever the appropriate plant material is available, the approach described saves time compared to the generation of an F2 population. In addition, we provide clues for planning similar experiments with regard to pool size and the sequencing depth required.

Selective DNA Pooling for Determination of Linkage between a Molecular Marker and a Quantitative Trait Locus

PubMed Central

Darvasi, A.; Soller, M.

1994-01-01

Selective genotyping is a method to reduce costs in marker-quantitative trait locus (QTL) linkage determination by genotyping only those individuals with extreme, and hence most informative, quantitative trait values. The DNA pooling strategy (termed: ``selective DNA pooling'') takes this one step further by pooling DNA from the selected individuals at each of the two phenotypic extremes, and basing the test for linkage on marker allele frequencies as estimated from the pooled samples only. This can reduce genotyping costs of marker-QTL linkage determination by up to two orders of magnitude. Theoretical analysis of selective DNA pooling shows that for experiments involving backcross, F(2) and half-sib designs, the power of selective DNA pooling for detecting genes with large effect, can be the same as that obtained by individual selective genotyping. Power for detecting genes with small effect, however, was found to decrease strongly with increase in the technical error of estimating allele frequencies in the pooled samples. The effect of technical error, however, can be markedly reduced by replication of technical procedures. It is also shown that a proportion selected of 0.1 at each tail will be appropriate for a wide range of experimental conditions. PMID:7896115

Microbial contributions to coupled arsenic and sulfur cycling in the acid-sulfide hot spring Champagne Pool, New Zealand.

PubMed

Hug, Katrin; Maher, William A; Stott, Matthew B; Krikowa, Frank; Foster, Simon; Moreau, John W

2014-01-01

Acid-sulfide hot springs are analogs of early Earth geothermal systems where microbial metal(loid) resistance likely first evolved. Arsenic is a metalloid enriched in the acid-sulfide hot spring Champagne Pool (Waiotapu, New Zealand). Arsenic speciation in Champagne Pool follows reaction paths not yet fully understood with respect to biotic contributions and coupling to biogeochemical sulfur cycling. Here we present quantitative arsenic speciation from Champagne Pool, finding arsenite dominant in the pool, rim and outflow channel (55-75% total arsenic), and dithio- and trithioarsenates ubiquitously present as 18-25% total arsenic. In the outflow channel, dimethylmonothioarsenate comprised ≤9% total arsenic, while on the outflow terrace thioarsenates were present at 55% total arsenic. We also quantified sulfide, thiosulfate, sulfate and elemental sulfur, finding sulfide and sulfate as major species in the pool and outflow terrace, respectively. Elemental sulfur concentration reached a maximum at the terrace. Phylogenetic analysis of 16S rRNA genes from metagenomic sequencing revealed the dominance of Sulfurihydrogenibium at all sites and an increased archaeal population at the rim and outflow channel. Several phylotypes were found closely related to known sulfur- and sulfide-oxidizers, as well as sulfur- and sulfate-reducers. Bioinformatic analysis revealed genes underpinning sulfur redox transformations, consistent with sulfur speciation data, and illustrating a microbial role in sulfur-dependent transformation of arsenite to thioarsenate. Metagenomic analysis also revealed genes encoding for arsenate reductase at all sites, reflecting the ubiquity of thioarsenate and a need for microbial arsenate resistance despite anoxic conditions. Absence of the arsenite oxidase gene, aio, at all sites suggests prioritization of arsenite detoxification over coupling to energy conservation. Finally, detection of methyl arsenic in the outflow channel, in conjunction with increased sequences from Aquificaceae, supports a role for methyltransferase in thermophilic arsenic resistance. Our study highlights microbial contributions to coupled arsenic and sulfur cycling at Champagne Pool, with implications for understanding the evolution of microbial arsenic resistance in sulfidic geothermal systems.

Gene expression changes in blood RNA after swimming in a chlorinated pool.

PubMed

Salas, Lucas A; Font-Ribera, Laia; Bustamante, Mariona; Sumoy, Lauro; Grimalt, Joan O; Bonnin, Sarah; Aguilar, Maria; Mattlin, Heidi; Hummel, Manuela; Ferrer, Anna; Kogevinas, Manolis; Villanueva, Cristina M

2017-08-01

Exposure to disinfection by-products (DBP) such as trihalomethanes (THM) in swimming pools has been linked to adverse health effects in humans, but their biological mechanisms are unclear. We evaluated short-term changes in blood gene expression of adult recreational swimmers after swimming in a chlorinated pool. Volunteers swam 40min in an indoor chlorinated pool. Blood samples were drawn and four THM (chloroform, bromodichloromethane, dibromochloromethane and bromoform) were measured in exhaled breath before and after swimming. Intensity of physical activity was measured as metabolic equivalents (METs). Gene expression in whole blood mRNA was evaluated using IlluminaHumanHT-12v3 Expression-BeadChip. Linear mixed models were used to evaluate the relationship between gene expression changes and THM exposure. Thirty-seven before-after pairs were analyzed. The median increase from baseline to after swimming were: 0.7 to 2.3 for MET, and 1.4 to 7.1μg/m 3 for exhaled total THM (sum of the four THM). Exhaled THM increased on average 0.94μg/m 3 per 1 MET. While 1643 probes were differentially expressed post-exposure. Of them, 189 were also associated with exhaled levels of individual/total THM or MET after False Discovery Rate. The observed associations with the exhaled THM were low to moderate (Log-fold change range: -0.17 to 0.15). In conclusion, we identified short-term gene expression changes associated with swimming in a pool that were minor in magnitude and their biological meaning was unspecific. The high collinearity between exhaled THM levels and intensity of physical activity precluded mutually adjusted models with both covariates. These exploratory results should be validated in future studies. Copyright © 2017. Published by Elsevier B.V.

Protocols for Biotechnological Interventions in Improvement of Vanilla (Vanilla planifolia Andrews.).

PubMed

Divakaran, Minoo; Babu, K Nirmal; Peter, K V

2016-01-01

Vanilla (Vanilla planifolia Andrews (syn. V. fragrans Salisb.), a native of Central America, is the primary source of natural vanillin and plays a major role in the global economy. The gene pool of vanilla is threatened by deforestation and overcollection that has resulted in disappearance of natural habitats and wild species. Continuous vegetative propagation and lack of natural seed set and sufficient variations in the gene pool hamper crop improvement programs. In vitro techniques, one of the key tools of plant biotechnology, can be employed for overcoming specific problems, viz. production of disease-free clones, inducing somaclonal variations, developing hybrids, gene pool conservation, incorporating desired traits by distant hybridization, genetic engineering, etc. However, realization of these objectives necessitates standardization of protocols. This chapter describes the various protocols optimized for crop improvement in Vanilla species.

High-Resolution Analysis of Human Y-Chromosome Variation Shows a Sharp Discontinuity and Limited Gene Flow between Northwestern Africa and the Iberian Peninsula

PubMed Central

Bosch, Elena; Calafell, Francesc; Comas, David; Oefner, Peter J.; Underhill, Peter A.; Bertranpetit, Jaume

2001-01-01

In the present study we have analyzed 44 Y-chromosome biallelic polymorphisms in population samples from northwestern (NW) Africa and the Iberian Peninsula, which allowed us to place each chromosome unequivocally in a phylogenetic tree based on >150 polymorphisms. The most striking results are that contemporary NW African and Iberian populations were found to have originated from distinctly different patrilineages and that the Strait of Gibraltar seems to have acted as a strong (although not complete) barrier to gene flow. In NW African populations, an Upper Paleolithic colonization that probably had its origin in eastern Africa contributed 75% of the current gene pool. In comparison, ∼78% of contemporary Iberian Y chromosomes originated in an Upper Paleolithic expansion from western Asia, along the northern rim of the Mediterranean basin. Smaller contributions to these gene pools (constituting 13% of Y chromosomes in NW Africa and 10% of Y chromosomes in Iberia) came from the Middle East during the Neolithic and, during subsequent gene flow, from Sub-Saharan to NW Africa. Finally, bidirectional gene flow across the Strait of Gibraltar has been detected: the genetic contribution of European Y chromosomes to the NW African gene pool is estimated at 4%, and NW African populations may have contributed 7% of Iberian Y chromosomes. The Islamic rule of Spain, which began in a.d. 711 and lasted almost 8 centuries, left only a minor contribution to the current Iberian Y-chromosome pool. The high-resolution analysis of the Y chromosome allows us to separate successive migratory components and to precisely quantify each historical layer. PMID:11254456

Clinicopathologic features of patients with non-small cell lung cancer harboring the EML4-ALK fusion gene: a meta-analysis.

PubMed

Wang, Ying; Wang, Shumin; Xu, Shiguang; Qu, Jiaqi; Liu, Bo

2014-01-01

The frequencies of EML4-ALK fusion gene in non-small cell lung cancer (NSCLC) with different clinicopathologic features described by previous studies are inconsistent. The key demographic and pathologic features associated with EML4-ALK fusion gene have not been definitively established. This meta-analysis was conducted to compare the frequency of the EML4-ALK fusion gene in patients with different clinicopathologic features and to identify an enriched population of patients with NSCLC harboring EML4-ALK fusion gene. The Pubmed and Embase databases for all studies on EML4-ALK fusion gene in NSCLC patients were searched up to July 2014. A criteria list and exclusion criteria were established to screen the studies. The frequency of the EML4-ALK fusion gene and the clinicopathologic features, including smoking status, pathologic type, gender, and EGFR status were abstracted. Seventeen articles consisting of 4511 NSCLC cases were included in this meta-analysis. A significant lower EML4-ALK fusion gene positive rate was associated with smokers (pooled OR = 0.40, 95% CI = 0.30-0.54, P<0.00001). A significantly higher EML4-ALK fusion gene positivity rate was associated with adenocarcinomas (pooled OR = 2.53, 95% CI = 1.66-3.86, P<0.0001) and female (pooled OR = 0.61, 95% CI = 0.41-0.90, P = 0.01). We found that a significantly lower EML4-ALK fusion gene positivity rate was associated with EGFR mutation (pooled OR = 0.07, 95% CI = 0.03-0.19, P<0.00001). No publication bias was observed in any meta-analysis (all P value of Egger's test >0.05); however, because of the small sample size, no results were in the meta-analysis regarding EGFR gene status. This meta-analysis revealed that the EML4-ALK fusion gene is highly correlated with a never/light smoking history, female and the pathologic type of adenocarcinoma, and is largely mutually exclusive of EGFR.

Clinicopathologic Features of Patients with Non-Small Cell Lung Cancer Harboring the EML4-ALK Fusion Gene: A Meta-Analysis

PubMed Central

Wang, Shumin; Xu, Shiguang; Qu, Jiaqi

2014-01-01

Background The frequencies of EML4-ALK fusion gene in non-small cell lung cancer (NSCLC) with different clinicopathologic features described by previous studies are inconsistent. The key demographic and pathologic features associated with EML4-ALK fusion gene have not been definitively established. This meta-analysis was conducted to compare the frequency of the EML4-ALK fusion gene in patients with different clinicopathologic features and to identify an enriched population of patients with NSCLC harboring EML4-ALK fusion gene. Methods The Pubmed and Embase databases for all studies on EML4-ALK fusion gene in NSCLC patients were searched up to July 2014. A criteria list and exclusion criteria were established to screen the studies. The frequency of the EML4-ALK fusion gene and the clinicopathologic features, including smoking status, pathologic type, gender, and EGFR status were abstracted. Results Seventeen articles consisting of 4511 NSCLC cases were included in this meta-analysis. A significant lower EML4-ALK fusion gene positive rate was associated with smokers (pooled OR = 0.40, 95% CI = 0.30–0.54, P<0.00001). A significantly higher EML4-ALK fusion gene positivity rate was associated with adenocarcinomas (pooled OR = 2.53, 95% CI = 1.66–3.86, P<0.0001) and female (pooled OR = 0.61, 95% CI = 0.41–0.90, P = 0.01). We found that a significantly lower EML4-ALK fusion gene positivity rate was associated with EGFR mutation (pooled OR = 0.07, 95% CI = 0.03–0.19, P<0.00001). No publication bias was observed in any meta-analysis (all P value of Egger's test >0.05); however, because of the small sample size, no results were in the meta-analysis regarding EGFR gene status. Conclusion This meta-analysis revealed that the EML4-ALK fusion gene is highly correlated with a never/light smoking history, female and the pathologic type of adenocarcinoma, and is largely mutually exclusive of EGFR. PMID:25360721

Exome Pool-Seq in neurodevelopmental disorders.

PubMed

Popp, Bernt; Ekici, Arif B; Thiel, Christian T; Hoyer, Juliane; Wiesener, Antje; Kraus, Cornelia; Reis, André; Zweier, Christiane

2017-12-01

High throughput sequencing has greatly advanced disease gene identification, especially in heterogeneous entities. Despite falling costs this is still an expensive and laborious technique, particularly when studying large cohorts. To address this problem we applied Exome Pool-Seq as an economic and fast screening technology in neurodevelopmental disorders (NDDs). Sequencing of 96 individuals can be performed in eight pools of 12 samples on less than one Illumina sequencer lane. In a pilot study with 96 cases we identified 27 variants, likely or possibly affecting function. Twenty five of these were identified in 923 established NDD genes (based on SysID database, status November 2016) (ACTB, AHDC1, ANKRD11, ATP6V1B2, ATRX, CASK, CHD8, GNAS, IFIH1, KCNQ2, KMT2A, KRAS, MAOA, MED12, MED13L, RIT1, SETD5, SIN3A, TCF4, TRAPPC11, TUBA1A, WAC, ZBTB18, ZMYND11), two in 543 (SysID) candidate genes (ZNF292, BPTF), and additionally a de novo loss-of-function variant in LRRC7, not previously implicated in NDDs. Most of them were confirmed to be de novo, but we also identified X-linked or autosomal-dominantly or autosomal-recessively inherited variants. With a detection rate of 28%, Exome Pool-Seq achieves comparable results to individual exome analyses but reduces costs by >85%. Compared with other large scale approaches using Molecular Inversion Probes (MIP) or gene panels, it allows flexible re-analysis of data. Exome Pool-Seq is thus well suited for large-scale, cost-efficient and flexible screening in characterized but heterogeneous entities like NDDs.

Registry in a tube: multiplexed pools of retrievable parts for genetic design space exploration

PubMed Central

Woodruff, Lauren B. A.; Gorochowski, Thomas E.; Roehner, Nicholas; Densmore, Douglas; Gordon, D. Benjamin; Nicol, Robert

2017-01-01

Abstract Genetic designs can consist of dozens of genes and hundreds of genetic parts. After evaluating a design, it is desirable to implement changes without the cost and burden of starting the construction process from scratch. Here, we report a two-step process where a large design space is divided into deep pools of composite parts, from which individuals are retrieved and assembled to build a final construct. The pools are built via multiplexed assembly and sequenced using next-generation sequencing. Each pool consists of ∼20 Mb of up to 5000 unique and sequence-verified composite parts that are barcoded for retrieval by PCR. This approach is applied to a 16-gene nitrogen fixation pathway, which is broken into pools containing a total of 55 848 composite parts (71.0 Mb). The pools encompass an enormous design space (1043 possible 23 kb constructs), from which an algorithm-guided 192-member 4.5 Mb library is built. Next, all 1030 possible genetic circuits based on 10 repressors (NOR/NOT gates) are encoded in pools where each repressor is fused to all permutations of input promoters. These demonstrate that multiplexing can be applied to encompass entire design spaces from which individuals can be accessed and evaluated. PMID:28007941

Modular design of synthetic gene circuits with biological parts and pools.

PubMed

Marchisio, Mario Andrea

2015-01-01

Synthetic gene circuits can be designed in an electronic fashion by displaying their basic components-Standard Biological Parts and Pools of molecules-on the computer screen and connecting them with hypothetical wires. This procedure, achieved by our add-on for the software ProMoT, was successfully applied to bacterial circuits. Recently, we have extended this design-methodology to eukaryotic cells. Here, highly complex components such as promoters and Pools of mRNA contain hundreds of species and reactions whose calculation demands a rule-based modeling approach. We showed how to build such complex modules via the joint employment of the software BioNetGen (rule-based modeling) and ProMoT (modularization). In this chapter, we illustrate how to utilize our computational tool for synthetic biology with the in silico implementation of a simple eukaryotic gene circuit that performs the logic AND operation.

Detecting differentially expressed genes in heterogeneous diseases using half Student's t-test.

PubMed

Hsu, Chun-Lun; Lee, Wen-Chung

2010-12-01

Microarray technology provides information about hundreds and thousands of gene-expression data in a single experiment. To search for disease-related genes, researchers test for those genes that are differentially expressed between the case subjects and the control subjects. The authors propose a new test, the 'half Student's t-test', specifically for detecting differentially expressed genes in heterogeneous diseases. Monte-Carlo simulation shows that the test maintains the nominal α level quite well for both normal and non-normal distributions. Power of the half Student's t is higher than that of the conventional 'pooled' Student's t when there is heterogeneity in the disease under study. The power gain by using the half Student's t can reach ∼10% when the standard deviation of the case group is 50% larger than that of the control group. Application to a colon cancer data reveals that when the false discovery rate (FDR) is controlled at 0.05, the half Student's t can detect 344 differentially expressed genes, whereas the pooled Student's t can detect only 65 genes. Or alternatively, if only 50 genes are to be selected, the FDR for the pooled Student's t has to be set at 0.0320 (false positive rate of ∼3%), but for the half Student's t, it can be at as low as 0.0001 (false positive rate of about one per ten thousands). The half Student's t-test is to be recommended for the detection of differentially expressed genes in heterogeneous diseases.

Meta-analysis of six genes (BDNF, DRD1, DRD3, DRD4, GRIN2B and MAOA) involved in neuroplasticity and the risk for alcohol dependence.

PubMed

Forero, Diego A; López-León, Sandra; Shin, Hyoung Doo; Park, Byung Lae; Kim, Dai-Jin

2015-04-01

Alcohol-related problems have a large impact on human health, accounting for around 4% of deaths and 4.5% of disability-adjusted life-years around the world. Genetic factors could explain a significant fraction of the risk for alcohol dependence (AD). Recent meta-analyses have found significant pooled odds ratios (ORs) for variants in the ADH1B, ADH1C, DRD2 and HTR2A genes. In the present study, we carried out a meta-analysis of common variants in 6 candidate genes involved in neurotransmission and neuroplasticity: BDNF, DRD1, DRD3, DRD4, GRIN2B and MAOA. We carried out a systematic search for published association studies that analyzed the genes of interest. Relevant articles were retrieved and demographic and genetic data were extracted. Pooled ORs were calculated using a random-effects model using the Meta-Analyst program. Dominant, recessive and allelic models were tested and analyses were also stratified by ethnicity. Forty two published studies were included in the current meta-analysis: BDNF-rs6265 (nine studies), DRD1-rs4532 (four studies), DRD3-rs6280 (eleven studies), DRD4-VNTR (seven studies), GRIN2B-rs1806201 (three studies) and MAOA-uVNTR (eight studies). We did not find significant pooled ORs for any of the six genes, under different models and stratifying for ethnicity. In terms of the number of candidate genes included, this is one of the most comprehensive meta-analyses for genetics of AD. Pooled ORs did not support consistent associations with any of the six candidate genes tested. Future studies of novel genes of functional relevance and meta-analyses of quantitative endophenotypes could identify further susceptibility molecular factors for AD. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Antibiotic Administration Routes Significantly Influence the Levels of Antibiotic Resistance in Gut Microbiota

PubMed Central

Zhang, Lu; Huang, Ying; Zhou, Yang; Buckley, Timothy

2013-01-01

This study examined the impact of oral exposure to antibiotic-resistant bacteria and antibiotic administration methods on antibiotic resistance (AR) gene pools and the profile of resistant bacteria in host gastrointestinal (GI) tracts using C57BL/6J mice with natural gut microbiota. Mice inoculated with a mixture of tet(M)-carrying Enterococcus spp. or blaCMY-2-carrying Escherichia coli were treated with different doses of tetracycline hydrochloride (Tet) or ampicillin sodium (Amp) and delivered via either feed or intravenous (i.v.) injection. Quantitative PCR assessment of mouse fecal samples revealed that (i) AR gene pools were below the detection limit in mice without prior inoculation of AR gene carriers regardless of subsequent exposure to corresponding antibiotics; (ii) oral exposure to high doses of Tet and Amp in mice inoculated with AR gene carriers led to rapid enrichment of corresponding AR gene pools in feces; (iii) significantly less or delayed development of AR in the GI tract of the AR carrier-inoculated mice was observed when the same doses of antibiotics were administered via i.v. injection rather than oral administration; and (iv) antibiotic dosage, and maybe the excretion route, affected AR in the GI tract. The shift of dominant AR bacterial populations in the gut microbiota was consistent with the dynamics of AR gene pools. The emergence of endogenous resistant bacteria in the gut microbiota corresponding to drug exposure was also observed. Together, these data suggest that oral administration of antibiotics has a prominent effect on AR amplification and development in gut microbiota, which may be minimized by alternative drug administration approaches, as illustrated by i.v. injection in this study and proper drug selection. PMID:23689712

Antibiotic administration routes significantly influence the levels of antibiotic resistance in gut microbiota.

PubMed

Zhang, Lu; Huang, Ying; Zhou, Yang; Buckley, Timothy; Wang, Hua H

2013-08-01

This study examined the impact of oral exposure to antibiotic-resistant bacteria and antibiotic administration methods on antibiotic resistance (AR) gene pools and the profile of resistant bacteria in host gastrointestinal (GI) tracts using C57BL/6J mice with natural gut microbiota. Mice inoculated with a mixture of tet(M)-carrying Enterococcus spp. or blaCMY-2-carrying Escherichia coli were treated with different doses of tetracycline hydrochloride (Tet) or ampicillin sodium (Amp) and delivered via either feed or intravenous (i.v.) injection. Quantitative PCR assessment of mouse fecal samples revealed that (i) AR gene pools were below the detection limit in mice without prior inoculation of AR gene carriers regardless of subsequent exposure to corresponding antibiotics; (ii) oral exposure to high doses of Tet and Amp in mice inoculated with AR gene carriers led to rapid enrichment of corresponding AR gene pools in feces; (iii) significantly less or delayed development of AR in the GI tract of the AR carrier-inoculated mice was observed when the same doses of antibiotics were administered via i.v. injection rather than oral administration; and (iv) antibiotic dosage, and maybe the excretion route, affected AR in the GI tract. The shift of dominant AR bacterial populations in the gut microbiota was consistent with the dynamics of AR gene pools. The emergence of endogenous resistant bacteria in the gut microbiota corresponding to drug exposure was also observed. Together, these data suggest that oral administration of antibiotics has a prominent effect on AR amplification and development in gut microbiota, which may be minimized by alternative drug administration approaches, as illustrated by i.v. injection in this study and proper drug selection.

Whole Genome Association Study in a Homogenous Population in Shandong Peninsula of China Reveals JARID2 as a Susceptibility Gene for Schizophrenia

PubMed Central

Liu, Yang; Chen, Gang; Norton, Nadine; Liu, Wenmin; Zhu, Haining; Zhou, Peng; Luan, Meng; Yang, Shulin; Chen, Xing; Carroll, Liam; Williams, Nigel M.; O'Donovan, Michael C.; Kirov, George; Owen, Michael J.

2009-01-01

DNA pooling can provide an economic and efficient way to detect susceptibility loci to complex diseases. We carried out a genome screen with 400 microsatellite markers spaced at approximately 10 cm in two DNA pools consisting of 119 schizophrenia (SZ) patients and 119 controls recruited from a homogenous population in the Chang Le area of the Shandong peninsula of China. Association of D6S289, a dinucleotide repeat polymorphism in the JARID2 gene with SZ, was found and confirmed by individual genotyping (X2 = 17.89; P = .047). In order to refine the signal, we genotyped 14 single nucleotide polymorphisms (SNPs) covering JARID2 and the neighboring gene, DNTBP1, in an extended sample of 309 cases and 309 controls from Shandong peninsula (including the samples from the pools). However, rs2235258 and rs9654600 in JARID2 showed association in allelic, genotypic and haplotypic tests with SZ patients from Chang Le area. This was not replicates in the extended sample, we conclude that JARID2 could be a susceptibility gene for SZ. PMID:19884986

Association between maternal, fetal and paternal MTHFR gene C677T and A1298C polymorphisms and risk of recurrent pregnancy loss: a comprehensive evaluation.

PubMed

Yang, Yi; Luo, Yunyao; Yuan, Jing; Tang, Yidan; Xiong, Lang; Xu, MangMang; Rao, XuDong; Liu, Hao

2016-06-01

Numerous studies have investigated the associations between methylenetetrahydrofolate reductase (MTHFR) gene C677T and A1298C polymorphisms and risk of recurrent pregnancy loss (RPL); however, the results remain controversial. The aim of this study is to drive a more precise estimation of association between MTHFR gene polymorphisms and risk of RPL. We searched PubMed, EMBASE, Cochrane library, Web of Science and China Knowledge Resource Integrated Database for papers on MTHFR gene C677T and A1298C polymorphisms and RPL risk. The pooled odds ratios (ORs) with 95 % confidence intervals (CIs) were used to assess the strength of association in the homozygous model, heterozygous model, dominant model, recessive model and an additive model. The software STATA (Version 13.0) was used for statistical analysis. Overall, 57 articles were included in the final meta-analysis. In maternal group the MTHFR C677T polymorphism showed pooled odds ratios for the homozygous comparison [OR = 2.285, 95 % CI (1.702, 3.067)] and the MTHFR A1298C polymorphism showed pooled odds ratios for recessive model [OR = 1.594, 95 % CI (1.136, 2.238)]. In fetal group the MTHFR C677T polymorphism showed pooled odds ratios for dominant model [OR = 1.037, 95 % CI (0.567, 1.894)] and the MTHFR A1298C polymorphism showed pooled odds ratios for dominant model [OR = 1.495, 95 % CI (1.102, 2.026)]. In summary, the results of our meta-analysis indicate that maternal and paternal MTHFR gene C677T and A1298C polymorphisms are associated with RPL. We also observed a significant association between fetal MTHFR A1298C polymorphism and RPL but not C677T.

Association between SLC2A9 (GLUT9) gene polymorphisms and gout susceptibility: an updated meta-analysis.

PubMed

Zhang, Xu; Yang, Xiao; Wang, Mengmeng; Li, Xiaona; Xia, Qing; Xu, Shengqian; Xu, Jianhua; Cai, Guoqi; Wang, Li; Xin, Lihong; Zou, Yanfeng; Pan, Faming

2016-08-01

The relationship between the SLC2A9 (solute carrier family 2, member 9) gene polymorphisms and gout was still inconsistent among the individual genetic association studies. Therefore, this present research was aimed to systematically evaluate the association between SLC2A9 gene polymorphisms and gout susceptibility. Relevant studies were enrolled by searching databases systematically. The pooled odds ratios (ORs) with 95 % confidence intervals (CIs) were used to assess the associations. The heterogeneity between each of the studies was calculated by using the Q statistic methods, and Begg's funnel plot and Egger's tests were performed to evaluate publication bias. A total of 13 studies investigated four single nucleotide polymorphisms (SNPs) in SLC2A9 were included. In this study, we found that the allele C of rs3733591 was higher in patients than in controls in both all-pooled population [C vs. T: OR (95 % CI) = 1.432 (1.213-1.691)] and Asians-pooled population [C vs. T: OR (95 % CI) = 1.583 (1.365-1.835)]. The allele frequency C of s6449213 was lower in the gout patients than in controls in both all-pooled population and Caucasians-pooled population. Additionally, the allele frequency T of rs16890979 and the allele frequency C of rs1014290 were lower in gout patients than in controls. This study demonstrated that the genetic susceptibility for gout is associated with the SLC2A9 gene polymorphisms. Four of them except for the rs3733591 are protective SNPs in Caucasians, and rs16890979 and rs1014290 are protective SNPs in both Caucasians and Asians, while rs3733591 may be susceptibility SNP in Asians.

Comparative use of InDel and SSR markers in deciphering the interspecific structure of cultivated citrus genetic diversity: a perspective for genetic association studies.

PubMed

García-Lor, Andrés; Luro, François; Navarro, Luis; Ollitrault, Patrick

2012-01-01

Genetic stratification associated with domestication history is a key parameter for estimating the pertinence of genetic association study within a gene pool. Previous molecular and phenotypic studies have shown that most of the diversity of cultivated citrus results from recombination between three main species: C. medica (citron), C. reticulata (mandarin) and C. maxima (pummelo). However, the precise contribution of each of these basic species to the genomes of secondary cultivated species, such as C. sinensis (sweet orange), C. limon (lemon), C. aurantium (sour orange), C. paradisi (grapefruit) and recent hybrids is unknown. Our study focused on: (1) the development of insertion-deletion (InDel) markers and their comparison with SSR markers for use in genetic diversity and phylogenetic studies; (2) the analysis of the contributions of basic taxa to the genomes of secondary species and modern cultivars and (3) the description of the organisation of the Citrus gene pool, to evaluate how genetic association studies should be done at the cultivated Citrus gene pool level. InDel markers appear to be better phylogenetic markers for tracing the contributions of the three ancestral species, whereas SSR markers are more useful for intraspecific diversity analysis. Most of the genetic organisation of the Citrus gene pool is related to the differentiation between C. reticulata, C. maxima and C. medica. High and generalised LD was observed, probably due to the initial differentiation between the basic species and a limited number of interspecific recombinations. This structure precludes association genetic studies at the genus level without developing additional recombinant populations from interspecific hybrids. Association genetic studies should also be affordable at intraspecific level in a less structured pool such as C. reticulata.

Registry in a tube: multiplexed pools of retrievable parts for genetic design space exploration.

PubMed

Woodruff, Lauren B A; Gorochowski, Thomas E; Roehner, Nicholas; Mikkelsen, Tarjei S; Densmore, Douglas; Gordon, D Benjamin; Nicol, Robert; Voigt, Christopher A

2017-02-17

Genetic designs can consist of dozens of genes and hundreds of genetic parts. After evaluating a design, it is desirable to implement changes without the cost and burden of starting the construction process from scratch. Here, we report a two-step process where a large design space is divided into deep pools of composite parts, from which individuals are retrieved and assembled to build a final construct. The pools are built via multiplexed assembly and sequenced using next-generation sequencing. Each pool consists of ∼20 Mb of up to 5000 unique and sequence-verified composite parts that are barcoded for retrieval by PCR. This approach is applied to a 16-gene nitrogen fixation pathway, which is broken into pools containing a total of 55 848 composite parts (71.0 Mb). The pools encompass an enormous design space (1043 possible 23 kb constructs), from which an algorithm-guided 192-member 4.5 Mb library is built. Next, all 1030 possible genetic circuits based on 10 repressors (NOR/NOT gates) are encoded in pools where each repressor is fused to all permutations of input promoters. These demonstrate that multiplexing can be applied to encompass entire design spaces from which individuals can be accessed and evaluated. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Pooled Enrichment Sequencing Identifies Diversity and Evolutionary Pressures at NLR Resistance Genes within a Wild Tomato Population

PubMed Central

Stam, Remco; Scheikl, Daniela; Tellier, Aurélien

2016-01-01

Nod-like receptors (NLRs) are nucleotide-binding domain and leucine-rich repeats containing proteins that are important in plant resistance signaling. Many of the known pathogen resistance (R) genes in plants are NLRs and they can recognize pathogen molecules directly or indirectly. As such, divergence and copy number variants at these genes are found to be high between species. Within populations, positive and balancing selection are to be expected if plants coevolve with their pathogens. In order to understand the complexity of R-gene coevolution in wild nonmodel species, it is necessary to identify the full range of NLRs and infer their evolutionary history. Here we investigate and reveal polymorphism occurring at 220 NLR genes within one population of the partially selfing wild tomato species Solanum pennellii. We use a combination of enrichment sequencing and pooling ten individuals, to specifically sequence NLR genes in a resource and cost-effective manner. We focus on the effects which different mapping and single nucleotide polymorphism calling software and settings have on calling polymorphisms in customized pooled samples. Our results are accurately verified using Sanger sequencing of polymorphic gene fragments. Our results indicate that some NLRs, namely 13 out of 220, have maintained polymorphism within our S. pennellii population. These genes show a wide range of πN/πS ratios and differing site frequency spectra. We compare our observed rate of heterozygosity with expectations for this selfing and bottlenecked population. We conclude that our method enables us to pinpoint NLR genes which have experienced natural selection in their habitat. PMID:27189991

Rapid Creation and Quantitative Monitoring of High Coverage shRNA Libraries

PubMed Central

Bassik, Michael C.; Lebbink, Robert Jan; Churchman, L. Stirling; Ingolia, Nicholas T.; Patena, Weronika; LeProust, Emily M.; Schuldiner, Maya; Weissman, Jonathan S.; McManus, Michael T.

2009-01-01

Short hairpin RNA (shRNA) libraries are limited by the low efficacy of many shRNAs, giving false negatives, and off-target effects, giving false positives. Here we present a strategy for rapidly creating expanded shRNA pools (∼30 shRNAs/gene) that are analyzed by deep-sequencing (EXPAND). This approach enables identification of multiple effective target-specific shRNAs from a complex pool, allowing a rigorous statistical evaluation of whether a gene is a true hit. PMID:19448642

MISSION LentiPlex pooled shRNA library screening in mammalian cells.

PubMed

Coussens, Matthew J; Corman, Courtney; Fischer, Ashley L; Sago, Jack; Swarthout, John

2011-12-21

RNA interference (RNAi) is an intrinsic cellular mechanism for the regulation of gene expression. Harnessing the innate power of this system enables us to knockdown gene expression levels in loss of gene function studies. There are two main methods for performing RNAi. The first is the use of small interfering RNAs (siRNAs) that are chemically synthesized, and the second utilizes short-hairpin RNAs (shRNAs) encoded within plasmids. The latter can be transfected into cells directly or packaged into replication incompetent lentiviral particles. The main advantages of using lentiviral shRNAs is the ease of introduction into a wide variety of cell types, their ability to stably integrate into the genome for long term gene knockdown and selection, and their efficacy in conducting high-throughput loss of function screens. To facilitate this we have created the LentiPlex pooled shRNA library. The MISSION LentiPlex Human shRNA Pooled Library is a genome-wide lentiviral pool produced using a proprietary process. The library consists of over 75,000 shRNA constructs from the TRC collection targeting 15,000+ human genes. Each library is tested for shRNA representation before product release to ensure robust library coverage. The library is provided in a ready-to-use lentiviral format at titers of at least 5 x 10(8) TU/ml via p24 assay and is pre-divided into ten subpools of approximately 8,000 shRNA constructs each. Amplification and sequencing primers are also provided for downstream target identification. Previous studies established a synergistic antitumor activity of TRAIL when combined with Paclitaxel in A549 cells, a human lung carcinoma cell line. In this study we demonstrate the application of a pooled LentiPlex shRNA library to rapidly conduct a positive selection screen for genes involved in the cytotoxicity of A549 cells when exposed to TRAIL and Paclitaxel. One barrier often encountered with high-throughput screens is the cost and difficulty in deconvolution; we also detail a cost-effective polyclonal approach utilizing traditional sequencing.

Genetic Association and Gene-Gene Interaction Analyses in African American Dialysis Patients With Nondiabetic Nephropathy

PubMed Central

Bostrom, Meredith A.; Kao, W.H. Linda; Li, Man; Abboud, Hanna E.; Adler, Sharon G.; Iyengar, Sudha K.; Kimmel, Paul L.; Hanson, Robert L.; Nicholas, Susanne B.; Rasooly, Rebekah S.; Sedor, John R.; Coresh, Josef; Kohn, Orly F.; Leehey, David J.; Thornley-Brown, Denyse; Bottinger, Erwin P.; Lipkowitz, Michael S.; Meoni, Lucy A.; Klag, Michael J.; Lu, Lingyi; Hicks, Pamela J.; Langefeld, Carl D.; Parekh, Rulan S.; Bowden, Donald W.; Freedman, Barry I.

2011-01-01

Background African Americans (AAs) have increased susceptibility to non-diabetic nephropathy relative to European Americans. Study Design Follow-up of a pooled genome-wide association study (GWAS) in AA dialysis patients with nondiabetic nephropathy; novel gene-gene interaction analyses. Setting & Participants Wake Forest sample: 962 AA nondiabetic nephropathy cases; 931 non-nephropathy controls. Replication sample: 668 Family Investigation of Nephropathy and Diabetes (FIND) AA nondiabetic nephropathy cases; 804 non-nephropathy controls. Predictors Individual genotyping of top 1420 pooled GWAS-associated single nucleotide polymorphisms (SNPs) and 54 SNPs in six nephropathy susceptibility genes. Outcomes APOL1 genetic association and additional candidate susceptibility loci interacting with, or independently from, APOL1. Results The strongest GWAS associations included two non-coding APOL1 SNPs, rs2239785 (odds ratio [OR], 0.33; dominant; p = 5.9 × 10−24) and rs136148 (OR, 0.54; additive; p = 1.1 × 10−7) with replication in FIND (p = 5.0 × 10−21 and 1.9 × 10−05, respectively). Rs2239785 remained significantly associated after controlling for the APOL1 G1 and G2 coding variants. Additional top hits included a CFH SNP(OR from meta-analysis in above 3367 AA cases and controls, 0.81; additive; p = 6.8 × 10−4). The 1420 SNPs were tested for interaction with APOL1 G1 and G2 variants. Several interactive SNPs were detected, the most significant was rs16854341 in the podocin gene (NPHS2) (p = 0.0001). Limitations Non-pooled GWAS have not been performed in AA nondiabetic nephropathy. Conclusions This follow-up of a pooled GWAS provides additional and independent evidence that APOL1 variants contribute to nondiabetic nephropathy in AAs and identified additional associated and interactive non-diabetic nephropathy susceptibility genes. PMID:22119407

Gene expression profiling of pre-eclamptic placentae by RNA sequencing.

PubMed

Kaartokallio, Tea; Cervera, Alejandra; Kyllönen, Anjuska; Laivuori, Krista; Kere, Juha; Laivuori, Hannele

2015-09-21

Pre-eclampsia is a common and complex pregnancy disorder that often involves impaired placental development. In order to identify altered gene expression in pre-eclamptic placenta, we sequenced placental transcriptomes of nine pre-eclamptic and nine healthy pregnant women in pools of three. The differential gene expression was tested both by including all the pools in the analysis and by excluding some of the pools based on phenotypic characteristics. From these analyses, we identified altogether 53 differently expressed genes, a subset of which was validated by qPCR in 20 cases and 19 controls. Furthermore, we conducted pathway and functional analyses which revealed disturbed vascular function and immunological balance in pre-eclamptic placenta. Some of the genes identified in our study have been reported by numerous microarray studies (BHLHE40, FSTL3, HK2, HTRA4, LEP, PVRL4, SASH1, SIGLEC6), but many have been implicated in only few studies or have not previously been linked to pre-eclampsia (ARMS2, BTNL9, CCSAP, DIO2, FER1L4, HPSE, LOC100129345, LYN, MYO7B, NCMAP, NDRG1, NRIP1, PLIN2, SBSPON, SERPINB9, SH3BP5, TET3, TPBG, ZNF175). Several of the molecules produced by these genes may have a role in the pathogenesis of pre-eclampsia, and some could qualify as biomarkers for prediction or detection of this pregnancy complication.

Gene expression profiling of pre-eclamptic placentae by RNA sequencing

PubMed Central

Kaartokallio, Tea; Cervera, Alejandra; Kyllönen, Anjuska; Laivuori, Krista; Laivuori, Hannele; Heinonen, Seppo; Kajantie, Eero; Kere, Juha; Kivinen, Katja; Pouta, Anneli

2015-01-01

Pre-eclampsia is a common and complex pregnancy disorder that often involves impaired placental development. In order to identify altered gene expression in pre-eclamptic placenta, we sequenced placental transcriptomes of nine pre-eclamptic and nine healthy pregnant women in pools of three. The differential gene expression was tested both by including all the pools in the analysis and by excluding some of the pools based on phenotypic characteristics. From these analyses, we identified altogether 53 differently expressed genes, a subset of which was validated by qPCR in 20 cases and 19 controls. Furthermore, we conducted pathway and functional analyses which revealed disturbed vascular function and immunological balance in pre-eclamptic placenta. Some of the genes identified in our study have been reported by numerous microarray studies (BHLHE40, FSTL3, HK2, HTRA4, LEP, PVRL4, SASH1, SIGLEC6), but many have been implicated in only few studies or have not previously been linked to pre-eclampsia (ARMS2, BTNL9, CCSAP, DIO2, FER1L4, HPSE, LOC100129345, LYN, MYO7B, NCMAP, NDRG1, NRIP1, PLIN2, SBSPON, SERPINB9, SH3BP5, TET3, TPBG, ZNF175). Several of the molecules produced by these genes may have a role in the pathogenesis of pre-eclampsia, and some could qualify as biomarkers for prediction or detection of this pregnancy complication. PMID:26388242

Rapid protein production from stable CHO cell pools using plasmid vector and the cumate gene-switch.

PubMed

Poulain, Adeline; Perret, Sylvie; Malenfant, Félix; Mullick, Alaka; Massie, Bernard; Durocher, Yves

2017-08-10

To rapidly produce large amounts of recombinant proteins, the generation of stable Chinese Hamster Ovary (CHO) cell pools represents a useful alternative to large-scale transient gene expression (TGE). We have developed a cell line (CHO BRI/rcTA ) allowing the inducible expression of recombinant proteins, based on the cumate gene switch. After the identification of optimal plasmid DNA topology (supercoiled vs linearized plasmid) for PEIpro™ mediated transfection and of optimal conditions for methionine sulfoximine (MSX) selection, we were able to generate CHO BRI/rcTA pools producing high levels of recombinant proteins. Volumetric productivities of up to 900mg/L were reproducibly achieved for a Fc fusion protein and up to 350mg/L for an antibody after 14days post-induction in non-optimized fed-batch cultures. In addition, we show that CHO pool volumetric productivities are not affected by a freeze-thaw cycle or following maintenance in culture for over one month in the presence of MSX. Finally, we demonstrate that volumetric protein production with the CR5 cumate-inducible promoter is three- to four-fold higher than with the human CMV or hybrid EF1α-HTLV constitutive promoters. These results suggest that the cumate-inducible CHO BRI/rcTA stable pool platform is a powerful and robust system for the rapid production of gram amounts of recombinant proteins. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

Origin of year-long bean (Phaseolus dumosus Macfady, Fabaceae) from reticulated hybridization events between multiple Phaseolus species.

PubMed

Mina-Vargas, Angela M; McKeown, Peter C; Flanagan, Nicola S; Debouck, Daniel G; Kilian, Andrzej; Hodkinson, Trevor R; Spillane, Charles

2016-08-06

Improved understanding of the secondary gene pools of crops is essential for advancing genetic gain in breeding programmes. Common bean, Phaseolus vulgaris, is a staple crop with several wild relatives in its secondary gene pool. The year-long bean, P. dumosus, an important crop in Guatemala, is considered particularly closely related to P. vulgaris and a potential source of novel variation. However, the genetic diversity and relationship to other Phaseolus species of P. dumosus remain unclear. We conducted the first comprehensive investigation of P. dumosus genetic diversity using both nuclear and chloroplast genome markers. Our nuclear marker set included over 700 markers present within the Phaseolus DArT (Diversity Arrays Technology) array, which we applied to P. dumosus and other relatives of P. vulgaris (including every secondary gene pool species: P. acutifolius, P. albescens, P. coccineus and P. costaricensis). Phaseolus dumosus arose from hybridization of P. vulgaris and P. coccineus, followed by at least two later hybridizations with sympatric congener populations. Existing P. dumosus collections have low genetic diversity. The under-utilized crop P. dumosus has a complex hybrid origin. Further sampling in the region in which it arose may uncover additional germplasm for introgressing favourable traits into crops within the P. vulgaris gene pool. © The Author 2016. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Bacteria from Animals as a Pool of Antimicrobial Resistance Genes

PubMed Central

Argudín, Maria Angeles; Deplano, Ariane; Meghraoui, Alaeddine; Dodémont, Magali; Heinrichs, Amelie; Denis, Olivier; Nonhoff, Claire; Roisin, Sandrine

2017-01-01

Antimicrobial agents are used in both veterinary and human medicine. The intensive use of antimicrobials in animals may promote the fixation of antimicrobial resistance genes in bacteria, which may be zoonotic or capable to transfer these genes to human-adapted pathogens or to human gut microbiota via direct contact, food or the environment. This review summarizes the current knowledge of the use of antimicrobial agents in animal health and explores the role of bacteria from animals as a pool of antimicrobial resistance genes for human bacteria. This review focused in relevant examples within the ESC(K)APE (Enterococcus faecium, Staphylococcus aureus, Clostridium difficile (Klebsiella pneumoniae), Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacteriaceae) group of bacterial pathogens that are the leading cause of nosocomial infections throughout the world. PMID:28587316

Genetic relationships among American donkey populations: insights into the process of colonization.

PubMed

Jordana, J; Ferrando, A; Miró, J; Goyache, F; Loarca, A; Martínez López, O R; Canelón, J L; Stemmer, A; Aguirre, L; Lara, M A C; Álvarez, L A; Llambí, S; Gómez, N; Gama, L T; Nóvoa, M F; Martínez, R D; Pérez, E; Sierra, A; Contreras, M A; Guastella, A M; Marletta, D; Arsenos, G; Curik, I; Landi, V; Martínez, A; Delgado, J V

2016-04-01

This study presents the first insights into the genetic diversity and structure of the American donkey metapopulation. The primary objectives were to detect the main structural features underlying variability among American donkey populations, identify boundaries between differentiated gene pools, and draw the main colonization pathways since the introduction of donkeys into America in the 15th century. A panel of 14 microsatellite markers was applied for genotyping 350 American donkeys from 13 countries. The genetic structure of this metapopulation was analysed using descriptive statistics and Bayesian model-based methods. These populations were then compared to a database containing information on 476 individuals from 11 European breeds to identify the most likely ancestral donor populations. Results showed the presence of two distinct genetic pools, with confluence of the two in Colombia. The southern pool showed a unique genetic signature subsequent to an older founder event, but lacked any significant influence of modern gene flow from Europe. The northern pool, conversely, may have retained more ancestral polymorphisms and/or have experienced modern gene flow from Spanish breeds. The Andalusian and, to a lesser extent, the Catalan breeds have left a more pronounced footprint in some of the American donkey populations analysed. © 2015 Blackwell Verlag GmbH.

From the ORFeome concept to highly comprehensive, full-genome screening libraries.

PubMed

Rid, Raphaela; Abdel-Hadi, Omar; Maier, Richard; Wagner, Martin; Hundsberger, Harald; Hintner, Helmut; Bauer, Johann; Onder, Kamil

2013-02-01

Recombination-based cloning techniques have in recent times facilitated the establishment of genome-scale single-gene ORFeome repositories. Their further handling and downstream application in systematic fashion is, however, practically impeded because of logistical plus economic challenges. At this juncture, simultaneously transferring entire gene collections in compiled pool format could represent an advanced compromise between systematic ORFeome (an organism's entire set of protein-encoding open reading frames) projects and traditional random library approaches, but has not yet been considered in great detail. In our endeavor to merge the comprehensiveness of ORFeomes with a basically simple, streamlined, and easily executable single-tube design, we have here produced five different pooled screening-ready libraries for both Staphylococcus aureus and Homo sapiens. By evaluating the parallel transfer efficiencies of differentially sized genes from initial polymerase chain reaction (PCR) product amplification to entry and final destination library construction via quantitative real-time PCR, we found that the complexity of the gene population is fairly stably maintained once an entry resource has been successfully established, and that no apparent size-selection bias loss of large inserts takes place. Recombinational transfer processes are hence robust enough for straightforwardly achieving such pooled screening libraries.

Microbial life in Champagne Pool, a geothermal spring in Waiotapu, New Zealand.

PubMed

Hetzer, Adrian; Morgan, Hugh W; McDonald, Ian R; Daughney, Christopher J

2007-07-01

Surveys of Champagne Pool, one of New Zealand's largest terrestrial hot springs and rich in arsenic ions and compounds, have been restricted to geological and geochemical descriptions, and a few microbiological studies applying culture-independent methods. In the current investigation, a combination of culture and culture-independent approaches were chosen to determine microbial density and diversity in Champagne Pool. Recovered total DNA and adenosine 5'-triphosphate (ATP) content of spring water revealed relatively low values compared to other geothermal springs within New Zealand and are in good agreement with low cell numbers of 5.6 +/- 0.5 x 10(6) cells/ml obtained for Champagne Pool water samples by 4',6-diamidino-2-phenylindole (DAPI) staining. Denaturing Gradient Gel Electrophoresis (DGGE) and 16S rRNA (small-subunit ribosomal nucleic acid) gene clone library analyses of environmental DNA indicated the abundance of Sulfurihydrogenibium, Sulfolobus, and Thermofilum-like populations in Champagne Pool. From these results, media were selected to target the enrichment of hydrogen-oxidizing and sulfur-dependent microorganisms. Three isolates were successfully obtained having 16S rRNA gene sequences with similarities of approximately 98% to Thermoanaerobacter tengcongensis, 94% to Sulfurihydrogenibium azorense, and 99% to Thermococcus waiotapuensis, respectively.

Pooled Enrichment Sequencing Identifies Diversity and Evolutionary Pressures at NLR Resistance Genes within a Wild Tomato Population.

PubMed

Stam, Remco; Scheikl, Daniela; Tellier, Aurélien

2016-06-02

Nod-like receptors (NLRs) are nucleotide-binding domain and leucine-rich repeats containing proteins that are important in plant resistance signaling. Many of the known pathogen resistance (R) genes in plants are NLRs and they can recognize pathogen molecules directly or indirectly. As such, divergence and copy number variants at these genes are found to be high between species. Within populations, positive and balancing selection are to be expected if plants coevolve with their pathogens. In order to understand the complexity of R-gene coevolution in wild nonmodel species, it is necessary to identify the full range of NLRs and infer their evolutionary history. Here we investigate and reveal polymorphism occurring at 220 NLR genes within one population of the partially selfing wild tomato species Solanum pennellii. We use a combination of enrichment sequencing and pooling ten individuals, to specifically sequence NLR genes in a resource and cost-effective manner. We focus on the effects which different mapping and single nucleotide polymorphism calling software and settings have on calling polymorphisms in customized pooled samples. Our results are accurately verified using Sanger sequencing of polymorphic gene fragments. Our results indicate that some NLRs, namely 13 out of 220, have maintained polymorphism within our S. pennellii population. These genes show a wide range of πN/πS ratios and differing site frequency spectra. We compare our observed rate of heterozygosity with expectations for this selfing and bottlenecked population. We conclude that our method enables us to pinpoint NLR genes which have experienced natural selection in their habitat. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Genetic diversity, population structure and subdivision of local Balkan pig breeds in Austria, Croatia, Serbia and Bosnia-Herzegovina and its practical value in conservation programs.

PubMed

Druml, Thomas; Salajpal, Kresimir; Dikic, Maria; Urosevic, Miroslav; Grilz-Seger, Gertrud; Baumung, Roswitha

2012-03-01

At present the Croatian Turopolje pig population comprises about 157 breeding animals. In Austria, 324 Turopolje pigs originating from six Croatian founder animals are registered. Multiple bottlenecks have occurred in this population, one major one rather recently and several more older and moderate ones. In addition, it has been subdivided into three subpopulations, one in Austria and two in Croatia, with restricted gene flow. These specificities explain the delicate situation of this endangered Croatian lard-type pig breed. In order to identify candidate breeding animals or gene pools for future conservation breeding programs, we studied the genetic diversity and population structure of this breed using microsatellite data from 197 individuals belonging to five different breeds. The genetic diversity of the Turopolje pig is dramatically low with observed heterozygosities values ranging from 0.38 to 0.57. Split into three populations since 1994, two genetic clusters could be identified: one highly conserved Croatian gene pool in Turopoljski Lug and the"Posavina" gene pool mainly present in the Austrian population. The second Croatian subpopulation in Lonjsko Polje in the Posavina region shows a constant gene flow from the Turopoljski Lug animals. One practical conclusion is that it is necessary to develop a "Posavina" boar line to preserve the "Posavina" gene pool and constitute a corresponding population in Croatia. Animals of the highly inbred herd in Turopoljski Lug should not be crossed with animals of other populations since they represent a specific phenotype-genotype combination. However to increase the genetic diversity of this herd, a program to optimize its sex ratio should be carried out, as was done in the Austrian population where the level of heterozygosity has remained moderate despite its heavy bottleneck in 1994. © 2012 Druml et al; licensee BioMed Central Ltd.

Mining a database of single amplified genomes from Red Sea brine pool extremophiles—improving reliability of gene function prediction using a profile and pattern matching algorithm (PPMA)

PubMed Central

Grötzinger, Stefan W.; Alam, Intikhab; Ba Alawi, Wail; Bajic, Vladimir B.; Stingl, Ulrich; Eppinger, Jörg

2014-01-01

Reliable functional annotation of genomic data is the key-step in the discovery of novel enzymes. Intrinsic sequencing data quality problems of single amplified genomes (SAGs) and poor homology of novel extremophile's genomes pose significant challenges for the attribution of functions to the coding sequences identified. The anoxic deep-sea brine pools of the Red Sea are a promising source of novel enzymes with unique evolutionary adaptation. Sequencing data from Red Sea brine pool cultures and SAGs are annotated and stored in the Integrated Data Warehouse of Microbial Genomes (INDIGO) data warehouse. Low sequence homology of annotated genes (no similarity for 35% of these genes) may translate into false positives when searching for specific functions. The Profile and Pattern Matching (PPM) strategy described here was developed to eliminate false positive annotations of enzyme function before progressing to labor-intensive hyper-saline gene expression and characterization. It utilizes InterPro-derived Gene Ontology (GO)-terms (which represent enzyme function profiles) and annotated relevant PROSITE IDs (which are linked to an amino acid consensus pattern). The PPM algorithm was tested on 15 protein families, which were selected based on scientific and commercial potential. An initial list of 2577 enzyme commission (E.C.) numbers was translated into 171 GO-terms and 49 consensus patterns. A subset of INDIGO-sequences consisting of 58 SAGs from six different taxons of bacteria and archaea were selected from six different brine pool environments. Those SAGs code for 74,516 genes, which were independently scanned for the GO-terms (profile filter) and PROSITE IDs (pattern filter). Following stringent reliability filtering, the non-redundant hits (106 profile hits and 147 pattern hits) are classified as reliable, if at least two relevant descriptors (GO-terms and/or consensus patterns) are present. Scripts for annotation, as well as for the PPM algorithm, are available through the INDIGO website. PMID:24778629

Construction of CRISPR Libraries for Functional Screening.

PubMed

Carstens, Carsten P; Felts, Katherine A; Johns, Sarah E

2018-01-01

Identification of gene function has been aided by the ability to generate targeted gene knockouts or transcriptional repression using the CRISPR/CAS9 system. Using pooled libraries of guide RNA expression vectors that direct CAS9 to a specific genomic site allows identification of genes that are either enriched or depleted in response to a selection scheme, thus linking the affected gene to the chosen phenotype. The quality of the data generated by the screening is dependent on the quality of the guide RNA delivery library with regards to error rates and especially evenness of distribution of the guides. Here, we describe a method for constructing complex plasmid libraries based on pooled designed oligomers with high representation and tight distributions. The procedure allows construction of plasmid libraries of >60,000 members with a 95th/5th percentile ratio of less than 3.5.

AST: an automated sequence-sampling method for improving the taxonomic diversity of gene phylogenetic trees.

PubMed

Zhou, Chan; Mao, Fenglou; Yin, Yanbin; Huang, Jinling; Gogarten, Johann Peter; Xu, Ying

2014-01-01

A challenge in phylogenetic inference of gene trees is how to properly sample a large pool of homologous sequences to derive a good representative subset of sequences. Such a need arises in various applications, e.g. when (1) accuracy-oriented phylogenetic reconstruction methods may not be able to deal with a large pool of sequences due to their high demand in computing resources; (2) applications analyzing a collection of gene trees may prefer to use trees with fewer operational taxonomic units (OTUs), for instance for the detection of horizontal gene transfer events by identifying phylogenetic conflicts; and (3) the pool of available sequences is biased towards extensively studied species. In the past, the creation of subsamples often relied on manual selection. Here we present an Automated sequence-Sampling method for improving the Taxonomic diversity of gene phylogenetic trees, AST, to obtain representative sequences that maximize the taxonomic diversity of the sampled sequences. To demonstrate the effectiveness of AST, we have tested it to solve four problems, namely, inference of the evolutionary histories of the small ribosomal subunit protein S5 of E. coli, 16 S ribosomal RNAs and glycosyl-transferase gene family 8, and a study of ancient horizontal gene transfers from bacteria to plants. Our results show that the resolution of our computational results is almost as good as that of manual inference by domain experts, hence making the tool generally useful to phylogenetic studies by non-phylogeny specialists. The program is available at http://csbl.bmb.uga.edu/~zhouchan/AST.php.

AST: An Automated Sequence-Sampling Method for Improving the Taxonomic Diversity of Gene Phylogenetic Trees

PubMed Central

Zhou, Chan; Mao, Fenglou; Yin, Yanbin; Huang, Jinling; Gogarten, Johann Peter; Xu, Ying

2014-01-01

A challenge in phylogenetic inference of gene trees is how to properly sample a large pool of homologous sequences to derive a good representative subset of sequences. Such a need arises in various applications, e.g. when (1) accuracy-oriented phylogenetic reconstruction methods may not be able to deal with a large pool of sequences due to their high demand in computing resources; (2) applications analyzing a collection of gene trees may prefer to use trees with fewer operational taxonomic units (OTUs), for instance for the detection of horizontal gene transfer events by identifying phylogenetic conflicts; and (3) the pool of available sequences is biased towards extensively studied species. In the past, the creation of subsamples often relied on manual selection. Here we present an Automated sequence-Sampling method for improving the Taxonomic diversity of gene phylogenetic trees, AST, to obtain representative sequences that maximize the taxonomic diversity of the sampled sequences. To demonstrate the effectiveness of AST, we have tested it to solve four problems, namely, inference of the evolutionary histories of the small ribosomal subunit protein S5 of E. coli, 16 S ribosomal RNAs and glycosyl-transferase gene family 8, and a study of ancient horizontal gene transfers from bacteria to plants. Our results show that the resolution of our computational results is almost as good as that of manual inference by domain experts, hence making the tool generally useful to phylogenetic studies by non-phylogeny specialists. The program is available at http://csbl.bmb.uga.edu/~zhouchan/AST.php. PMID:24892935

Adaptive genomic divergence under high gene flow between freshwater and brackish-water ecotypes of prickly sculpin (Cottus asper) revealed by Pool-Seq.

PubMed

Dennenmoser, Stefan; Vamosi, Steven M; Nolte, Arne W; Rogers, Sean M

2017-01-01

Understanding the genomic basis of adaptive divergence in the presence of gene flow remains a major challenge in evolutionary biology. In prickly sculpin (Cottus asper), an abundant euryhaline fish in northwestern North America, high genetic connectivity among brackish-water (estuarine) and freshwater (tributary) habitats of coastal rivers does not preclude the build-up of neutral genetic differentiation and emergence of different life history strategies. Because these two habitats present different osmotic niches, we predicted high genetic differentiation at known teleost candidate genes underlying salinity tolerance and osmoregulation. We applied whole-genome sequencing of pooled DNA samples (Pool-Seq) to explore adaptive divergence between two estuarine and two tributary habitats. Paired-end sequence reads were mapped against genomic contigs of European Cottus, and the gene content of candidate regions was explored based on comparisons with the threespine stickleback genome. Genes showing signals of repeated differentiation among brackish-water and freshwater habitats included functions such as ion transport and structural permeability in freshwater gills, which suggests that local adaptation to different osmotic niches might contribute to genomic divergence among habitats. Overall, the presence of both repeated and unique signatures of differentiation across many loci scattered throughout the genome is consistent with polygenic adaptation from standing genetic variation and locally variable selection pressures in the early stages of life history divergence. © 2016 John Wiley & Sons Ltd.

Novel Two-Step Hierarchical Screening of Mutant Pools Reveals Mutants under Selection in Chicks

PubMed Central

Yang, Hee-Jeong; Bogomolnaya, Lydia M.; Elfenbein, Johanna R.; Endicott-Yazdani, Tiana; Reynolds, M. Megan; Porwollik, Steffen; Cheng, Pui; Xia, Xiao-Qin

2016-01-01

Contaminated chicken/egg products are major sources of human salmonellosis, yet the strategies used by Salmonella to colonize chickens are poorly understood. We applied a novel two-step hierarchical procedure to identify new genes important for colonization and persistence of Salmonella enterica serotype Typhimurium in chickens. A library of 182 S. Typhimurium mutants each containing a targeted deletion of a group of contiguous genes (for a total of 2,069 genes deleted) was used to identify regions under selection at 1, 3, and 9 days postinfection in chicks. Mutants in 11 regions were under selection at all assayed times (colonization mutants), and mutants in 15 regions were under selection only at day 9 (persistence mutants). We assembled a pool of 92 mutants, each deleted for a single gene, representing nearly all genes in nine regions under selection. Twelve single gene deletion mutants were under selection in this assay, and we confirmed 6 of 9 of these candidate mutants via competitive infections and complementation analysis in chicks. STM0580, STM1295, STM1297, STM3612, STM3615, and STM3734 are needed for Salmonella to colonize and persist in chicks and were not previously associated with this ability. One of these key genes, STM1297 (selD), is required for anaerobic growth and supports the ability to utilize formate under these conditions, suggesting that metabolism of formate is important during infection. We report a hierarchical screening strategy to interrogate large portions of the genome during infection of animals using pools of mutants of low complexity. Using this strategy, we identified six genes not previously known to be needed during infection in chicks, and one of these (STM1297) suggests an important role for formate metabolism during infection. PMID:26857572

Gene Flow within and between Catchments in the Threatened Riparian Plant Myricaria germanica

PubMed Central

Werth, Silke; Scheidegger, Christoph

2014-01-01

One of the major distinctions of riparian habitats is their linearity. In linear habitats, gene flow is predicted to follow a one-dimensional stepping stone model, characterized by bidirectional gene flow between neighboring populations. Here, we studied the genetic structure of Myricaria germanica, a threatened riparian shrub which is capable of both wind and water dispersal. Our data led us to reject the ‘one catchment – one gene pool’ hypothesis as we found support for two gene pools, rather than four as expected in a study area including four catchments. This result also implies that in the history of the studied populations, dispersal across catchments has occurred. Two contemporary catchment-crossing migration events were detected, albeit between spatially proximate catchments. Allelic richness and inbreeding coefficients differed substantially between gene pools. There was significant isolation by distance, and our data confirmed the one-dimensional stepping-stone model of gene flow. Contemporary migration was bidirectional within the studied catchments, implying that dispersal vectors other than water are important for M. germanica. PMID:24932520

Genetic structure of the date palm (Phoenix dactylifera) in the Old World reveals a strong differentiation between eastern and western populations

PubMed Central

Zehdi-Azouzi, Salwa; Cherif, Emira; Moussouni, Souhila; Gros-Balthazard, Muriel; Abbas Naqvi, Summar; Ludeña, Bertha; Castillo, Karina; Chabrillange, Nathalie; Bouguedoura, Nadia; Bennaceur, Malika; Si-Dehbi, Farida; Abdoulkader, Sabira; Daher, Abdourahman; Terral, Jean-Frederic; Santoni, Sylvain; Ballardini, Marco; Mercuri, Antonio; Ben Salah, Mohamed; Kadri, Karim; Othmani, Ahmed; Littardi, Claudio; Salhi-Hannachi, Amel; Pintaud, Jean-Christophe; Aberlenc-Bertossi, Frédérique

2015-01-01

Background and Aims Date palms (Phoenix dactylifera, Arecaceae) are of great economic and ecological value to the oasis agriculture of arid and semi-arid areas. However, despite the availability of a large date palm germplasm spreading from the Atlantic shores to Southern Asia, improvement of the species is being hampered by a lack of information on global genetic diversity and population structure. In order to contribute to the varietal improvement of date palms and to provide new insights on the influence of geographic origins and human activity on the genetic structure of the date palm, this study analysed the diversity of the species. Methods Genetic diversity levels and population genetic structure were investigated through the genotyping of a collection of 295 date palm accessions ranging from Mauritania to Pakistan using a set of 18 simple sequence repeat (SSR) markers and a plastid minisatellite. Key Results Using a Bayesian clustering approach, the date palm genotypes can be structured into two different gene pools: the first, termed the Eastern pool, consists of accessions from Asia and Djibouti, whilst the second, termed the Western pool, consists of accessions from Africa. These results confirm the existence of two ancient gene pools that have contributed to the current date palm diversity. The presence of admixed genotypes is also noted, which points at gene flows between eastern and western origins, mostly from east to west, following a human-mediated diffusion of the species. Conclusions This study assesses the distribution and level of genetic diversity of accessible date palm resources, provides new insights on the geographic origins and genetic history of the cultivated component of this species, and confirms the existence of at least two domestication origins. Furthermore, the strong genetic structure clearly established here is a prerequisite for any breeding programme exploiting the effective polymorphism related to each gene pool. PMID:26113618

Gene Flow Among Different Teosinte Taxa and Into the Domesticated Maize Gene Pool

USDA-ARS?s Scientific Manuscript database

Maize (Zea mays ssp. mays) was domesticated from one wild species ancestor, the Balsas teosinte (Zea mays ssp. parviglumis) about 9000 years ago. Higher levels of gene diversity are found in teosinte taxa compared to maize following domestication and selection bottlenecks. Diversity in maize can b...

Ulk4 Regulates Neural Stem Cell Pool.

PubMed

Liu, Min; Guan, Zhenlong; Shen, Qin; Flinter, Frances; Domínguez, Laura; Ahn, Joo Wook; Collier, David A; O'Brien, Timothy; Shen, Sanbing

2016-09-01

The size of neural stem cell (NSC) pool at birth determines the starting point of adult neurogenesis. Aberrant neurogenesis is associated with major mental illness, in which ULK4 is proposed as a rare risk factor. Little is known about factors regulating the NSC pool, or function of the ULK4. Here, we showed that Ulk4(tm1a/tm1a) mice displayed a dramatically reduced NSC pool at birth. Ulk4 was expressed in a cell cycle-dependent manner and peaked in G2/M phases. Targeted disruption of the Ulk4 perturbed mid-neurogenesis and significantly reduced cerebral cortex in postnatal mice. Pathway analyses of dysregulated genes in Ulk4(tm1a/tm1a) mice revealed Ulk4 as a key regulator of cell cycle and NSC proliferation, partially through regulation of the Wnt signaling. In addition, we identified hemizygous deletion of ULK4 gene in 1.2/1,000 patients with pleiotropic symptoms including severe language delay and learning difficulties. ULK4, therefore, may significantly contribute to neurodevelopmental, neuropsychiatric, and neurodegenerative disorders. Stem Cells 2016;34:2318-2331. © 2016 AlphaMed Press.

Two endornaviruses show differential infection patterns between gene pools of Phaseolus vulgaris.

PubMed

Khankhum, Surasak; Valverde, Rodrigo A; Pastor-Corrales, Marcial A; Osorno, Juan M; Sabanadzovic, Sead

2015-04-01

We investigated the occurrence of two plant endornaviruses, Phaseolus vulgaris endornavirus 1 and Phaseolus vulgaris endornavirus 2, in breeding lines, cultivars, landraces, and wild genotypes of common bean (Phaseolus vulgaris) collected from the two centers of common bean domestication: Mesoamerica and the Andes. The two endornaviruses were detected in many genotypes of Mesoamerican origin but rarely in genotypes of Andean origin. The results suggest that these two endornaviruses were introduced into the Mesoamerican modern genotypes during common bean domestication and provide more evidence for the existence of two divergent gene pools of common bean.

Development of innovative techniques and principles that may be used as models to improve plant performance. Progress report

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

Hanna, W.W.; Burton, G.W.

1984-06-01

Dominant immunity to rust (Puccinia) and Piricularia leaf spot, a new stable A/sub 1/ cytoplasm and yield genes have been discovered in a single accession of P. monodii, a subspecies of pearl millet Pennisetum americanum. The useful genes were transferred to the best pearl millet inbreds using a backcrossing and screening system enhanced by controlling daylength, temperature, and breaking seed dormancy. Genes for fertility restoration, plant morphology, head characteristics and grain yield were transferred from the A' genome of P. purpureum (secondary gene pool) to pearl millet. This was made possible by doubling the chromosome number of the sterile triploidmore » between pearl millet and P. purpureum to produce fertile hexaploids. A number of hexaploids were backcrossed to pearl millet to find hexaploids that produced A' gametes to produce AA' 2n = 14 pearl millet plants. The value of the genes on the A' genome were not recognized before the AA' plants were produced because the B genome masked the characteristics on the A' genome in P. purpureum. In the tertiary gene pool, male and female fertile apomictic BC/sub 1/ plants were produced between pearl millet and P. orientale or P. squamulatum by manipulating chromosome levels or by producing trispecific hybrids. The development of fertile backcross plants in this gene pool is encouraging for further germplasm transfer research with emphasis on apomixis. The cytoplasm of P. schweinfurthii has been transferred to pearl millet. Mutations for early maturity and daylength insensitivity induced with mutagens have resulted in the development and release of early maturing inbreds Tift 23A/sub 1/E/sub 1/ and Tift 23B/sub 1/E/sub 1/.« less

Environmental genomics of "Haloquadratum walsbyi" in a saltern crystallizer indicates a large pool of accessory genes in an otherwise coherent species

PubMed Central

Legault, Boris A; Lopez-Lopez, Arantxa; Alba-Casado, Jose Carlos; Doolittle, W Ford; Bolhuis, Henk; Rodriguez-Valera, Francisco; Papke, R Thane

2006-01-01

Background Mature saturated brine (crystallizers) communities are largely dominated (>80% of cells) by the square halophilic archaeon "Haloquadratum walsbyi". The recent cultivation of the strain HBSQ001 and thesequencing of its genome allows comparison with the metagenome of this taxonomically simplified environment. Similar studies carried out in other extreme environments have revealed very little diversity in gene content among the cell lineages present. Results The metagenome of the microbial community of a crystallizer pond has been analyzed by end sequencing a 2000 clone fosmid library and comparing the sequences obtained with the genome sequence of "Haloquadratum walsbyi". The genome of the sequenced strain was retrieved nearly complete within this environmental DNA library. However, many ORF's that could be ascribed to the "Haloquadratum" metapopulation by common genome characteristics or scaffolding to the strain genome were not present in the specific sequenced isolate. Particularly, three regions of the sequenced genome were associated with multiple rearrangements and the presence of different genes from the metapopulation. Many transposition and phage related genes were found within this pool which, together with the associated atypical GC content in these areas, supports lateral gene transfer mediated by these elements as the most probable genetic cause of this variability. Additionally, these sequences were highly enriched in putative regulatory and signal transduction functions. Conclusion These results point to a large pan-genome (total gene repertoire of the genus/species) even in this highly specialized extremophile and at a single geographic location. The extensive gene repertoire is what might be expected of a population that exploits a diverse nutrient pool, resulting from the degradation of biomass produced at lower salinities. PMID:16820057

The Fate of Spermatogonial Stem Cells in the Cryptorchid Testes of RXFP2 Deficient Mice

PubMed Central

Ferguson, Lydia; How, Javier J.; Agoulnik, Alexander I.

2013-01-01

The environmental niche of the spermatogonial stem cell pool is critical to ensure the continued generation of the germ cell population. To study the consequences of an aberrant testicular environment in cryptorchidism we used a mouse model with a deletion of Rxfp2 gene resulting in a high intra-abdominal testicular position. Mutant males were infertile with the gross morphology of the cryptorchid testis progressively deteriorating with age. Few spermatogonia were identifiable in 12 month old cryptorchid testes. Gene expression analysis showed no difference between mutant and control testes at postnatal day 10. In three month old males a decrease in expression of spermatogonial stem cell (SSC) markers Id4, Nanos2, and Ret was shown. The direct counting of ID4+ cells supported a significant decrease of SSCs. In contrast, the expression of Plzf, a marker for undifferentiated and differentiating spermatogonia was not reduced, and the number of PLZF+ cells in the cryptorchid testis was higher in three month old testes, but equal to control in six month old mutants. The PLZF+ cells did not show a higher rate of apoptosis in cryptorchid testis. The expression of the Sertoli cell FGF2 gene required for SSC maintenance was significantly reduced in mutant testis. Based on these findings we propose that the deregulation of somatic and germ cell genes in the cryptorchid testis, directs the SSCs towards the differentiation pathway. This leads to a depletion of the SSC pool and an increase in the number of PLZF+ spermatogonial cells, which too, eventually decreases with the exhaustion of the stem cell pool. Such a dynamic suggests that an early correction of cryptorchidism is critical for the retention of the SSC pool. PMID:24098584

Beans in Europe: origin and structure of the European landraces of Phaseolus vulgaris L.

PubMed

Angioi, S A; Rau, D; Attene, G; Nanni, L; Bellucci, E; Logozzo, G; Negri, V; Spagnoletti Zeuli, P L; Papa, R

2010-09-01

This study focuses on the expansion of Phaseolus vulgaris in Europe. The pathways of distribution of beans into and across Europe were very complex, with several introductions from the New World that were combined with direct exchanges between European and other Mediterranean countries. We have analyzed here six chloroplast microsatellite (cpSSR) loci and two unlinked nuclear loci (for phaseolin types and Pv-shatterproof1). We have assessed the genetic structure and level of diversity of a large collection of European landraces of P. vulgaris (307) in comparison to 94 genotypes from the Americas that are representative of the Andean and Mesoamerican gene pools. First, we show that most of the European common bean landraces (67%) are of Andean origin, and that there are no strong differences across European regions for the proportions of the Andean and Mesoamerican gene pools. Moreover, cytoplasmic diversity is evenly distributed across European regions. Secondly, the cytoplasmic bottleneck that was due to the introduction of P. vulgaris into the Old World was very weak or nearly absent. This is in contrast to evidence from nuclear analyses that have suggested a bottleneck of greater intensity. Finally, we estimate that a relatively high proportion of the European bean germplasm (about 44%) was derived from hybridization between the Andean and Mesoamerican gene pools. Moreover, although hybrids are present everywhere in Europe, they show an uneven distribution, with high frequencies in central Europe, and low frequencies in Spain and Italy. On the basis of these data, we suggest that the entire European continent and not only some of the countries therein can be regarded as a secondary diversification center for P. vulgaris. Finally, we outline the relevance of these inter-gene pool hybrids for plant breeding.

Detection of new HLA-DPB1 alleles generated by interallelic gene conversion using PCR amplification of DPB1 second exon sequences from sperm

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

Erlich, H.; Zangenberg, G.; Bugawan, T.

The rate at which allelic diversity at the HLA class I and class II loci evolves has been the subject of considerable controversy as have the mechanisms which generate new alleles. The patchwork pattern of polymorphism, particularly within the second exon of the HLA-DPB1 locus where the polymorphic sequence motifs are localized to 6 discrete regions, is consistent with the hypothesis that much of the allelic sequence variation may have been generated by segmental exchange (gene conversion). To measure the rate of new DPB1 variant generation, we have developed a strategy in which DPB1 second exon sequences are amplified frommore » pools of FACS-sorted sperm (n=50) from a heterozygous sperm donor. Pools of sperm from these heterozygous individuals are amplified with an allele-specific primer for one allele and analyzed with sequence-specific oligonucleotide probes (SSOP) complementary to the other allele. This screening procedure, which is capable of detecting a single variant molecule in a pool of parental alleles, allows the identification of new variants that have been generated by recombination and/or gene conversion between the two parental alleles. To control for potential PCR artifacts, the same screening procedure was carried out with mixtures of sperm from DPB1 *0301/*0301 and DPB1 *0401/ 0401 individuals. Pools containing putative new variants DPB1 alleles were analyzed further by cloning into M13 and sequencing the M13 clones. Our current estimate is that about 1/10,000 sperm from these heterozygous individuals represents a new DPB1 allele generated by micro-gene conversion within the second exon.« less

Uniparental genetic heritage of belarusians: encounter of rare middle eastern matrilineages with a central European mitochondrial DNA pool.

PubMed

Kushniarevich, Alena; Sivitskaya, Larysa; Danilenko, Nina; Novogrodskii, Tadeush; Tsybovsky, Iosif; Kiseleva, Anna; Kotova, Svetlana; Chaubey, Gyaneshwer; Metspalu, Ene; Sahakyan, Hovhannes; Bahmanimehr, Ardeshir; Reidla, Maere; Rootsi, Siiri; Parik, Jüri; Reisberg, Tuuli; Achilli, Alessandro; Hooshiar Kashani, Baharak; Gandini, Francesca; Olivieri, Anna; Behar, Doron M; Torroni, Antonio; Davydenko, Oleg; Villems, Richard

2013-01-01

Ethnic Belarusians make up more than 80% of the nine and half million people inhabiting the Republic of Belarus. Belarusians together with Ukrainians and Russians represent the East Slavic linguistic group, largest both in numbers and territory, inhabiting East Europe alongside Baltic-, Finno-Permic- and Turkic-speaking people. Till date, only a limited number of low resolution genetic studies have been performed on this population. Therefore, with the phylogeographic analysis of 565 Y-chromosomes and 267 mitochondrial DNAs from six well covered geographic sub-regions of Belarus we strove to complement the existing genetic profile of eastern Europeans. Our results reveal that around 80% of the paternal Belarusian gene pool is composed of R1a, I2a and N1c Y-chromosome haplogroups - a profile which is very similar to the two other eastern European populations - Ukrainians and Russians. The maternal Belarusian gene pool encompasses a full range of West Eurasian haplogroups and agrees well with the genetic structure of central-east European populations. Our data attest that latitudinal gradients characterize the variation of the uniparentally transmitted gene pools of modern Belarusians. In particular, the Y-chromosome reflects movements of people in central-east Europe, starting probably as early as the beginning of the Holocene. Furthermore, the matrilineal legacy of Belarusians retains two rare mitochondrial DNA haplogroups, N1a3 and N3, whose phylogeographies were explored in detail after de novo sequencing of 20 and 13 complete mitogenomes, respectively, from all over Eurasia. Our phylogeographic analyses reveal that two mitochondrial DNA lineages, N3 and N1a3, both of Middle Eastern origin, might mark distinct events of matrilineal gene flow to Europe: during the mid-Holocene period and around the Pleistocene-Holocene transition, respectively.

“Every Gene Is Everywhere but the Environment Selects”: Global Geolocalization of Gene Sharing in Environmental Samples through Network Analysis

PubMed Central

Fondi, Marco; Karkman, Antti; Tamminen, Manu V.; Bosi, Emanuele; Virta, Marko; Fani, Renato; Alm, Eric; McInerney, James O.

2016-01-01

The spatial distribution of microbes on our planet is famously formulated in the Baas Becking hypothesis as “everything is everywhere but the environment selects.” While this hypothesis does not strictly rule out patterns caused by geographical effects on ecology and historical founder effects, it does propose that the remarkable dispersal potential of microbes leads to distributions generally shaped by environmental factors rather than geographical distance. By constructing sequence similarity networks from uncultured environmental samples, we show that microbial gene pool distributions are not influenced nearly as much by geography as ecology, thus extending the Bass Becking hypothesis from whole organisms to microbial genes. We find that gene pools are shaped by their broad ecological niche (such as sea water, fresh water, host, and airborne). We find that freshwater habitats act as a gene exchange bridge between otherwise disconnected habitats. Finally, certain antibiotic resistance genes deviate from the general trend of habitat specificity by exhibiting a high degree of cross-habitat mobility. The strong cross-habitat mobility of antibiotic resistance genes is a cause for concern and provides a paradigmatic example of the rate by which genes colonize new habitats when new selective forces emerge. PMID:27190206

A flexible and economical barcoding approach for highly multiplexed amplicon sequencing of diverse target genes

PubMed Central

Herbold, Craig W.; Pelikan, Claus; Kuzyk, Orest; Hausmann, Bela; Angel, Roey; Berry, David; Loy, Alexander

2015-01-01

High throughput sequencing of phylogenetic and functional gene amplicons provides tremendous insight into the structure and functional potential of complex microbial communities. Here, we introduce a highly adaptable and economical PCR approach to barcoding and pooling libraries of numerous target genes. In this approach, we replace gene- and sequencing platform-specific fusion primers with general, interchangeable barcoding primers, enabling nearly limitless customized barcode-primer combinations. Compared to barcoding with long fusion primers, our multiple-target gene approach is more economical because it overall requires lower number of primers and is based on short primers with generally lower synthesis and purification costs. To highlight our approach, we pooled over 900 different small-subunit rRNA and functional gene amplicon libraries obtained from various environmental or host-associated microbial community samples into a single, paired-end Illumina MiSeq run. Although the amplicon regions ranged in size from approximately 290 to 720 bp, we found no significant systematic sequencing bias related to amplicon length or gene target. Our results indicate that this flexible multiplexing approach produces large, diverse, and high quality sets of amplicon sequence data for modern studies in microbial ecology. PMID:26236305

Distinct effects of struvite and biochar amendment on the class 1 integron antibiotic resistance gene cassettes in phyllosphere and rhizosphere.

PubMed

An, Xin-Li; Chen, Qing-Lin; Zhu, Dong; Su, Jian-Qiang

2018-08-01

Struvite recovered from wastewater is promising for recycling phosphorus into soil as fertilizers. However, struvite application may prompt the proliferation of antibiotic resistance in soil and plant. This study examined the impacts of struvite application and biochar amendment on integrons abundance and gene cassette contexts in rhizosphere soil and phyllosphere using quantitative PCR and clone library analysis. Microcosm experiments revealed that class 1 integron was the most prevalent in all samples, with higher concentration and higher relative abundance in rhizosphere than those in phyllosphere. The majority of resistance gene cassettes were associated with genes encoding resistance to aminoglycosides, beta-lactams and chloramphenicols. Struvite application significantly increased the genetic diversity of antibiotic resistance gene cassettes in both rhizosphere and phyllosphere. However, biochar amendment attenuated the increasing effect of struvite application exerting on the class 1 integron antibiotic resistance gene cassette pool in phyllosphere. These findings highlighted human activities to be the source of integron gene cassette pool and raised the possibility of using biochar amendment as an alternative mean for mitigating antibiotic resistance in environments. Copyright © 2018 Elsevier B.V. All rights reserved.

Serial analysis of gene expression (SAGE) in bovine trypanotolerance: preliminary results

PubMed Central

2003-01-01

In Africa, trypanosomosis is a tsetse-transmitted disease which represents the most important constraint to livestock production. Several indigenous West African taurine (Bos taurus) breeds, such as the Longhorn (N'Dama) cattle are well known to control trypanosome infections. This genetic ability named "trypanotolerance" results from various biological mechanisms under multigenic control. The methodologies used so far have not succeeded in identifying the complete pool of genes involved in trypanotolerance. New post genomic biotechnologies such as transcriptome analyses are efficient in characterising the pool of genes involved in the expression of specific biological functions. We used the serial analysis of gene expression (SAGE) technique to construct, from Peripheral Blood Mononuclear Cells of an N'Dama cow, 2 total mRNA transcript libraries, at day 0 of a Trypanosoma congolense experimental infection and at day 10 post-infection, corresponding to the peak of parasitaemia. Bioinformatic comparisons in the bovine genomic databases allowed the identification of 187 up- and down- regulated genes, EST and unknown functional genes. Identification of the genes involved in trypanotolerance will allow to set up specific microarray sets for further metabolic and pharmacological studies and to design field marker-assisted selection by introgression programmes. PMID:12927079

Serial analysis of gene expression (SAGE) in bovine trypanotolerance: preliminary results.

PubMed

Berthier, David; Quéré, Ronan; Thevenon, Sophie; Belemsaga, Désiré; Piquemal, David; Marti, Jacques; Maillard, Jean-Charles

2003-01-01

In Africa, trypanosomosis is a tsetse-transmitted disease which represents the most important constraint to livestock production. Several indigenous West African taurine Bos taurus) breeds, such as the Longhorn (N'Dama) cattle are well known to control trypanosome infections. This genetic ability named "trypanotolerance" results from various biological mechanisms under multigenic control. The methodologies used so far have not succeeded in identifying the complete pool of genes involved in trypanotolerance. New post genomic biotechnologies such as transcriptome analyses are efficient in characterising the pool of genes involved in the expression of specific biological functions. We used the serial analysis of gene expression (SAGE) technique to construct, from Peripheral Blood Mononuclear Cells of an N'Dama cow, 2 total mRNA transcript libraries, at day 0 of a Trypanosoma congolense experimental infection and at day 10 post-infection, corresponding to the peak of parasitaemia. Bioinformatic comparisons in the bovine genomic databases allowed the identification of 187 up- and down- regulated genes, EST and unknown functional genes. Identification of the genes involved in trypanotolerance will allow to set up specific microarray sets for further metabolic and pharmacological studies and to design field marker-assisted selection by introgression programmes.

Balinese Y-chromosome perspective on the peopling of Indonesia: genetic contributions from pre-neolithic hunter-gatherers, Austronesian farmers, and Indian traders.

PubMed

Karafet, Tatiana M; Lansing, J S; Redd, Alan J; Reznikova, Svetlana; Watkins, Joseph C; Surata, S P K; Arthawiguna, W A; Mayer, Laura; Bamshad, Michael; Jorde, Lynn B; Hammer, Michael F

2005-02-01

The island of Bali lies near the center of the southern chain of islands in the Indonesian archipelago, which served as a stepping-stone for early migrations of hunter-gatherers to Melanesia and Australia and for more recent migrations of Austronesian farmers from mainland Southeast Asia to the Pacific. Bali is the only Indonesian island with a population that currently practices the Hindu religion and preserves various other Indian cultural, linguistic, and artistic traditions (Lansing 1983). Here, we examine genetic variation on the Y chromosomes of 551 Balinese men to investigate the relative contributions of Austronesian farmers and pre-Neolithic hunter-gatherers to the contemporary Balinese paternal gene pool and to test the hypothesis of recent paternal gene flow from the Indian subcontinent. Seventy-one Y-chromosome binary polymorphisms (single nucleotide polymorphisms, SNPs) and 10 Y-chromosome-linked short tandem repeats (STRs) were genotyped on a sample of 1,989 Y chromosomes from 20 populations representing Indonesia (including Bali), southern China, Southeast Asia, South Asia, the Near East, and Oceania. SNP genotyping revealed 22 Balinese lineages, 3 of which (O-M95, O-M119, and O-M122) account for nearly 83.7% of Balinese Y chromosomes. Phylogeographic analyses suggest that all three major Y-chromosome haplogroups migrated to Bali with the arrival of Austronesian speakers; however, STR diversity patterns associated with these haplogroups are complex and may be explained by multiple waves of Austronesian expansion to Indonesia by different routes. Approximately 2.2% of contemporary Balinese Y chromosomes (i.e., K-M9*, K-M230, and M lineages) may represent the pre-Neolithic component of the Indonesian paternal gene pool. In contrast, eight other haplogroups (e.g., within H, J, L, and R), making up approximately 12% of the Balinese paternal gene pool, appear to have migrated to Bali from India. These results indicate that the Austronesian expansion had a profound effect on the composition of the Balinese paternal gene pool and that cultural transmission from India to Bali was accompanied by substantial levels of gene flow.

Pooled-DNA sequencing identifies genomic regions of selection in Nigerian isolates of Plasmodium falciparum.

PubMed

Oyebola, Kolapo M; Idowu, Emmanuel T; Olukosi, Yetunde A; Awolola, Taiwo S; Amambua-Ngwa, Alfred

2017-06-29

The burden of falciparum malaria is especially high in sub-Saharan Africa. Differences in pressure from host immunity and antimalarial drugs lead to adaptive changes responsible for high level of genetic variations within and between the parasite populations. Population-specific genetic studies to survey for genes under positive or balancing selection resulting from drug pressure or host immunity will allow for refinement of interventions. We performed a pooled sequencing (pool-seq) of the genomes of 100 Plasmodium falciparum isolates from Nigeria. We explored allele-frequency based neutrality test (Tajima's D) and integrated haplotype score (iHS) to identify genes under selection. Fourteen shared iHS regions that had at least 2 SNPs with a score > 2.5 were identified. These regions code for genes that were likely to have been under strong directional selection. Two of these genes were the chloroquine resistance transporter (CRT) on chromosome 7 and the multidrug resistance 1 (MDR1) on chromosome 5. There was a weak signature of selection in the dihydrofolate reductase (DHFR) gene on chromosome 4 and MDR5 genes on chromosome 13, with only 2 and 3 SNPs respectively identified within the iHS window. We observed strong selection pressure attributable to continued chloroquine and sulfadoxine-pyrimethamine use despite their official proscription for the treatment of uncomplicated malaria. There was also a major selective sweep on chromosome 6 which had 32 SNPs within the shared iHS region. Tajima's D of circumsporozoite protein (CSP), erythrocyte-binding antigen (EBA-175), merozoite surface proteins - MSP3 and MSP7, merozoite surface protein duffy binding-like (MSPDBL2) and serine repeat antigen (SERA-5) were 1.38, 1.29, 0.73, 0.84 and 0.21, respectively. We have demonstrated the use of pool-seq to understand genomic patterns of selection and variability in P. falciparum from Nigeria, which bears the highest burden of infections. This investigation identified known genomic signatures of selection from drug pressure and host immunity. This is evidence that P. falciparum populations explore common adaptive strategies that can be targeted for the development of new interventions.

Nuclear Calcium Signaling Controls Expression of a Large Gene Pool: Identification of a Gene Program for Acquired Neuroprotection Induced by Synaptic Activity

PubMed Central

Zhang, Sheng-Jia; Zou, Ming; Lu, Li; Lau, David; Ditzel, Désirée A. W.; Delucinge-Vivier, Celine; Aso, Yoshinori; Descombes, Patrick; Bading, Hilmar

2009-01-01

Synaptic activity can boost neuroprotection through a mechanism that requires synapse-to-nucleus communication and calcium signals in the cell nucleus. Here we show that in hippocampal neurons nuclear calcium is one of the most potent signals in neuronal gene expression. The induction or repression of 185 neuronal activity-regulated genes is dependent upon nuclear calcium signaling. The nuclear calcium-regulated gene pool contains a genomic program that mediates synaptic activity-induced, acquired neuroprotection. The core set of neuroprotective genes consists of 9 principal components, termed Activity-regulated Inhibitor of Death (AID) genes, and includes Atf3, Btg2, GADD45β, GADD45γ, Inhibin β-A, Interferon activated gene 202B, Npas4, Nr4a1, and Serpinb2, which strongly promote survival of cultured hippocampal neurons. Several AID genes provide neuroprotection through a common process that renders mitochondria more resistant to cellular stress and toxic insults. Stereotaxic delivery of AID gene-expressing recombinant adeno-associated viruses to the hippocampus confers protection in vivo against seizure-induced brain damage. Thus, treatments that enhance nuclear calcium signaling or supplement AID genes represent novel therapies to combat neurodegenerative conditions and neuronal cell loss caused by synaptic dysfunction, which may be accompanied by a deregulation of calcium signal initiation and/or propagation to the cell nucleus. PMID:19680447

Cellular choline and glycine betaine pools impact osmoprotection and phospholipase C production in Pseudomonas aeruginosa.

PubMed

Fitzsimmons, Liam F; Hampel, Ken J; Wargo, Matthew J

2012-09-01

Choline is abundantly produced by eukaryotes and plays an important role as a precursor of the osmoprotectant glycine betaine. In Pseudomonas aeruginosa, glycine betaine has additional roles as a nutrient source and an inducer of the hemolytic phospholipase C, PlcH. The multiple functions for glycine betaine suggested that the cytoplasmic pool of glycine betaine is regulated in P. aeruginosa. We used (13)C nuclear magnetic resonance ((13)C-NMR) to demonstrate that P. aeruginosa maintains both choline and glycine betaine pools under a variety of conditions, in contrast to the transient glycine betaine pool reported for most bacteria. We were able to experimentally manipulate the choline and glycine betaine pools by overexpression of the cognate catabolic genes. Depletion of either the choline or glycine betaine pool reduced phospholipase production, a result unexpected for choline depletion. Depletion of the glycine betaine pool, but not the choline pool, inhibited growth under conditions of high salt with glucose as the primary carbon source. Depletion of the choline pool inhibited growth under high-salt conditions with choline as the sole carbon source, suggesting a role for the choline pool under these conditions. Here we have described the presence of a choline pool in P. aeruginosa and other pseudomonads that, with the glycine betaine pool, regulates osmoprotection and phospholipase production and impacts growth under high-salt conditions. These findings suggest that the levels of both pools are actively maintained and that perturbation of either pool impacts P. aeruginosa physiology.

Cellular Choline and Glycine Betaine Pools Impact Osmoprotection and Phospholipase C Production in Pseudomonas aeruginosa

PubMed Central

Fitzsimmons, Liam F.; Hampel, Ken J.

2012-01-01

Choline is abundantly produced by eukaryotes and plays an important role as a precursor of the osmoprotectant glycine betaine. In Pseudomonas aeruginosa, glycine betaine has additional roles as a nutrient source and an inducer of the hemolytic phospholipase C, PlcH. The multiple functions for glycine betaine suggested that the cytoplasmic pool of glycine betaine is regulated in P. aeruginosa. We used 13C nuclear magnetic resonance (13C-NMR) to demonstrate that P. aeruginosa maintains both choline and glycine betaine pools under a variety of conditions, in contrast to the transient glycine betaine pool reported for most bacteria. We were able to experimentally manipulate the choline and glycine betaine pools by overexpression of the cognate catabolic genes. Depletion of either the choline or glycine betaine pool reduced phospholipase production, a result unexpected for choline depletion. Depletion of the glycine betaine pool, but not the choline pool, inhibited growth under conditions of high salt with glucose as the primary carbon source. Depletion of the choline pool inhibited growth under high-salt conditions with choline as the sole carbon source, suggesting a role for the choline pool under these conditions. Here we have described the presence of a choline pool in P. aeruginosa and other pseudomonads that, with the glycine betaine pool, regulates osmoprotection and phospholipase production and impacts growth under high-salt conditions. These findings suggest that the levels of both pools are actively maintained and that perturbation of either pool impacts P. aeruginosa physiology. PMID:22753069

ENCoRE: an efficient software for CRISPR screens identifies new players in extrinsic apoptosis.

PubMed

Trümbach, Dietrich; Pfeiffer, Susanne; Poppe, Manuel; Scherb, Hagen; Doll, Sebastian; Wurst, Wolfgang; Schick, Joel A

2017-11-25

As CRISPR/Cas9 mediated screens with pooled guide libraries in somatic cells become increasingly established, an unmet need for rapid and accurate companion informatics tools has emerged. We have developed a lightweight and efficient software to easily manipulate large raw next generation sequencing datasets derived from such screens into informative relational context with graphical support. The advantages of the software entitled ENCoRE (Easy NGS-to-Gene CRISPR REsults) include a simple graphical workflow, platform independence, local and fast multithreaded processing, data pre-processing and gene mapping with custom library import. We demonstrate the capabilities of ENCoRE to interrogate results from a pooled CRISPR cellular viability screen following Tumor Necrosis Factor-alpha challenge. The results not only identified stereotypical players in extrinsic apoptotic signaling but two as yet uncharacterized members of the extrinsic apoptotic cascade, Smg7 and Ces2a. We further validated and characterized cell lines containing mutations in these genes against a panel of cell death stimuli and involvement in p53 signaling. In summary, this software enables bench scientists with sensitive data or without access to informatic cores to rapidly interpret results from large scale experiments resulting from pooled CRISPR/Cas9 library screens.

The role of the megagametophyte in maintaining loblolly pine (Pinus taeda L.) seedling arginase gene expression in vitro.

PubMed

Todd, Christopher D; Gifford, David J

2002-05-01

Following loblolly pine (Pinus taeda L.) seed germination, storage-protein breakdown in the megagametophyte and in the seedling results in a large increase in the seedling's free amino acid pool. A substantial portion of both the storage proteins and the amino acid pool is arginine, a very efficient nitrogen-storage compound. Free arginine is hydrolyzed in the seedling by the enzyme arginase (EC 3.5.3.1), which is under strong developmental control. At present, regulation of arginase in conifers is not well understood. Here we report the utilization of an in vitro culture system to address the separate impacts of the seedling and megagametophyte tissues on arginase enzyme activity, protein levels and patterns of gene expression. We also describe the generation of an anti-arginase antibody prepared from a histidine-tagged loblolly pine arginase fusion protein expressed in Escherichia coli. Our results indicate that arginase gene expression in the seedling is initiated by the seedling itself and then maintained or up-regulated by the megagametophyte. The contribution of storage-protein breakdown and the free amino acid pool, particularly arginine, in this regulation is also addressed.

Genetics of resistance to the geminivirus, Bean dwarf mosaic virus, and the role of the hypersensitive response in common bean.

PubMed

Seo, Y-S; Gepts, P; Gilbertson, R L

2004-03-01

Bean dwarf mosaic virus (BDMV) is a single-stranded DNA virus (genus: Begomovirus, family: Geminiviridae) that infects common bean ( Phaseolus vulgaris L.) and causes stunted plant growth, and mosaic and mottle symptoms in leaves. BDMV shows differential pathogenicity in common bean, infecting germplasm of the Andean gene pool (e.g., the snap bean cultivar Topcrop), but not that of the Middle American gene pool (e.g., the pinto bean cultivar Othello). Resistance to BDMV in Othello is associated with development of a hypersensitive response (HR) in vascular (phloem) tissues. In this study, Middle American germplasm representing the four recognized races (i.e., Durango, Guatemala, Jalisco, and Mesoamerica) and the parents of Othello were inoculated with BDMV and a BDMV-green fluorescent protein (GFP) reporter. All genotypes showed partial or complete resistance to BDMV and BDMV-GFP, indicating the widespread distribution of resistance in the Middle American gene pool. A number of BDMV-resistant germplasm did not show the HR, indicating it is not correlated with resistance. In the F(1), F(2), and F(3) of reciprocal crosses between Othello and Topcrop, a single dominant allele, Bdm, conferred BDMV resistance.

Nanobiotechnologies in the System of Farm Animals' Gene Pool Preservation

NASA Astrophysics Data System (ADS)

Kovtun, S. I.; Galagan, N. P.; Shcherbak, O. V.; Klymenko, N. Y.; Osypchuk, O. S.

It has been determined that sperm viability and mobility upon long storage in liquid nitrogen is being reduced. This issue is relevant for gene pool objects' preservation and for reproductive medicine. The use of nanomaterials (NM) in reproductive biotechnology can expand to the methodology of the rational gene pool preservation technology, especially of valuable biomaterial. Use of nanoparticles, based on ultrafine silica (UFS) with different agents located on its surface gives a positive effect on the thawed sperm mobility, increasing its activity and survival rate and leading to increased fertilization ability. After thawing, the sperm of bulls had an average activity level of 47 %. This activity index of gametes in control was being maintained for 30 min. In the experimental groups after 30 min, the most active gametes were those with UFS + sucrose (52 %). A lower activity compared with the control and UFS + sucrose had gametes with UFS + D-galactosamine. Sperm activity with UFS + D-galactosamine decreased by 14 % compared with the control and by 19 % compared to the UFS + sucrose. So, the feasibility of adding NM with UFS + sucrose at a concentration of 0.001 % to thawed bull sperm that has been cryopreserved for a long time was proved.

Allele mining in the gene pool of wild Solanum species for homologues of late blight resistance gene RB/Rpi-blb1

USDA-ARS?s Scientific Manuscript database

Solanum bulbocastanum comprising a CC-NBS-LRR gene RB/Rpi-blb1 confers broad-spectrum resistance to Phytophthora infestans and is currently employed in potato breeding for durable late blight (LB) resistance. Genomes of several Solanum species were reported to contain RB homologues with confirmed b...

Next generation semiconductor based-sequencing of a nutrigenetics target gene (GPR120) and association with growth rate in Italian Large White pigs.

PubMed

Fontanesi, Luca; Bertolini, Francesca; Scotti, Emilio; Schiavo, Giuseppina; Colombo, Michela; Trevisi, Paolo; Ribani, Anisa; Buttazzoni, Luca; Russo, Vincenzo; Dall'Olio, Stefania

2015-01-01

The GPR120 gene (also known as FFAR4 or O3FAR1) encodes for a functional omega-3 fatty acid receptor/sensor that mediates potent insulin sensitizing effects by repressing macrophage-induced tissue inflammation. For its functional role, GPR120 could be considered a potential target gene in animal nutrigenetics. In this work we resequenced the porcine GPR120 gene by high throughput Ion Torrent semiconductor sequencing of amplified fragments obtained from 8 DNA pools derived, on the whole, from 153 pigs of different breeds/populations (two Italian Large White pools, Italian Duroc, Italian Landrace, Casertana, Pietrain, Meishan, and wild boars). Three single nucleotide polymorphisms (SNPs), two synonymous substitutions and one in the putative 3'-untranslated region (g.114765469C > T), were identified and their allele frequencies were estimated by sequencing reads count. The g.114765469C > T SNP was also genotyped by PCR-RFLP confirming estimated frequency in Italian Large White pools. Then, this SNP was analyzed in two Italian Large White cohorts using a selective genotyping approach based on extreme and divergent pigs for back fat thickness (BFT) estimated breeding value (EBV) and average daily gain (ADG) EBV. Significant differences of allele and genotype frequencies distribution was observed between the extreme ADG-EBV groups (P < 0.001) whereas this marker was not associated with BFT-EBV.

Advances in Arachis genomics for peanut improvement

USDA-ARS?s Scientific Manuscript database

Peanut genomics is very challenging due to its inherent problem of genetic architecture. Blockage of gene flow from diploid wild relatives to tetraploid cultivated peanut, recent polyploidization combined with self pollination and narrow genetic base of primary gene pool resulted in low genetic dive...

Magic Pools: Parallel Assessment of Transposon Delivery Vectors in Bacteria

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

Liu, Hualan; Price, Morgan N.; Waters, Robert Jordan

Transposon mutagenesis coupled to next-generation sequencing (TnSeq) is a powerful approach for discovering the functions of bacterial genes. However, the development of a suitable TnSeq strategy for a given bacterium can be costly and time-consuming. To meet this challenge, we describe a part-based strategy for constructing libraries of hundreds of transposon delivery vectors, which we term “magic pools.” Within a magic pool, each transposon vector has a different combination of upstream sequences (promoters and ribosome binding sites) and antibiotic resistance markers as well as a random DNA barcode sequence, which allows the tracking of each vector during mutagenesis experiments. Tomore » identify an efficient vector for a given bacterium, we mutagenize it with a magic pool and sequence the resulting insertions; we then use this efficient vector to generate a large mutant library. We used the magic pool strategy to construct transposon mutant libraries in five genera of bacteria, including three genera of the phylumBacteroidetes. IMPORTANCEMolecular genetics is indispensable for interrogating the physiology of bacteria. However, the development of a functional genetic system for any given bacterium can be time-consuming. Here, we present a streamlined approach for identifying an effective transposon mutagenesis system for a new bacterium. Our strategy first involves the construction of hundreds of different transposon vector variants, which we term a “magic pool.” The efficacy of each vector in a magic pool is monitored in parallel using a unique DNA barcode that is introduced into each vector design. Using archived DNA “parts,” we next reassemble an effective vector for making a whole-genome transposon mutant library that is suitable for large-scale interrogation of gene function using competitive growth assays. Here, we demonstrate the utility of the magic pool system to make mutant libraries in five genera of bacteria.« less

Magic Pools: Parallel Assessment of Transposon Delivery Vectors in Bacteria

DOE PAGES

Liu, Hualan; Price, Morgan N.; Waters, Robert Jordan; ...

2018-01-16

Transposon mutagenesis coupled to next-generation sequencing (TnSeq) is a powerful approach for discovering the functions of bacterial genes. However, the development of a suitable TnSeq strategy for a given bacterium can be costly and time-consuming. To meet this challenge, we describe a part-based strategy for constructing libraries of hundreds of transposon delivery vectors, which we term “magic pools.” Within a magic pool, each transposon vector has a different combination of upstream sequences (promoters and ribosome binding sites) and antibiotic resistance markers as well as a random DNA barcode sequence, which allows the tracking of each vector during mutagenesis experiments. Tomore » identify an efficient vector for a given bacterium, we mutagenize it with a magic pool and sequence the resulting insertions; we then use this efficient vector to generate a large mutant library. We used the magic pool strategy to construct transposon mutant libraries in five genera of bacteria, including three genera of the phylumBacteroidetes. IMPORTANCEMolecular genetics is indispensable for interrogating the physiology of bacteria. However, the development of a functional genetic system for any given bacterium can be time-consuming. Here, we present a streamlined approach for identifying an effective transposon mutagenesis system for a new bacterium. Our strategy first involves the construction of hundreds of different transposon vector variants, which we term a “magic pool.” The efficacy of each vector in a magic pool is monitored in parallel using a unique DNA barcode that is introduced into each vector design. Using archived DNA “parts,” we next reassemble an effective vector for making a whole-genome transposon mutant library that is suitable for large-scale interrogation of gene function using competitive growth assays. Here, we demonstrate the utility of the magic pool system to make mutant libraries in five genera of bacteria.« less

High-throughput, pooled sequencing identifies mutations in NUBPL and FOXRED1 in human complex I deficiency

PubMed Central

Calvo, Sarah E; Tucker, Elena J; Compton, Alison G; Kirby, Denise M; Crawford, Gabriel; Burtt, Noel P; Rivas, Manuel A; Guiducci, Candace; Bruno, Damien L; Goldberger, Olga A; Redman, Michelle C; Wiltshire, Esko; Wilson, Callum J; Altshuler, David; Gabriel, Stacey B; Daly, Mark J; Thorburn, David R; Mootha, Vamsi K

2010-01-01

Discovering the molecular basis of mitochondrial respiratory chain disease is challenging given the large number of both mitochondrial and nuclear genes involved. We report a strategy of focused candidate gene prediction, high-throughput sequencing, and experimental validation to uncover the molecular basis of mitochondrial complex I (CI) disorders. We created five pools of DNA from a cohort of 103 patients and then performed deep sequencing of 103 candidate genes to spotlight 151 rare variants predicted to impact protein function. We used confirmatory experiments to establish genetic diagnoses in 22% of previously unsolved cases, and discovered that defects in NUBPL and FOXRED1 can cause CI deficiency. Our study illustrates how large-scale sequencing, coupled with functional prediction and experimental validation, can reveal novel disease-causing mutations in individual patients. PMID:20818383

Population genetic diversity and structure of a naturally isolated plant species, Rhodiola dumulosa (Crassulaceae).

PubMed

Hou, Yan; Lou, Anru

2011-01-01

Rhodiola dumulosa (Crassulaceae) is a perennial diploid species found in high-montane areas. It is distributed in fragmented populations across northern, central and northwestern China. In this study, we aimed to (i) measure the genetic diversity of this species and that of its populations; (ii) describe the genetic structure of these populations across the entire distribution range in China; and (iii) evaluate the extent of gene flow among the naturally fragmented populations. Samples from 1089 individuals within 35 populations of R. dumulosa were collected, covering as much of the entire distribution range of this species within China as possible. Population genetic diversity and structure were analyzed using AFLP molecular markers. Gene flow among populations was estimated according to the level of population differentiation. The total genetic diversity of R. dumulosa was high but decreased with increasing altitude. Population-structure analysis indicated that the most closely related populations were geographically restricted and occurred in close proximity to each other. A significant isolation-by-distance pattern, caused by the naturally fragmented population distribution, was observed. At least two distinct gene pools were found in the 35 sampled populations, one composed of populations in northern China and the other composed of populations in central and northwestern China. The calculation of Nei's gene diversity index revealed that the genetic diversity in the northern China pool (0.1972) was lower than that in the central and northwestern China pool (0.2216). The populations were significantly isolated, and gene flow was restricted throughout the entire distribution. However, gene flow among populations on the same mountain appears to be unrestricted, as indicated by the weak genetic isolation among these populations.

Population Genetic Diversity and Structure of a Naturally Isolated Plant Species, Rhodiola dumulosa (Crassulaceae)

PubMed Central

Hou, Yan; Lou, Anru

2011-01-01

Aims Rhodiola dumulosa (Crassulaceae) is a perennial diploid species found in high-montane areas. It is distributed in fragmented populations across northern, central and northwestern China. In this study, we aimed to (i) measure the genetic diversity of this species and that of its populations; (ii) describe the genetic structure of these populations across the entire distribution range in China; and (iii) evaluate the extent of gene flow among the naturally fragmented populations. Methods Samples from 1089 individuals within 35 populations of R. dumulosa were collected, covering as much of the entire distribution range of this species within China as possible. Population genetic diversity and structure were analyzed using AFLP molecular markers. Gene flow among populations was estimated according to the level of population differentiation. Important Findings The total genetic diversity of R. dumulosa was high but decreased with increasing altitude. Population-structure analysis indicated that the most closely related populations were geographically restricted and occurred in close proximity to each other. A significant isolation-by-distance pattern, caused by the naturally fragmented population distribution, was observed. At least two distinct gene pools were found in the 35 sampled populations, one composed of populations in northern China and the other composed of populations in central and northwestern China. The calculation of Nei's gene diversity index revealed that the genetic diversity in the northern China pool (0.1972) was lower than that in the central and northwestern China pool (0.2216). The populations were significantly isolated, and gene flow was restricted throughout the entire distribution. However, gene flow among populations on the same mountain appears to be unrestricted, as indicated by the weak genetic isolation among these populations. PMID:21909437

Ion Torrent sequencing as a tool for mutation discovery in the flax (Linum usitatissimum L.) genome.

PubMed

Galindo-González, Leonardo; Pinzón-Latorre, David; Bergen, Erik A; Jensen, Dustin C; Deyholos, Michael K

2015-01-01

Detection of induced mutations is valuable for inferring gene function and for developing novel germplasm for crop improvement. Many reverse genetics approaches have been developed to identify mutations in genes of interest within a mutagenized population, including some approaches that rely on next-generation sequencing (e.g. exome capture, whole genome resequencing). As an alternative to these genome or exome-scale methods, we sought to develop a scalable and efficient method for detection of induced mutations that could be applied to a small number of target genes, using Ion Torrent technology. We developed this method in flax (Linum usitatissimum), to demonstrate its utility in a crop species. We used an amplicon-based approach in which DNA samples from an ethyl methanesulfonate (EMS)-mutagenized population were pooled and used as template in PCR reactions to amplify a region of each gene of interest. Barcodes were incorporated during PCR, and the pooled amplicons were sequenced using an Ion Torrent PGM. A pilot experiment with known SNPs showed that they could be detected at a frequency > 0.3% within the pools. We then selected eight genes for which we wanted to discover novel mutations, and applied our approach to screen 768 individuals from the EMS population, using either the Ion 314 or Ion 316 chips. Out of 29 potential mutations identified after processing the NGS reads, 16 mutations were confirmed using Sanger sequencing. The methodology presented here demonstrates the utility of Ion Torrent technology in detecting mutation variants in specific genome regions for large populations of a species such as flax. The methodology could be scaled-up to test >100 genes using the higher capacity chips now available from Ion Torrent.

Sex Bias and Maternal Contribution to Gene Expression Divergence in Drosophila Blastoderm Embryos

PubMed Central

Paris, Mathilde; Villalta, Jacqueline E.; Eisen, Michael B.; Lott, Susan E.

2015-01-01

Early embryogenesis is a unique developmental stage where genetic control of development is handed off from mother to zygote. Yet the contribution of this transition to the evolution of gene expression is poorly understood. Here we study two aspects of gene expression specific to early embryogenesis in Drosophila: sex-biased gene expression prior to the onset of canonical X chromosomal dosage compensation, and the contribution of maternally supplied mRNAs. We sequenced mRNAs from individual unfertilized eggs and precisely staged and sexed blastoderm embryos, and compared levels between D. melanogaster, D. yakuba, D. pseudoobscura and D. virilis. First, we find that mRNA content is highly conserved for a given stage and that studies relying on pooled embryos likely systematically overstate the degree of gene expression divergence. Unlike studies done on larvae and adults where most species show a larger proportion of genes with male-biased expression, we find that transcripts in Drosophila embryos are largely female-biased in all species, likely due to incomplete dosage compensation prior to the activation of the canonical dosage compensation mechanism. The divergence of sex-biased gene expression across species is observed to be often due to lineage-specific decrease of expression; the most drastic example of which is the overall reduction of male expression from the neo-X chromosome in D. pseudoobscura, leading to a pervasive female-bias on this chromosome. We see no evidence for a faster evolution of expression on the X chromosome in embryos (no “faster-X” effect), unlike in adults, and contrary to a previous study on pooled non-sexed embryos. Finally, we find that most genes are conserved in regard to their maternal or zygotic origin of transcription, and present evidence that differences in maternal contribution to the blastoderm transcript pool may be due to species-specific divergence of transcript degradation rates. PMID:26485701

Climate-driven range shifts explain the distribution of extant gene pools and predict future loss of unique lineages in a marine brown alga.

PubMed

Assis, J; Serrão, E A; Claro, B; Perrin, C; Pearson, G A

2014-06-01

The climate-driven dynamics of species ranges is a critical research question in evolutionary ecology. We ask whether present intraspecific diversity is determined by the imprint of past climate. This is an ongoing debate requiring interdisciplinary examination of population genetic pools and persistence patterns across global ranges. Previously, contrasting inferences and predictions have resulted from distinct genomic coverage and/or geographical information. We aim to describe and explain the causes of geographical contrasts in genetic diversity and their consequences for the future baseline of the global genetic pool, by comparing present geographical distribution of genetic diversity and differentiation with predictive species distribution modelling (SDM) during past extremes, present time and future climate scenarios for a brown alga, Fucus vesiculosus. SDM showed that both atmospheric and oceanic variables shape the global distribution of intertidal species, revealing regions of persistence, extinction and expansion during glacial and postglacial periods. These explained the distribution and structure of present genetic diversity, consisting of differentiated genetic pools with maximal diversity in areas of long-term persistence. Most of the present species range comprises postglacial expansion zones and, in contrast to highly dispersive marine organisms, expansions involved only local fronts, leaving distinct genetic pools at rear edges. Besides unravelling a complex phylogeographical history and showing congruence between genetic diversity and persistent distribution zones, supporting the hypothesis of niche conservatism, range shifts and loss of unique genetic diversity at the rear edge were predicted for future climate scenarios, impoverishing the global gene pool. © 2014 John Wiley & Sons Ltd.

Genetic structure of the date palm (Phoenix dactylifera) in the Old World reveals a strong differentiation between eastern and western populations.

PubMed

Zehdi-Azouzi, Salwa; Cherif, Emira; Moussouni, Souhila; Gros-Balthazard, Muriel; Abbas Naqvi, Summar; Ludeña, Bertha; Castillo, Karina; Chabrillange, Nathalie; Bouguedoura, Nadia; Bennaceur, Malika; Si-Dehbi, Farida; Abdoulkader, Sabira; Daher, Abdourahman; Terral, Jean-Frederic; Santoni, Sylvain; Ballardini, Marco; Mercuri, Antonio; Ben Salah, Mohamed; Kadri, Karim; Othmani, Ahmed; Littardi, Claudio; Salhi-Hannachi, Amel; Pintaud, Jean-Christophe; Aberlenc-Bertossi, Frédérique

2015-07-01

Date palms (Phoenix dactylifera, Arecaceae) are of great economic and ecological value to the oasis agriculture of arid and semi-arid areas. However, despite the availability of a large date palm germplasm spreading from the Atlantic shores to Southern Asia, improvement of the species is being hampered by a lack of information on global genetic diversity and population structure. In order to contribute to the varietal improvement of date palms and to provide new insights on the influence of geographic origins and human activity on the genetic structure of the date palm, this study analysed the diversity of the species. Genetic diversity levels and population genetic structure were investigated through the genotyping of a collection of 295 date palm accessions ranging from Mauritania to Pakistan using a set of 18 simple sequence repeat (SSR) markers and a plastid minisatellite. Using a Bayesian clustering approach, the date palm genotypes can be structured into two different gene pools: the first, termed the Eastern pool, consists of accessions from Asia and Djibouti, whilst the second, termed the Western pool, consists of accessions from Africa. These results confirm the existence of two ancient gene pools that have contributed to the current date palm diversity. The presence of admixed genotypes is also noted, which points at gene flows between eastern and western origins, mostly from east to west, following a human-mediated diffusion of the species. This study assesses the distribution and level of genetic diversity of accessible date palm resources, provides new insights on the geographic origins and genetic history of the cultivated component of this species, and confirms the existence of at least two domestication origins. Furthermore, the strong genetic structure clearly established here is a prerequisite for any breeding programme exploiting the effective polymorphism related to each gene pool. © The Author 2015. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Integrated platform for genome-wide screening and construction of high-density genetic interaction maps in mammalian cells

PubMed Central

Kampmann, Martin; Bassik, Michael C.; Weissman, Jonathan S.

2013-01-01

A major challenge of the postgenomic era is to understand how human genes function together in normal and disease states. In microorganisms, high-density genetic interaction (GI) maps are a powerful tool to elucidate gene functions and pathways. We have developed an integrated methodology based on pooled shRNA screening in mammalian cells for genome-wide identification of genes with relevant phenotypes and systematic mapping of all GIs among them. We recently demonstrated the potential of this approach in an application to pathways controlling the susceptibility of human cells to the toxin ricin. Here we present the complete quantitative framework underlying our strategy, including experimental design, derivation of quantitative phenotypes from pooled screens, robust identification of hit genes using ultra-complex shRNA libraries, parallel measurement of tens of thousands of GIs from a single double-shRNA experiment, and construction of GI maps. We describe the general applicability of our strategy. Our pooled approach enables rapid screening of the same shRNA library in different cell lines and under different conditions to determine a range of different phenotypes. We illustrate this strategy here for single- and double-shRNA libraries. We compare the roles of genes for susceptibility to ricin and Shiga toxin in different human cell lines and reveal both toxin-specific and cell line-specific pathways. We also present GI maps based on growth and ricin-resistance phenotypes, and we demonstrate how such a comparative GI mapping strategy enables functional dissection of physical complexes and context-dependent pathways. PMID:23739767

Variation in chromosome constitution of the Xiaoyan series partial amphiploids and its relations to stripe rust and stem rust resistance

USDA-ARS?s Scientific Manuscript database

In the tertiary gene pool of wheat, tall wheatgrass Thinopyrum ponticum (2n = 10x = 70) is an excellent source of resistance genes against numerous wheat diseases. The creation of wheat-Th. ponticum partial amphiploids is an intermediate step for transferring the useful genes from Th. ponticum to w...

Seasonal and Cyclical Changes in Genetic Composition of the Marine Intertidal Rock Pool Copepod Tigriopus brevicornis.

PubMed

Van Wormhoudt, Alain

2015-06-01

Tigriopus brevicornis is a marine rock pool copepod widely distributed along Atlantic coasts. Due to the absence of a known dispersal mechanism by free swimming stages, exchanges between populations over long distances are questionable. In order to analyse the evolution of an isolated supralittoral rock pool population, sampling of the copepod was performed monthly during 1 year and compared to samplings over 5 years in the same rock pool, as well as from other rock pools. Using ITS1 analysis, cyclical changes in genetic composition were detected. Our results give clear indications concerning the segregation of the rock pool population and a lack of gene flow among outside populations. A network analysis shows the presence of several shared dominant haplotypes and also singletons differing by one mutation point. F(st) analyses indicate that the main changes occur in autumn and winter. The few analogies of ITS1 sequences with nearby populations may indicate that new migrants must re-colonise the pools from surrounding rock crevices in the intertidal habitat where they may have found a refuge after bad weather conditions.

"Every Gene Is Everywhere but the Environment Selects": Global Geolocalization of Gene Sharing in Environmental Samples through Network Analysis.

PubMed

Fondi, Marco; Karkman, Antti; Tamminen, Manu V; Bosi, Emanuele; Virta, Marko; Fani, Renato; Alm, Eric; McInerney, James O

2016-05-13

The spatial distribution of microbes on our planet is famously formulated in the Baas Becking hypothesis as "everything is everywhere but the environment selects." While this hypothesis does not strictly rule out patterns caused by geographical effects on ecology and historical founder effects, it does propose that the remarkable dispersal potential of microbes leads to distributions generally shaped by environmental factors rather than geographical distance. By constructing sequence similarity networks from uncultured environmental samples, we show that microbial gene pool distributions are not influenced nearly as much by geography as ecology, thus extending the Bass Becking hypothesis from whole organisms to microbial genes. We find that gene pools are shaped by their broad ecological niche (such as sea water, fresh water, host, and airborne). We find that freshwater habitats act as a gene exchange bridge between otherwise disconnected habitats. Finally, certain antibiotic resistance genes deviate from the general trend of habitat specificity by exhibiting a high degree of cross-habitat mobility. The strong cross-habitat mobility of antibiotic resistance genes is a cause for concern and provides a paradigmatic example of the rate by which genes colonize new habitats when new selective forces emerge. © The Author(s) 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

TRAM (Transcriptome Mapper): database-driven creation and analysis of transcriptome maps from multiple sources

PubMed Central

2011-01-01

Background Several tools have been developed to perform global gene expression profile data analysis, to search for specific chromosomal regions whose features meet defined criteria as well as to study neighbouring gene expression. However, most of these tools are tailored for a specific use in a particular context (e.g. they are species-specific, or limited to a particular data format) and they typically accept only gene lists as input. Results TRAM (Transcriptome Mapper) is a new general tool that allows the simple generation and analysis of quantitative transcriptome maps, starting from any source listing gene expression values for a given gene set (e.g. expression microarrays), implemented as a relational database. It includes a parser able to assign univocal and updated gene symbols to gene identifiers from different data sources. Moreover, TRAM is able to perform intra-sample and inter-sample data normalization, including an original variant of quantile normalization (scaled quantile), useful to normalize data from platforms with highly different numbers of investigated genes. When in 'Map' mode, the software generates a quantitative representation of the transcriptome of a sample (or of a pool of samples) and identifies if segments of defined lengths are over/under-expressed compared to the desired threshold. When in 'Cluster' mode, the software searches for a set of over/under-expressed consecutive genes. Statistical significance for all results is calculated with respect to genes localized on the same chromosome or to all genome genes. Transcriptome maps, showing differential expression between two sample groups, relative to two different biological conditions, may be easily generated. We present the results of a biological model test, based on a meta-analysis comparison between a sample pool of human CD34+ hematopoietic progenitor cells and a sample pool of megakaryocytic cells. Biologically relevant chromosomal segments and gene clusters with differential expression during the differentiation toward megakaryocyte were identified. Conclusions TRAM is designed to create, and statistically analyze, quantitative transcriptome maps, based on gene expression data from multiple sources. The release includes FileMaker Pro database management runtime application and it is freely available at http://apollo11.isto.unibo.it/software/, along with preconfigured implementations for mapping of human, mouse and zebrafish transcriptomes. PMID:21333005

Monitoring and source tracking of tetracycline resistance genes in lagoons and groundwater adjacent to swine production facilities over a 3-year period

USGS Publications Warehouse

Koike, S.; Krapac, I.G.; Oliver, H.D.; Yannarell, A.C.; Chee-Sanford, J. C.; Aminov, R.I.; Mackie, R.I.

2007-01-01

To monitor the dissemination of resistance genes into the environment, we determined the occurrence of tetracycline resistance (Tcr) genes in groundwater underlying two swine confinement operations. Monitoring well networks (16 wells at site A and 6 wells at site C) were established around the lagoons at each facility. Groundwater (n = 124) and lagoon (n = 12) samples were collected from the two sites at six sampling times from 2000 through 2003. Total DNA was extracted, and PCR was used to detect seven Tcr genes [tet(M), tet(O), tet(Q), tet(W), tet(C), tet(H), and tet(Z)]. The concentration of Tcr genes was quantified by real-time quantitative PCR. To confirm the Tcr gene source in groundwater, comparative analysis of tet(W) gene sequences was performed on groundwater and lagoon samples. All seven Tcr genes were continually detected in groundwater during the 3-year monitoring period at both sites. At site A, elevated detection frequency and concentration of Tcr genes were observed in the wells located down-gradient of the lagoon. Comparative analysis of tet(W) sequences revealed that the impacted groundwater contained gene sequences almost identical (99.8% identity) to those in the lagoon, but these genes were not found in background libraries. Novel sequence clusters and unique indigenous resistance gene pools were also found in the groundwater. Thus, antibiotic resistance genes in groundwater are affected by swine manure, but they are also part of the indigenous gene pool. Copyright ?? 2007, American Society for Microbiology. All Rights Reserved.

Monitoring and Source Tracking of Tetracycline Resistance Genes in Lagoons and Groundwater Adjacent to Swine Production Facilities over a 3-Year Period▿

PubMed Central

Koike, S.; Krapac, I. G.; Oliver, H. D.; Yannarell, A. C.; Chee-Sanford, J. C.; Aminov, R. I.; Mackie, R. I.

2007-01-01

To monitor the dissemination of resistance genes into the environment, we determined the occurrence of tetracycline resistance (Tcr) genes in groundwater underlying two swine confinement operations. Monitoring well networks (16 wells at site A and 6 wells at site C) were established around the lagoons at each facility. Groundwater (n = 124) and lagoon (n = 12) samples were collected from the two sites at six sampling times from 2000 through 2003. Total DNA was extracted, and PCR was used to detect seven Tcr genes [tet(M), tet(O), tet(Q), tet(W), tet(C), tet(H), and tet(Z)]. The concentration of Tcr genes was quantified by real-time quantitative PCR. To confirm the Tcr gene source in groundwater, comparative analysis of tet(W) gene sequences was performed on groundwater and lagoon samples. All seven Tcr genes were continually detected in groundwater during the 3-year monitoring period at both sites. At site A, elevated detection frequency and concentration of Tcr genes were observed in the wells located down-gradient of the lagoon. Comparative analysis of tet(W) sequences revealed that the impacted groundwater contained gene sequences almost identical (99.8% identity) to those in the lagoon, but these genes were not found in background libraries. Novel sequence clusters and unique indigenous resistance gene pools were also found in the groundwater. Thus, antibiotic resistance genes in groundwater are affected by swine manure, but they are also part of the indigenous gene pool. PMID:17545324

Toward an animal model of cystic fibrosis: Targeted interruption of exon 10 of the cystic fibrosis transmembrane regulator gene in embryonic stem cells

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

Koller, B.H.; Hyungsuk Kim; Latour, A.M.

1991-12-01

A gene-targeting construct was made containing 7.8 kilobases of DNA spanning exon 10 of the mouse cystic fibrosis transmembrane regulator (CFTR) gene in which part of the exon has been replaced by two neomycin-resistance (Neo) genes driven by different promoters. (This replacement introduces a chain-termination codon at amino acid position 489 in the CFTR sequence). A herpes simplex thymidine kinase gene was on each end of the construct, which was electroporated into embryonic stem (ES) cells. Colonies resistant to G418, or to G418 plus ganciclovir, were selected and screened by Southern blotting or by PCR amplification. Five pools of G418-resistantmore » cells gave PCR products diagnostic of targeting. Four independent clones of ES cells with a disrupted CFTR gene have been isolated from these pools. The frequency of targeting was 1/2500 G418-resistant colonies. This low frequency is not the consequence of marginal expression of the Neo genes in the targeted cells. The CFTR targeting events were clustered among our experiments in a manner suggesting that some unidentified factor(s), possible passage number, influences the recovery of CFTR-targeted cells.« less

Myosin Heavy Chain Gene Expression in Developing Neonatal Skeletal Muscle: Involvement of the Nerve, Gravity, and Thyroid State

NASA Technical Reports Server (NTRS)

Baldwin, K. M.; Adams, G.; Haddad, F.; Zeng, M.; Qin, A.; Qin, L.; McCue, S.; Bodell, P.

1999-01-01

The myosin heavy chain (MHC) gene family encodes at least six MHC proteins (herein designated as neonatal, embryonic, slow type I (beta), and fast IIa, IIx, and IIb) that are expressed in skeletal muscle in a muscle-specific and developmentally-regulated fashion. At birth, both antigravity (e.g. soleus) and locomotor (e.g., plantaris) skeletal muscles are undifferentiated relative to the adult MHC phenotype such that the neonatal and embryonic MHC isoforms account for 80 - 90% of the MHC pool in a fast locomotor muscle; whereas, the embryonic and slow, type I isoforms account for approx. 90% of the pool in a typical antigravity muscle. The goal of this study was to investigate the role of an intact nerve, gravity and thyroid hormone (T3), as well as certain interactions of these interventions, on MHC gene expression in developing neonatal skeletal muscles of rodents.

High-Throughput Parallel Sequencing to Measure Fitness of Leptospira interrogans Transposon Insertion Mutants during Acute Infection

PubMed Central

Matsunaga, James; Haake, David A.

2016-01-01

Pathogenic species of Leptospira are the causative agents of leptospirosis, a zoonotic disease that causes mortality and morbidity worldwide. The understanding of the virulence mechanisms of Leptospira spp is still at an early stage due to the limited number of genetic tools available for this microorganism. The development of random transposon mutagenesis in pathogenic strains a decade ago has contributed to the identification of several virulence factors. In this study, we used the transposon sequencing (Tn-Seq) technique, which combines transposon mutagenesis with massive parallel sequencing, to study the in vivo fitness of a pool of Leptospira interrogans mutants. We infected hamsters with a pool of 42 mutants (input pool), which included control mutants with insertions in four genes previously analyzed by virulence testing (loa22, ligB, flaA1, and lic20111) and 23 mutants with disrupted signal transduction genes. We quantified the mutants in different tissues (blood, kidney and liver) at 4 days post-challenge by high-throughput sequencing and compared the frequencies of mutants recovered from tissues to their frequencies in the input pool. Control mutants that were less fit in the Tn-Seq experiment were attenuated for virulence when tested separately in the hamster model of lethal leptospirosis. Control mutants with unaltered fitness were as virulent as the wild-type strain. We identified two mutants with the transposon inserted in the same putative adenylate/guanylate cyclase gene (lic12327) that had reduced in vivo fitness in blood, kidney and liver. Both lic12327 mutants were attenuated for virulence when tested individually in hamsters. Growth of the control mutants and lic12327 mutants in culture medium were similar to that of the wild-type strain. These results demonstrate the feasibility of screening large pools of L. interrogans transposon mutants for those with altered fitness, and potentially attenuated virulence, by transposon sequencing. PMID:27824878

High-resolution mapping and sequence analysis of 597 cDNA clones transcribed from the 1 Mb region in human chromosome 4q16.3 containing Huntington disease gene

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

Hadano, S.; Ishida, Y.; Tomiyasu, H.

1994-09-01

To complete a transcription map of the 1 Mb region in human chromosome 4p16.3 containing the Huntington disease (HD) gene, the isolation of cDNA clones are being performed throughout. Our method relies on a direct screening of the cDNA libraries probed with single copy microclones from 3 YAC clones spanning 1 Mbp of the HD gene region. AC-DNAs were isolated by a preparative pulsed-field gel electrophoresis, amplified by both a single unique primer (SUP)-PCR and a linker ligation PCR, and 6 microclone-DNA libraries were generated. Then, 8,640 microclones from these libraries were independently amplified by PCR, and arrayed onto themore » membranes. 800-900 microclones that were not cross-hybridized with total human and yeast genomic DNA, TAC vector DNA, and ribosomal cDNA on a dot hybridization (putatively carrying single copy sequences) were pooled to make 9 probe pools. A total of {approximately}1.8x10{sup 7} plaques from the human brain cDNA libraries was screened with 9 pool-probes, and then 672 positive cDNA clones were obtained. So far, 597 cDNA clones were defined and arrayed onto a map of the 1 Mbp of the HD gene region by hybridization with HD region-specific cosmid contigs and YAC clones. Further characterization including a DNA sequencing and Northern blot analysis is currently underway.« less

A DNA pooling based system to detect Escherichia coli virulence factors in fecal and wastewater samples

PubMed Central

Luz María Chacón, J; Lizeth Taylor, C; Carmen Valiente, A; Irene Alvarado, P; Ximena Cortés, B

2012-01-01

The availability of a useful tool for simple and timely detection of the most important virulent varieties of Escherichia coli is indispensable. To this end, bacterial DNA pools which had previously been categorized were obtained from isolated colonies as well as selected in terms of utilized phenotype; the pools were assessed by two PCR Multiplex for the detection of virulent E. coli eaeA, bfpA, stx1, stx2, ipaH, ST, LT, and aatA genes, with the 16S gene used as DNA control. The system was validated with 66 fecal samples and 44 wastewater samples. At least one positive isolate was detected by a virulent gene among the 20 that were screened. The analysis of fecal samples from children younger than 6 years of age detected frequencies of 25% LT positive strains, 8.3% eae, 8.3% bfpA, 16.7% ipaH, as well as 12.5 % aatA and ST. On the other hand, wastewater samples revealed frequencies of 25.7% eaeA positive, 30.3% stx1, 15.1% LT and 19.7% aatA. This study is an initial step toward carrying out epidemiological field research that will reveal the presence of these bacterial varieties. PMID:24031959

Why barcode? High-throughput multiplex sequencing of mitochondrial genomes for molecular systematics.

PubMed

Timmermans, M J T N; Dodsworth, S; Culverwell, C L; Bocak, L; Ahrens, D; Littlewood, D T J; Pons, J; Vogler, A P

2010-11-01

Mitochondrial genome sequences are important markers for phylogenetics but taxon sampling remains sporadic because of the great effort and cost required to acquire full-length sequences. Here, we demonstrate a simple, cost-effective way to sequence the full complement of protein coding mitochondrial genes from pooled samples using the 454/Roche platform. Multiplexing was achieved without the need for expensive indexing tags ('barcodes'). The method was trialled with a set of long-range polymerase chain reaction (PCR) fragments from 30 species of Coleoptera (beetles) sequenced in a 1/16th sector of a sequencing plate. Long contigs were produced from the pooled sequences with sequencing depths ranging from ∼10 to 100× per contig. Species identity of individual contigs was established via three 'bait' sequences matching disparate parts of the mitochondrial genome obtained by conventional PCR and Sanger sequencing. This proved that assembly of contigs from the sequencing pool was correct. Our study produced sequences for 21 nearly complete and seven partial sets of protein coding mitochondrial genes. Combined with existing sequences for 25 taxa, an improved estimate of basal relationships in Coleoptera was obtained. The procedure could be employed routinely for mitochondrial genome sequencing at the species level, to provide improved species 'barcodes' that currently use the cox1 gene only.

Prevalence of tick-borne encephalitis virus in ixodid ticks collected from the republic of Korea during 2011-2012.

PubMed

Yun, Seok-Min; Song, Bong Gu; Choi, Wooyoung; Park, Won Il; Kim, Sung Yun; Roh, Jong Yul; Ryou, Jungsang; Ju, Young Ran; Park, Chan; Shin, E-Hyun

2012-12-01

In this study, we demonstrated that TBEV-infected ticks have been distributed in the ROK, combined with our previous results. These results suggest that TBEV may exist in the ROK, and H. longicornis, H. flava, and I. nipponensis may be potential vectors of TBEV. In addition, these results emphasize the need for further epidemiological research of TBEV. We examined for the presence of RNA of TBEV by reverse transcriptase-nested polymerase chain reaction (RT-nested PCR) using ixodid ticks captured in 25 localities of 10 provinces. Ticks were collected by the flagging and dragging method or using sentinel BG traps at forests, grass thickets, and grassland. A total of 13,053 ticks belonging to two genera and four species were collected and pooled (1292 pools), according to collection site, species of tick, and developmental stage. Among 1292 pools, the envelope (E) protein gene of TBEV was detected using RT-nested PCR in 10 pools (3 pools of the 1,331 adult ticks and 7 pools of the 11,169 nymph ticks) collected from Gangwon-do province, Jeonrabuk-do province, and Jeju Island. The minimum infection rates for TBEV of Haemaphysalis longicornis, Haemaphysalis flava, and Ixodes nipponensis were 0.06%, 0.17%, and 2.38%, respectively. Phylogenetic analysis based on the partial E protein gene was performed to identify relationships between the TBEV strains. This showed that 10 Korean strains clustered with the Western subtype. In this study, we investigated the prevalence of tick-borne encephalitis virus (TBEV) in ixodid ticks from various regions of the Republic of Korea (ROK) during 2011-2012 to identify whether TBEV is circulating and to determine the endemic regions of TBEV.

MafK/NF-E2 p18 is required for beta-globin genes activation by mediating the proximity of LCR and active beta-globin genes in MEL cell line.

PubMed

Du, Mei-Jun; Lv, Xiang; Hao, De-Long; Zhao, Guo-Wei; Wu, Xue-Song; Wu, Feng; Liu, De-Pei; Liang, Chih-Chuan

2008-01-01

Evidences indicate that locus control region (LCR) of beta-globin spatially closes to the downstream active gene promoter to mediate the transcriptional activation by looping. DNA binding proteins may play an important role in the looping formation. NF-E2 is one of the key transcription factors in beta-globin gene transcriptional activation. To shed light on whether NF-E2 is involved in this process, DS19MafKsiRNA cell pools were established by specifically knocked down the expression of MafK/NF-E2 p18, one subunit of NF-E2 heterodimer. In the above cell pools, it was observed that the occupancy efficiency of NF-E2 on beta-globin gene locus and the expression level of beta-globin genes were decreased. H3 acetylation, H3-K4 methylation and the deposition of RNA polymerase II, but not the recruitment of GATA-1, were also found reduced at the beta-globin gene cluster. Chromosome Conformation Capture (3C) assay showed that the cross-linking frequency between the main NF-E2 binding site HS2 and downstream structural genes was reduced compared to the normal cell. This result demonstrated that MafK/NF-E2 p18 recruitment was involved in the physical proximity of LCR and active beta-globin genes upon beta-globin gene transcriptional activation.

Uniparental Genetic Heritage of Belarusians: Encounter of Rare Middle Eastern Matrilineages with a Central European Mitochondrial DNA Pool

PubMed Central

Kushniarevich, Alena; Sivitskaya, Larysa; Danilenko, Nina; Novogrodskii, Tadeush; Tsybovsky, Iosif; Kiseleva, Anna; Kotova, Svetlana; Chaubey, Gyaneshwer; Metspalu, Ene; Sahakyan, Hovhannes; Bahmanimehr, Ardeshir; Reidla, Maere; Rootsi, Siiri; Parik, Jüri; Reisberg, Tuuli; Achilli, Alessandro; Hooshiar Kashani, Baharak; Gandini, Francesca; Olivieri, Anna; Behar, Doron M.; Torroni, Antonio; Davydenko, Oleg; Villems, Richard

2013-01-01

Ethnic Belarusians make up more than 80% of the nine and half million people inhabiting the Republic of Belarus. Belarusians together with Ukrainians and Russians represent the East Slavic linguistic group, largest both in numbers and territory, inhabiting East Europe alongside Baltic-, Finno-Permic- and Turkic-speaking people. Till date, only a limited number of low resolution genetic studies have been performed on this population. Therefore, with the phylogeographic analysis of 565 Y-chromosomes and 267 mitochondrial DNAs from six well covered geographic sub-regions of Belarus we strove to complement the existing genetic profile of eastern Europeans. Our results reveal that around 80% of the paternal Belarusian gene pool is composed of R1a, I2a and N1c Y-chromosome haplogroups – a profile which is very similar to the two other eastern European populations – Ukrainians and Russians. The maternal Belarusian gene pool encompasses a full range of West Eurasian haplogroups and agrees well with the genetic structure of central-east European populations. Our data attest that latitudinal gradients characterize the variation of the uniparentally transmitted gene pools of modern Belarusians. In particular, the Y-chromosome reflects movements of people in central-east Europe, starting probably as early as the beginning of the Holocene. Furthermore, the matrilineal legacy of Belarusians retains two rare mitochondrial DNA haplogroups, N1a3 and N3, whose phylogeographies were explored in detail after de novo sequencing of 20 and 13 complete mitogenomes, respectively, from all over Eurasia. Our phylogeographic analyses reveal that two mitochondrial DNA lineages, N3 and N1a3, both of Middle Eastern origin, might mark distinct events of matrilineal gene flow to Europe: during the mid-Holocene period and around the Pleistocene-Holocene transition, respectively. PMID:23785503

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

Culbert, A.A.; Wallis, G.A.; Kadler, K.E.

The brittleness of bone in people with lethal (type II) osteogenesis imperfecta, a heritable disorder caused by mutations in the type I collagen genes, arises from the deposition of abnormal collagen in the bone matrix. The inability of the abnormal collagen to participate in mineralization may be caused by its failure to interact with other bone proteins. Here, we have designed a strategy to isolate the genes important for mineralization of collagen during bone formation. Cells isolated from 16-day embryonic chick calvaria and seeded post-confluence in culture deposited a mineralized matrix over a period of 2 weeks. Chick skin fibroblastsmore » seeded and cultured under the same conditions did not mineralize. Using RT-PCR, we prepared short cDNAs ({approximately}300 bp) corresponding to the 3{prime} ends of mRNA from fibroblasts and separately from the mineralizing calvarial cells. Subtractive cDNA hybridization generated a pool of cDNAs that were specific to mineralizing calvarial cells but not to fibroblasts. Screening of 100,000 plaques of a chick bone ZAP Express cDNA library with this pool of mineralizing-specific cDNAs identified ten clones which comprised full-length cDNAs for the bone proteins osteopontin (eight of the ten positives), bone sialoprotein II (one of the ten positives), and cystatin (one of the ten positives). cDNAs for type I collagen, fibronectin, alkaline phosphatase, house-keeping genes, and other genes expressed in fibroblasts were not identified in this preliminary screen. The pool of short cDNAs is likely to comprise cDNAs for further bone-specific genes and will be used to screen the entire bone cDNA library of 4.2 million clones. 30 refs., 4 figs.« less

High Temperature Inhibits Ascorbate Recycling and Light Stimulation of the Ascorbate Pool in Tomato despite Increased Expression of Biosynthesis Genes

PubMed Central

Massot, Capucine; Bancel, Doriane; Lopez Lauri, Félicie; Truffault, Vincent; Baldet, Pierre; Stevens, Rebecca; Gautier, Hélène

2013-01-01

Understanding how the fruit microclimate affects ascorbate (AsA) biosynthesis, oxidation and recycling is a great challenge in improving fruit nutritional quality. For this purpose, tomatoes at breaker stage were harvested and placed in controlled environment conditions at different temperatures (12, 17, 23, 27 and 31°C) and irradiance regimes (darkness or 150 µmol m-2 s-1). Fruit pericarp tissue was used to assay ascorbate, glutathione, enzymes related to oxidative stress and the AsA/glutathione cycle and follow the expression of genes coding for 5 enzymes of the AsA biosynthesis pathway (GME, VTC2, GPP, L-GalDH, GLDH). The AsA pool size in pericarp tissue was significantly higher under light at temperatures below 27°C. In addition, light promoted glutathione accumulation at low and high temperatures. At 12°C, increased AsA content was correlated with the enhanced expression of all genes of the biosynthesis pathway studied, combined with higher DHAR and MDHAR activities and increased enzymatic activities related to oxidative stress (CAT and APX). In contrast, at 31°C, MDHAR and GR activities were significantly reduced under light indicating that enzymes of the AsA/glutathione cycle may limit AsA recycling and pool size in fruit pericarp, despite enhanced expression of genes coding for AsA biosynthesis enzymes. In conclusion, this study confirms the important role of fruit microclimate in the regulation of fruit pericarp AsA content, as under oxidative conditions (12°C, light) total fruit pericarp AsA content increased up to 71%. Moreover, it reveals that light and temperature interact to regulate both AsA biosynthesis gene expression in tomato fruits and AsA oxidation and recycling. PMID:24367665

High temperature inhibits ascorbate recycling and light stimulation of the ascorbate pool in tomato despite increased expression of biosynthesis genes.

PubMed

Massot, Capucine; Bancel, Doriane; Lopez Lauri, Félicie; Truffault, Vincent; Baldet, Pierre; Stevens, Rebecca; Gautier, Hélène

2013-01-01

Understanding how the fruit microclimate affects ascorbate (AsA) biosynthesis, oxidation and recycling is a great challenge in improving fruit nutritional quality. For this purpose, tomatoes at breaker stage were harvested and placed in controlled environment conditions at different temperatures (12, 17, 23, 27 and 31 °C) and irradiance regimes (darkness or 150 µmol m(-2) s(-1)). Fruit pericarp tissue was used to assay ascorbate, glutathione, enzymes related to oxidative stress and the AsA/glutathione cycle and follow the expression of genes coding for 5 enzymes of the AsA biosynthesis pathway (GME, VTC2, GPP, L-GalDH, GLDH). The AsA pool size in pericarp tissue was significantly higher under light at temperatures below 27 °C. In addition, light promoted glutathione accumulation at low and high temperatures. At 12 °C, increased AsA content was correlated with the enhanced expression of all genes of the biosynthesis pathway studied, combined with higher DHAR and MDHAR activities and increased enzymatic activities related to oxidative stress (CAT and APX). In contrast, at 31 °C, MDHAR and GR activities were significantly reduced under light indicating that enzymes of the AsA/glutathione cycle may limit AsA recycling and pool size in fruit pericarp, despite enhanced expression of genes coding for AsA biosynthesis enzymes. In conclusion, this study confirms the important role of fruit microclimate in the regulation of fruit pericarp AsA content, as under oxidative conditions (12 °C, light) total fruit pericarp AsA content increased up to 71%. Moreover, it reveals that light and temperature interact to regulate both AsA biosynthesis gene expression in tomato fruits and AsA oxidation and recycling.

Microbiological Analysis in Three Diverse Natural Geothermal Bathing Pools in Iceland

PubMed Central

Thorolfsdottir, Berglind Osk Th.; Marteinsson, Viggo Thor

2013-01-01

Natural thermal bathing pools contain geothermal water that is very popular to bathe in but the water is not sterilized, irradiated or treated in any way. Increasing tourism in Iceland will lead to increasing numbers of bath guests, which can in turn affect the microbial flora in the pools and therefore user safety. Today, there is no legislation that applies to natural geothermal pools in Iceland, as the water is not used for consumption and the pools are not defined as public swimming pools. In this study, we conducted a microbiological analysis on three popular but different natural pools in Iceland, located at Lýsuhóll, Hveravellir and Landmannalaugar. Total bacterial counts were performed by flow cytometry, and with plate count at 22 °C, 37 °C and 50 °C. The presence of viable coliforms, Enterococcus spp. and pseudomonads were investigated by growth experiments on selective media. All samples were screened for noroviruses by real time PCR. The results indicate higher fecal contamination in the geothermal pools where the geothermal water flow was low and bathing guest count was high during the day. The number of cultivated Pseudomonas spp. was high (13,000–40,000 cfu/100 mL) in the natural pools, and several strains were isolated and classified as opportunistic pathogens. Norovirus was not detected in the three pools. DNA was extracted from one-liter samples in each pool and analyzed by partial 16S rRNA gene sequencing. Microbial diversity analysis revealed different microbial communities between the pools and they were primarily composed of alpha-, beta- and gammaproteobacteria. PMID:23493033

Further evidence of an Amerindian contribution to the Polynesian gene pool on Easter Island.

PubMed

Thorsby, E; Flåm, S T; Woldseth, B; Dupuy, B M; Sanchez-Mazas, A; Fernandez-Vina, M A

2009-06-01

Available evidence suggests a Polynesian origin of the Easter Island population. We recently found that some native Easter Islanders also carried some common American Indian (Amerindian) human leukocyte antigen (HLA) alleles, which probably were introduced before Europeans discovered the island in 1722. In this study, we report molecular genetic investigations of 21 other selected native Easter Islanders. Analysis of mitochondrial DNA and Y chromosome markers showed no traces of an Amerindian contribution. However, high-resolution genomic HLA typing showed that two individuals carried some other common Amerindian HLA alleles, different from those found in our previous investigations. The new data support our previous evidence of an Amerindian contribution to the gene pool on Easter Island.

Method for nonlinear optimization for gas tagging and other systems

DOEpatents

Chen, Ting; Gross, Kenny C.; Wegerich, Stephan

1998-01-01

A method and system for providing nuclear fuel rods with a configuration of isotopic gas tags. The method includes selecting a true location of a first gas tag node, selecting initial locations for the remaining n-1 nodes using target gas tag compositions, generating a set of random gene pools with L nodes, applying a Hopfield network for computing on energy, or cost, for each of the L gene pools and using selected constraints to establish minimum energy states to identify optimal gas tag nodes with each energy compared to a convergence threshold and then upon identifying the gas tag node continuing this procedure until establishing the next gas tag node until all remaining n nodes have been established.

Method for nonlinear optimization for gas tagging and other systems

DOEpatents

Chen, T.; Gross, K.C.; Wegerich, S.

1998-01-06

A method and system are disclosed for providing nuclear fuel rods with a configuration of isotopic gas tags. The method includes selecting a true location of a first gas tag node, selecting initial locations for the remaining n-1 nodes using target gas tag compositions, generating a set of random gene pools with L nodes, applying a Hopfield network for computing on energy, or cost, for each of the L gene pools and using selected constraints to establish minimum energy states to identify optimal gas tag nodes with each energy compared to a convergence threshold and then upon identifying the gas tag node continuing this procedure until establishing the next gas tag node until all remaining n nodes have been established. 6 figs.

A family of LRR sequences in the vicinity of the Co-2 locus for anthracnose resistance in Phaseolus vulgaris and its potential use in marker-assisted selection.

PubMed

Geffroy, V; Creusot, F; Falquet, J; Sévignac, M; Adam-Blondon, A F; Bannerot, H; Gepts, P; Dron, M

1998-03-01

Molecular markers offer new opportunities for breeding for disease resistance. Resistance gene pyramiding in a single cultivar, as a strategy for durable resistance, can be facilitated by marker-assisted selection (MAS). A RAPD marker, ROH20(450), linked to the Mesoamerican Co-2 anthracnose resistance gene, was previously transformed into a SCAR marker, SCH20. In the present paper we have further characterized the relevance of the SCH20 SCAR marker in different genetic backgrounds. Since this SCAR marker was found to be useful mainly in the Andean gene pool, we identified a new PCR-based marker (SCAreoli) for indirect scoring of the presence of the Co-2 gene. The SCAreoli SCAR marker is polymorphic in the Mesoamerican as well as in the Andean gene pool and should be useful in MAS. We also report that PvH20, the cloned sequence corresponding to the 450-bp RAPD marker ROH20(450), contains six imperfect leucine-rich repeats, and reveals a family of related sequences in the vicinity of the Co-2 locus. These results are discussed in the context of the recent cloning of some plant resistance genes.

Spatial genetic structure in wild cardoon, the ancestor of cultivated globe artichoke: Limited gene flow, fragmentation and population history.

PubMed

Rau, D; Rodriguez, M; Rapposelli, E; Murgia, M L; Papa, R; Brown, A H D; Attene, G

2016-12-01

Nuclear and chloroplast markers and phenotypic characters were integrated to analyse the population genetic structure of wild cardoon, Cynara cardunculus var. sylvestris, the ancestor of cultivated globe artichoke, Cynara cardunculus var. scolymus on the island of Sardinia, Italy. The spatial scale ranged from a few metres to ∼200km. Wild cardoon appears to be genetically fragmented, with significant genetic divergence at various scales, indicating that gene flow is insufficient to counterbalance the effects of genetic drift or founder effects. Divergence between populations was higher for chloroplast (40%) than for nuclear markers (15%), suggesting that gene flow via seed was lower than via pollen. Two main genetic groups were detected; these correlated with differences in flowering time, capitula size, glossiness, and anthocyanin pigmentation. A complex population structure of wild cardoon emerged over small spatial scales, likely resulting from the interplay between gene dispersal, colonisation history and selective forces. Indeed, Sardinia appears to be a 'hybrid zone' of different gene pools. The island has unique diverse germplasm that has originated from hybridisation among different gene pools. The sampling of seeds from a few plants but from many sites is suggested as the best strategy to harvest the genetic diversity of wild cardoon. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Construction and Evaluation of Normalized cDNA Libraries Enriched with Full-Length Sequences for Rapid Discovery of New Genes from Sisal (Agave sisalana Perr.) Different Developmental Stages

PubMed Central

Zhou, Wen-Zhao; Zhang, Yan-Mei; Lu, Jun-Ying; Li, Jun-Feng

2012-01-01

To provide a resource of sisal-specific expressed sequence data and facilitate this powerful approach in new gene research, the preparation of normalized cDNA libraries enriched with full-length sequences is necessary. Four libraries were produced with RNA pooled from Agave sisalana multiple tissues to increase efficiency of normalization and maximize the number of independent genes by SMART™ method and the duplex-specific nuclease (DSN). This procedure kept the proportion of full-length cDNAs in the subtracted/normalized libraries and dramatically enhanced the discovery of new genes. Sequencing of 3875 cDNA clones of libraries revealed 3320 unigenes with an average insert length about 1.2 kb, indicating that the non-redundancy of libraries was about 85.7%. These unigene functions were predicted by comparing their sequences to functional domain databases and extensively annotated with Gene Ontology (GO) terms. Comparative analysis of sisal unigenes and other plant genomes revealed that four putative MADS-box genes and knotted-like homeobox (knox) gene were obtained from a total of 1162 full-length transcripts. Furthermore, real-time PCR showed that the characteristics of their transcripts mainly depended on the tight expression regulation of a number of genes during the leaf and flower development. Analysis of individual library sequence data indicated that the pooled-tissue approach was highly effective in discovering new genes and preparing libraries for efficient deep sequencing. PMID:23202944

Glycosylation-related genes in NS0 cells are insensitive to moderately elevated ammonium concentrations

PubMed Central

Brodsky, Arthur Nathan; Caldwell, Mary; Bae, Sooneon; Harcum, Sarah W.

2014-01-01

NS0 and Chinese hamster ovary (CHO) cell lines are used to produce recombinant proteins for human therapeutics; however, ammonium accumulation can negatively impact cell growth, recombinant protein production, and protein glycosylation. To improve product quality and decrease costs, the relationship between ammonium and protein glycosylation needs to be elucidated. While ammonium has been shown to adversely affect glycosylation-related gene expression in CHO cells, NS0 studies have not been performed. Therefore, this study sought to determine if glycosylation in NS0 cells were ammonium-sensitive at the gene expression level. Using a DNA microarray that contained mouse glycosylation-related and housekeeping genes, the of these genes was analysed in response to various culture conditions – elevated ammonium, elevated salt, and elevated ammonium with proline. Surprisingly, no significant differences in gene expression levels were observed between the control and these conditions. Further, the elevated ammonium cultures were analysed using real-time quantitative reverse transcriptase PCR (qRT-PCR) for key glycosylation genes, and the qRT-PCR results corroborated the DNA microarray results, demonstrating that NS0 cells are ammonium-insensitive at the gene expression level. Since NS0 are known to have elevated nucleotide sugar pools under ammonium stress, and none of the genes directly responsible for these metabolic pools were changed, consequently cellular control at the translational or substrate-level must be responsible for the universally observed decreased glycosylation quality under elevated ammonium. PMID:25062658

Prospects and challenges for fungal metatranscriptomics of complex communities

Treesearch

Cheryl R. Kuske; Cedar N. Hesse; Jean F. Challacombe; Daniel Cullen; Joshua R. Herr; Rebecca C. Mueller; Adrian Tsang; Rytas Vilgalys

2015-01-01

The ability to extract and purify messenger RNA directly from plants, decomposing organic matter and soil, followed by highthroughput sequencing of the pool of expressed genes, has spawned the emerging research area of metatranscriptomics. Each metatranscriptome provides a snapshot of the composition and relative abundance of actively transcribed genes, and thus...

Pre-breeding for diversification of primary gene pool and genetic enhancement of grain legumes

PubMed Central

Sharma, Shivali; Upadhyaya, H. D.; Varshney, R. K.; Gowda, C. L. L.

2013-01-01

The narrow genetic base of cultivars coupled with low utilization of genetic resources are the major factors limiting grain legume production and productivity globally. Exploitation of new and diverse sources of variation is needed for the genetic enhancement of grain legumes. Wild relatives with enhanced levels of resistance/tolerance to multiple stresses provide important sources of genetic diversity for crop improvement. However, their exploitation for cultivar improvement is limited by cross-incompatibility barriers and linkage drags. Pre-breeding provides a unique opportunity, through the introgression of desirable genes from wild germplasm into genetic backgrounds readily used by the breeders with minimum linkage drag, to overcome this. Pre-breeding activities using promising landraces, wild relatives, and popular cultivars have been initiated at International Crops Research Institute for the Semi-Arid Tropics (ICRISAT) to develop new gene pools in chickpea, pigeonpea, and groundnut with a high frequency of useful genes, wider adaptability, and a broad genetic base. The availability of molecular markers will greatly assist in reducing linkage drags and increasing the efficiency of introgression in pre-breeding programs. PMID:23970889

Extreme-phenotype genome-wide association study (XP-GWAS): a method for identifying trait-associated variants by sequencing pools of individuals selected from a diversity panel.

PubMed

Yang, Jinliang; Jiang, Haiying; Yeh, Cheng-Ting; Yu, Jianming; Jeddeloh, Jeffrey A; Nettleton, Dan; Schnable, Patrick S

2015-11-01

Although approaches for performing genome-wide association studies (GWAS) are well developed, conventional GWAS requires high-density genotyping of large numbers of individuals from a diversity panel. Here we report a method for performing GWAS that does not require genotyping of large numbers of individuals. Instead XP-GWAS (extreme-phenotype GWAS) relies on genotyping pools of individuals from a diversity panel that have extreme phenotypes. This analysis measures allele frequencies in the extreme pools, enabling discovery of associations between genetic variants and traits of interest. This method was evaluated in maize (Zea mays) using the well-characterized kernel row number trait, which was selected to enable comparisons between the results of XP-GWAS and conventional GWAS. An exome-sequencing strategy was used to focus sequencing resources on genes and their flanking regions. A total of 0.94 million variants were identified and served as evaluation markers; comparisons among pools showed that 145 of these variants were statistically associated with the kernel row number phenotype. These trait-associated variants were significantly enriched in regions identified by conventional GWAS. XP-GWAS was able to resolve several linked QTL and detect trait-associated variants within a single gene under a QTL peak. XP-GWAS is expected to be particularly valuable for detecting genes or alleles responsible for quantitative variation in species for which extensive genotyping resources are not available, such as wild progenitors of crops, orphan crops, and other poorly characterized species such as those of ecological interest. © 2015 The Authors The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.

Expression of growth hormone and its transcription factor, Pit-1, in early bovine development.

PubMed

Joudrey, E M; Lechniak, D; Petrik, J; King, W A

2003-03-01

During bovine embryogenesis, bovine growth hormone (bGH) contributes to proliferation, differentiation, and modulation of embryo metabolism. Pituitary-specific transcription factor-1 (Pit-1) is a transcription factor that binds to promoters of GH, prolactin (PRL), and thyroid-stimulating hormone-beta (TSHbeta) encoding genes. A polymorphism in the fifth exon of the bGH gene resulting in a leucine (Leu) to valine (Val) substitution provides an Alu I restriction site when the Leu allele is present. To determine the onset of embryonic expression of the bGH gene, oocytes derived from ovaries homozygous for Leu alleles were fertilized in vitro with spermatozoa obtained from a Val homozygote. For each developmental stage examined, three separate pools of embryos composed of approximately 100 cell samples underwent RNA isolation, reverse transcription to cDNA, and amplification by nested PCR (nPCR). Bovine GH gene transcripts were identified at 2- to 4-cell (n = 162), 8- to 16-cell (n = 73), morulae (n = 51), and blastocyst (n = 15) stages. Likewise, transcripts for Pit-1 were detected at 2-cell (n = 125), 4-cell (n = 114), 8-cell (n = 56), 12-to-32-cell (n = 32), morulae (n = 68), and blastocyst (n = 14) stages. After digestion with Alu1, bGH cDNA was genotyped by restriction fragment length polymorphism (RFLP) analysis. Bovine GH mRNA was present in all pools of stages examined. Both Leu and Val alleles (maternal and paternal) were only detected in pools of embryos that had reached 8- to 16-cell stage. Results suggest that transcription of the bGH gene begins at the 8- to 16-cell stage in bovine embryos, possibly under control of the transcription factor, Pit-1, and that RFLP analysis of the bGH gene can be used to determine parental origin of transcripts in early embryonic development. Copyright 2003 Wiley-Liss, Inc.

Association studies of 23 positional/functional candidate genes on chromosome 10 in late-onset Alzheimer's disease.

PubMed

Morgan, A R; Turic, D; Jehu, L; Hamilton, G; Hollingworth, P; Moskvina, V; Jones, L; Lovestone, S; Brayne, C; Rubinsztein, D C; Lawlor, B; Gill, M; O'Donovan, M C; Owen, M J; Williams, J

2007-09-05

Late-onset Alzheimer's disease (LOAD) is a common neurodegenerative disorder, with a complex etiology. APOE is the only confirmed susceptibility gene for LOAD. Others remain yet to be found. Evidence from linkage studies suggests that a gene (or genes) conferring susceptibility for LOAD resides on chromosome 10. We studied 23 positional/functional candidate genes from our linkage region on chromosome 10 (APBB1IP, ALOX5, AD037, SLC18A3, DKK1, ZWINT, ANK3, UBE2D1, CDC2, SIRT1, JDP1, NET7, SUPV3L1, NEN3, SAR1, SGPL1, SEC24C, CAMK2G, PP3CB, SNCG, CH25H, PLCE1, ANXV111) in the MRC genetic resource for LOAD. These candidates were screened for sequence polymorphisms in a sample of 14 LOAD subjects and detected polymorphisms tested for association with LOAD in a three-stage design involving two stages of genotyping pooled DNA samples followed by a third stage in which markers showing evidence for association in the first stages were subjected to individual genotyping. One hundred and twenty polymorphisms were identified and tested in stage 1 (4 case + 4 control pools totaling 366 case and 366 control individuals). Single nucleotide polymorphisms (SNPs) showing evidence of association with LOAD were then studied in stage 2 (8 case + 4 control pools totaling 1,001 case and 1,001 control individuals). Five SNPs, in four genes, showed evidence for association (P < 0.1) at stage 2 and were individually genotyped in the complete dataset, comprising 1,160 LOAD cases and 1,389 normal controls. Two SNPs in SGPL1 demonstrated marginal evidence of association, with uncorrected P values of 0.042 and 0.056, suggesting that variation in SGPL1 may confer susceptibility to LOAD. Copyright 2007 Wiley-Liss, Inc.

Pharmacokinetics and pharmacogenetics of the MEK1/2 inhibitor, selumetinib, in Asian and Western healthy subjects: a pooled analysis.

PubMed

Dymond, Angela W; Elks, Cathy; Martin, Paul; Carlile, David J; Mariani, Gabriella; Lovick, Susan; Huang, Yifan; Lorch, Ulrike; Brown, Helen; So, Karen

2017-06-01

Emerging data on selumetinib, a MEK1/2 inhibitor in clinical development, suggest a possible difference in pharmacokinetics (PK) between Japanese and Western patients. This pooled analysis sought to assess the effect of ethnicity on selumetinib exposure in healthy Western and Asian subjects, and to identify any association between genetic variants in the UGT1A1, CYP2C19 and ABCG2 genes and observed differences in selumetinib PK. A pooled analysis of data from ten Phase I studies, one in Asian subjects (encompassing Japanese, non-Japanese Asian and Indian Asian subjects) and nine in Western subjects, was conducted. Key findings were derived from the collective exposure data across doses of 25, 35, 50 and 75 mg selumetinib; primary variables were dose-normalized AUC and C max . PK data from 308 subjects (10 studies) were available for the pooled analysis; genetic data from 87 subjects (3 studies) were available for the pharmacogenetic analysis. Dose-normalized AUC and C max were 35% (95% CI: 25-47%) and 39% (95% CI: 24-56%) higher in the pooled Asian group, respectively, compared with Western subjects. PK exposure parameters were similar between the Japanese, non-Japanese Asian and Indian groups. There was no evidence that the polymorphisms assessed in the genes UGT1A1, CYP2C19 and ABCG2 account for observed PK differences. Selumetinib exposure was higher in healthy Asian subjects compared with Western subjects, and these data provide valuable insight for clinicians to consider when treating patients of Asian ethnicity with selumetinib.

Uneven colonization of the lymphoid periphery by T cells that undergo early TCR{alpha} rearrangements.

PubMed

Hendricks, Deborah W; Fink, Pamela J

2009-04-01

A sparse population of thymocytes undergoes TCRalpha gene rearrangement early in development, before the double-positive stage. The potential of these cells to contribute to the peripheral T cell pool is unknown. To examine the peripheral T cell compartment expressing a repertoire biased to early TCR gene rearrangements, we developed a mouse model in which TCRalpha rearrangements are restricted to the double-negative stage of thymocyte development. These mice carry floxed RAG2 alleles and a Cre transgene driven by the CD4 promoter. As expected, conventional T cell development is compromised in such Cre(+) RAG2(fl/fl) mice, and the TCRalphabeta(+) T cells that develop are limited in their TCRalpha repertoire, preferentially using early rearranging Valpha genes. In the gut, the Thy-1(+)TCRalphabeta(+) intraepithelial lymphocyte (IEL) compartment is surprisingly intact, whereas the Thy-1(-)TCRalphabeta(+) subset is almost completely absent. Thus, T cells expressing a TCRalpha repertoire that is the product of early gene rearrangements can preferentially populate distinct IEL compartments. Despite this capacity, Cre(+) RAG2(fl/fl) T cell progenitors cannot compete with wild-type T cell progenitors in mixed bone marrow chimeras, suggesting that in normal mice, there is only a small contribution to the peripheral T cell pool by cells that have undergone early TCRalpha rearrangements. In the absence of wild-type competitors, aggressive homeostatic proliferation in the IEL compartment can promote a relatively normal Thy-1(+) TCRalphabeta(+) T cell pool from the limited population derived from Cre(+) RAG2(fl/fl) progenitors.

Uneven colonization of the lymphoid periphery by T cells that undergo early TCRα rearrangements1

PubMed Central

Hendricks, Deborah W.; Fink, Pamela J.

2009-01-01

A sparse population of thymocytes undergoes TCRα gene rearrangement early in development, before the double positive stage. The potential of these cells to contribute to the peripheral T cell pool is unknown. To examine the peripheral T cell compartment expressing a repertoire biased to early TCR gene rearrangements, we developed a mouse model in which TCRα rearrangements are restricted to the double negative stage of thymocyte development. These mice carry floxed RAG2 alleles and a Cre transgene driven by the CD4 promoter. As expected, conventional T cell development is compromised in such Cre(+) RAG2fl/fl mice, and the TCRαβ+ T cells that develop are limited in their TCRα repertoire, preferentially utilizing early-rearranging Vα genes. In the gut, the Thy-1+TCRαβ+ intraepithelial lymphocyte (IEL) compartment is surprisingly intact, while the Thy-1−TCRαβ+ subset is almost completely absent. Thus, T cells expressing a TCRα repertoire that is the product of early gene rearrangements can preferentially populate distinct IEL compartments. Despite this capacity, Cre(+) RAG2fl/fl T cell progenitors cannot compete with wild-type (WT) T cell progenitors in mixed bone marrow chimeras, suggesting that in normal mice, there is only a small contribution to the peripheral T cell pool by cells that have undergone early TCRα rearrangements. In the absence of WT competitors, aggressive homeostatic proliferation in the IEL compartment can promote a relatively normal Thy-1+ TCRαβ+ T cell pool from the limited population derived from Cre(+) RAG2fl/fl progenitors. PMID:19299725

PTGER4 gene variant rs76523431 is a candidate risk factor for radiological joint damage in rheumatoid arthritis patients: a genetic study of six cohorts.

PubMed

Rodriguez-Rodriguez, Luis; Ivorra-Cortes, Jose; Carmona, F David; Martín, Javier; Balsa, Alejandro; van Steenbergen, Hanna W; van der Helm-van Mil, Annette H M; González-Álvaro, Isidoro; Fernandez-Gutiérrez, Benjamín

2015-11-05

Prostaglandin E receptor 4 (PTGER4) is implicated in immune regulation and bone metabolism. The aim of this study was to analyze its role in radiological joint damage in rheumatoid arthritis (RA). Six independent cohorts of patients with RA of European or North American descent were included, comprising 1789 patients with 5083 sets of X-rays. The Hospital Clínico San Carlos Rheumatoid Arthritis, Princesa Early Arthritis Register Longitudinal study, and Hospital Universitario de La Paz early arthritis (Spain) cohorts were used as discovery cohorts, and the Leiden Early Arthritis Clinic (The Netherlands), Wichita (United States), and National Databank for Rheumatic Diseases (United States and Canada) cohorts as replication cohorts. First, the PTGER4 rs6896969 single-nucleotide polymorphism (SNP) was genotyped using TaqMan assays and available Illumina Immunochip data and studied in the discovery and replication cohorts. Second, the PTGER4 gene and adjacent regions were analyzed using Immunochip genotyping data in the discovery cohorts. On the basis of pooled p values, linkage disequilibrium structure of the region, and location in regions with transcriptional properties, SNPs were selected for replication. The results from discovery, replication, and overall cohorts were pooled using inverse-variance-weighted meta-analysis. Influence of the polymorphisms on the overall radiological damage (constant effect) and on damage progression over time (time-varying effect) was analyzed. The rs6896969 polymorphism showed a significant association with radiological damage in the constant effect pooled analysis of the discovery cohorts, although no significant association was observed in the replication cohorts or the overall pooled analysis. Regarding the analysis of the PTGER4 region, 976 variants were analyzed in the discovery cohorts. From the constant and time-varying effect analyses, 12 and 20 SNPs, respectively, were selected for replication. Only the rs76523431 variant showed a significant association with radiographic progression in the time-varying effect pooled analysis of the discovery, replication, and overall cohorts. The overall pooled effect size was 1.10 (95 % confidence interval 1.05-1.14, p = 2.10 × 10(-5)), meaning that radiographic yearly progression was 10 % greater for each copy of the minor allele. The PTGER4 gene is a candidate risk factor for radiological progression in RA.

Molecular genetic studies of natives on Easter Island: evidence of an early European and Amerindian contribution to the Polynesian gene pool.

PubMed

Lie, B A; Dupuy, B M; Spurkland, A; Fernández-Viña, M A; Hagelberg, E; Thorsby, E

2007-01-01

Most archaeological and linguistic evidence suggest a Polynesian origin of the population of Easter Island (Rapanui), and this view has been supported by the identification of Polynesian mitochondrial DNA (mtDNA) polymorphisms in prehistoric skeletal remains. However, some evidence of an early South American contact also exists (the sweet potato, bottle gourd etc.), but genetic studies have so far failed to show an early Amerindian contribution to the gene pool on Easter Island. To address this issue, we analyzed mtDNA and Y chromosome markers and performed high-resolution human leukocyte antigen (HLA) genotyping of DNA harvested from previously collected sera of 48 reputedly nonadmixed native Easter Islanders. All individuals carried mtDNA types and HLA alleles previously found in Polynesia, and most men carried Y chromosome markers of Polynesian origin, providing further evidence of a Polynesian origin of the population of Easter Island. A few individuals carried HLA alleles and/or Y chromosome markers of European origin. More interestingly, some individuals carried the HLA alleles A*0212 and B*3905, which are of typical Amerindian origin. The genealogy of some of the individuals carrying these non-Polynesian HLA alleles and their haplotypic backgrounds suggest an introduction into Easter Island in the early 1800s, or earlier. Thus, there may have been an early European and Amerindian contribution to the Polynesian gene pool of Easter Island.

Dissecting maize diversity in lowland South America: genetic structure and geographic distribution models.

PubMed

Bracco, Mariana; Cascales, Jimena; Hernández, Julián Cámara; Poggio, Lidia; Gottlieb, Alexandra M; Lia, Verónica V

2016-08-26

Maize landraces from South America have traditionally been assigned to two main categories: Andean and Tropical Lowland germplasm. However, the genetic structure and affiliations of the lowland gene pools have been difficult to assess due to limited sampling and the lack of comparative analysis. Here, we examined SSR and Adh2 sequence variation in a diverse sample of maize landraces from lowland middle South America, and performed a comprehensive integrative analysis of population structure and diversity including already published data of archaeological and extant specimens from the Americas. Geographic distribution models were used to explore the relationship between environmental factors and the observed genetic structure. Bayesian and multivariate analyses of population structure showed the existence of two previously overlooked lowland gene pools associated with Guaraní indigenous communities of middle South America. The singularity of this germplasm was also evidenced by the frequency distribution of microsatellite repeat motifs of the Adh2 locus and the distinct spatial pattern inferred from geographic distribution models. Our results challenge the prevailing view that lowland middle South America is just a contact zone between Andean and Tropical Lowland germplasm and highlight the occurrence of a unique, locally adapted gene pool. This information is relevant for the conservation and utilization of maize genetic resources, as well as for a better understanding of environment-genotype associations.

Pooled Resequencing of 122 Ulcerative Colitis Genes in a Large Dutch Cohort Suggests Population-Specific Associations of Rare Variants in MUC2.

PubMed

Visschedijk, Marijn C; Alberts, Rudi; Mucha, Soren; Deelen, Patrick; de Jong, Dirk J; Pierik, Marieke; Spekhorst, Lieke M; Imhann, Floris; van der Meulen-de Jong, Andrea E; van der Woude, C Janneke; van Bodegraven, Adriaan A; Oldenburg, Bas; Löwenberg, Mark; Dijkstra, Gerard; Ellinghaus, David; Schreiber, Stefan; Wijmenga, Cisca; Rivas, Manuel A; Franke, Andre; van Diemen, Cleo C; Weersma, Rinse K

2016-01-01

Genome-wide association studies have revealed several common genetic risk variants for ulcerative colitis (UC). However, little is known about the contribution of rare, large effect genetic variants to UC susceptibility. In this study, we performed a deep targeted re-sequencing of 122 genes in Dutch UC patients in order to investigate the contribution of rare variants to the genetic susceptibility to UC. The selection of genes consists of 111 established human UC susceptibility genes and 11 genes that lead to spontaneous colitis when knocked-out in mice. In addition, we sequenced the promoter regions of 45 genes where known variants exert cis-eQTL-effects. Targeted pooled re-sequencing was performed on DNA of 790 Dutch UC cases. The Genome of the Netherlands project provided sequence data of 500 healthy controls. After quality control and prioritization based on allele frequency and pathogenicity probability, follow-up genotyping of 171 rare variants was performed on 1021 Dutch UC cases and 1166 Dutch controls. Single-variant association and gene-based analyses identified an association of rare variants in the MUC2 gene with UC. The associated variants in the Dutch population could not be replicated in a German replication cohort (1026 UC cases, 3532 controls). In conclusion, this study has identified a putative role for MUC2 on UC susceptibility in the Dutch population and suggests a population-specific contribution of rare variants to UC.

Cross-species transcriptomic approach reveals genes in hamster implantation sites.

PubMed

Lei, Wei; Herington, Jennifer; Galindo, Cristi L; Ding, Tianbing; Brown, Naoko; Reese, Jeff; Paria, Bibhash C

2014-12-01

The mouse model has greatly contributed to understanding molecular mechanisms involved in the regulation of progesterone (P4) plus estrogen (E)-dependent blastocyst implantation process. However, little is known about contributory molecular mechanisms of the P4-only-dependent blastocyst implantation process that occurs in species such as hamsters, guineapigs, rabbits, pigs, rhesus monkeys, and perhaps humans. We used the hamster as a model of P4-only-dependent blastocyst implantation and carried out cross-species microarray (CSM) analyses to reveal differentially expressed genes at the blastocyst implantation site (BIS), in order to advance the understanding of molecular mechanisms of implantation. Upregulation of 112 genes and downregulation of 77 genes at the BIS were identified using a mouse microarray platform, while use of the human microarray revealed 62 up- and 38 down-regulated genes at the BIS. Excitingly, a sizable number of genes (30 up- and 11 down-regulated genes) were identified as a shared pool by both CSMs. Real-time RT-PCR and in situ hybridization validated the expression patterns of several up- and down-regulated genes identified by both CSMs at the hamster and mouse BIS to demonstrate the merit of CSM findings across species, in addition to revealing genes specific to hamsters. Functional annotation analysis found that genes involved in the spliceosome, proteasome, and ubiquination pathways are enriched at the hamster BIS, while genes associated with tight junction, SAPK/JNK signaling, and PPARα/RXRα signalings are repressed at the BIS. Overall, this study provides a pool of genes and evidence of their participation in up- and down-regulated cellular functions/pathways at the hamster BIS. © 2014 Society for Reproduction and Fertility.

Musing on the Memes of Open and Distance Education

ERIC Educational Resources Information Center

Latchem, Colin

2014-01-01

Just as genes propagate themselves in the gene pool by leaping from body to body, so memes (ideas, behaviours, and actions) transmit cultural ideas or practices from one mind to another through writing, speech, or other imitable phenomena. This paper considers the memes that influence the evolution of open and distance education. If the…

De novo assembly and next-generation sequencing to analyse full-length gene variants from codon-barcoded libraries.

PubMed

Cho, Namjin; Hwang, Byungjin; Yoon, Jung-ki; Park, Sangun; Lee, Joongoo; Seo, Han Na; Lee, Jeewon; Huh, Sunghoon; Chung, Jinsoo; Bang, Duhee

2015-09-21

Interpreting epistatic interactions is crucial for understanding evolutionary dynamics of complex genetic systems and unveiling structure and function of genetic pathways. Although high resolution mapping of en masse variant libraries renders molecular biologists to address genotype-phenotype relationships, long-read sequencing technology remains indispensable to assess functional relationship between mutations that lie far apart. Here, we introduce JigsawSeq for multiplexed sequence identification of pooled gene variant libraries by combining a codon-based molecular barcoding strategy and de novo assembly of short-read data. We first validate JigsawSeq on small sub-pools and observed high precision and recall at various experimental settings. With extensive simulations, we then apply JigsawSeq to large-scale gene variant libraries to show that our method can be reliably scaled using next-generation sequencing. JigsawSeq may serve as a rapid screening tool for functional genomics and offer the opportunity to explore evolutionary trajectories of protein variants.

Possible ancestral structure in human populations.

PubMed

Plagnol, Vincent; Wall, Jeffrey D

2006-07-01

Determining the evolutionary relationships between fossil hominid groups such as Neanderthals and modern humans has been a question of enduring interest in human evolutionary genetics. Here we present a new method for addressing whether archaic human groups contributed to the modern gene pool (called ancient admixture), using the patterns of variation in contemporary human populations. Our method improves on previous work by explicitly accounting for recent population history before performing the analyses. Using sequence data from the Environmental Genome Project, we find strong evidence for ancient admixture in both a European and a West African population (p approximately 10(-7)), with contributions to the modern gene pool of at least 5%. While Neanderthals form an obvious archaic source population candidate in Europe, there is not yet a clear source population candidate in West Africa.

Impact and quantification of the sources of error in DNA pooling designs.

PubMed

Jawaid, A; Sham, P

2009-01-01

The analysis of genome wide variation offers the possibility of unravelling the genes involved in the pathogenesis of disease. Genome wide association studies are also particularly useful for identifying and validating targets for therapeutic intervention as well as for detecting markers for drug efficacy and side effects. The cost of such large-scale genetic association studies may be reduced substantially by the analysis of pooled DNA from multiple individuals. However, experimental errors inherent in pooling studies lead to a potential increase in the false positive rate and a loss in power compared to individual genotyping. Here we quantify various sources of experimental error using empirical data from typical pooling experiments and corresponding individual genotyping counts using two statistical methods. We provide analytical formulas for calculating these different errors in the absence of complete information, such as replicate pool formation, and for adjusting for the errors in the statistical analysis. We demonstrate that DNA pooling has the potential of estimating allele frequencies accurately, and adjusting the pooled allele frequency estimates for differential allelic amplification considerably improves accuracy. Estimates of the components of error show that differential allelic amplification is the most important contributor to the error variance in absolute allele frequency estimation, followed by allele frequency measurement and pool formation errors. Our results emphasise the importance of minimising experimental errors and obtaining correct error estimates in genetic association studies.

Investigation of Bartonella infection in ixodid ticks from California.

PubMed

Chang, Chao-chin; Hayashidani, Hideki; Pusterla, Nicola; Kasten, Rickie W; Madigan, John E; Chomel, Bruno B

2002-07-01

A total of 1253 ixodid ticks (254 tick pools) collected between the end of 1995 and the spring of 1997 from six California counties (El Dorado, Los Angeles, Orange, Santa Cruz, Shasta and Sonoma) were examined for the presence of Bartonella DNA by PCR of the citrate synthase gene. Of 1,119 adult Ixodes pacificus ticks tested, 26 (11.6%) of 224 pools, each containing five ticks, were positive (minimum percentage of ticks harboring detectable Bartonella DNA, 2.3%). Bartonella PCR-positive ticks were identified in five counties but none of the ticks from Los Angeles County was positive. Among 47 nymphal I. pacificus ticks collected in Sonoma County, one (10%) positive pool out of 10 pools was identified (minimum percentage of ticks harboring detectable Bartonella DNA, 2.1%). Among the 54 Dermacentor occidentalis grouped in 12 pools from Orange County, one pool (8.3%) was PCR positive for Bartonella and similarly one pool (14.3%) was positive among the 30 Dermacentor variabilis ticks grouped in seven pools. None of the three D. occidentalis from El Dorado County were positive. None of the nine tick pools positive for Ehrlichia phagocytophila were positive for Bartonella. Following our previous findings of Bartonella PCR-positive adult I. pacificus ticks in central coastal California, this is the first preliminary report of the presence of Bartonella DNA in I. pacificus nymphs and in Dermacentor sp. ticks. Distribution of Bartonella among ixodid ticks appears widespread in California.

Mass effects meet species sorting: transformations of microbial assemblages in epiphreatic subsurface karst water pools.

PubMed

Shabarova, Tanja; Widmer, Franco; Pernthaler, Jakob

2013-09-01

We investigated the transformations of the microbial communities in epiphreatic karst cave pools with different flooding frequencies. Fingerprinting of 16S rRNA genes was combined with microscopic and sequence analysis to examine if source water would transport comparable microbial inocula into the pools at consecutive flood events, and to assess possible effects of residence time on the microbial assemblages during stagnant periods. Variability in the concentrations of dissolved organic carbon and conductivity indicated differences between floods and changes of pool water over time. High numbers of Betaproteobacteria affiliated with Methylophilaceae and Comamonadaceae were introduced into the pools during floodings. While the former persisted in the pools, the latter exhibited considerable microdiversification. These Betaproteobacteria might thus represent core microbial groups in karst water. A decrease in the estimated total diversity of the remaining bacterial taxa was apparent after a few weeks of residence: Some were favoured by stagnant conditions, whereas the majority was rapidly outcompeted. Thus, the microbial communities consisted of different components governed by complementary assembly mechanisms (dispersal versus environmental filtering) upon introduction into the pools. High overlap of temporary and persistent community members between samplings from two winters, moreover, reflected the seasonal recurrence of the studied microbial assemblages. © 2013 John Wiley & Sons Ltd and Society for Applied Microbiology.

A Novel Strategy for Selection and Validation of Reference Genes in Dynamic Multidimensional Experimental Design in Yeast

PubMed Central

Cankorur-Cetinkaya, Ayca; Dereli, Elif; Eraslan, Serpil; Karabekmez, Erkan; Dikicioglu, Duygu; Kirdar, Betul

2012-01-01

Background Understanding the dynamic mechanism behind the transcriptional organization of genes in response to varying environmental conditions requires time-dependent data. The dynamic transcriptional response obtained by real-time RT-qPCR experiments could only be correctly interpreted if suitable reference genes are used in the analysis. The lack of available studies on the identification of candidate reference genes in dynamic gene expression studies necessitates the identification and the verification of a suitable gene set for the analysis of transient gene expression response. Principal Findings In this study, a candidate reference gene set for RT-qPCR analysis of dynamic transcriptional changes in Saccharomyces cerevisiae was determined using 31 different publicly available time series transcriptome datasets. Ten of the twelve candidates (TPI1, FBA1, CCW12, CDC19, ADH1, PGK1, GCN4, PDC1, RPS26A and ARF1) we identified were not previously reported as potential reference genes. Our method also identified the commonly used reference genes ACT1 and TDH3. The most stable reference genes from this pool were determined as TPI1, FBA1, CDC19 and ACT1 in response to a perturbation in the amount of available glucose and as FBA1, TDH3, CCW12 and ACT1 in response to a perturbation in the amount of available ammonium. The use of these newly proposed gene sets outperformed the use of common reference genes in the determination of dynamic transcriptional response of the target genes, HAP4 and MEP2, in response to relaxation from glucose and ammonium limitations, respectively. Conclusions A candidate reference gene set to be used in dynamic real-time RT-qPCR expression profiling in yeast was proposed for the first time in the present study. Suitable pools of stable reference genes to be used under different experimental conditions could be selected from this candidate set in order to successfully determine the expression profiles for the genes of interest. PMID:22675547

Selection of Salmonella enterica Serovar Typhi Genes Involved during Interaction with Human Macrophages by Screening of a Transposon Mutant Library

PubMed Central

Sabbagh, Sébastien C.; Lepage, Christine; McClelland, Michael; Daigle, France

2012-01-01

The human-adapted Salmonella enterica serovar Typhi (S. Typhi) causes a systemic infection known as typhoid fever. This disease relies on the ability of the bacterium to survive within macrophages. In order to identify genes involved during interaction with macrophages, a pool of approximately 105 transposon mutants of S. Typhi was subjected to three serial passages of 24 hours through human macrophages. Mutants recovered from infected macrophages (output) were compared to the initial pool (input) and those significantly underrepresented resulted in the identification of 130 genes encoding for cell membrane components, fimbriae, flagella, regulatory processes, pathogenesis, and many genes of unknown function. Defined deletions in 28 genes or gene clusters were created and mutants were evaluated in competitive and individual infection assays for uptake and intracellular survival during interaction with human macrophages. Overall, 26 mutants had defects in the competitive assay and 14 mutants had defects in the individual assay. Twelve mutants had defects in both assays, including acrA, exbDB, flhCD, fliC, gppA, mlc, pgtE, typA, waaQGP, SPI-4, STY1867-68, and STY2346. The complementation of several mutants by expression of plasmid-borne wild-type genes or gene clusters reversed defects, confirming that the phenotypic impairments within macrophages were gene-specific. In this study, 35 novel phenotypes of either uptake or intracellular survival in macrophages were associated with Salmonella genes. Moreover, these results reveal several genes encoding molecular mechanisms not previously known to be involved in systemic infection by human-adapted typhoidal Salmonella that will need to be elucidated. PMID:22574205

Identification of Differentially Expressed Genes in Blood Cells of Narcolepsy Patients

PubMed Central

Tanaka, Susumu; Honda, Yutaka; Honda, Makoto

2007-01-01

Study Objective: A close association between the human leukocyte antigen (HLA)-DRB1*1501/DQB1*0602 and abnormalities in some inflammatory cytokines have been demonstrated in narcolepsy. Specific alterations in the immune system have been suggested to occur in this disorder. We attempted to identify alterations in gene expression underlying the abnormalities in the blood cells of narcoleptic patients. Designs: Total RNA from 12 narcolepsy-cataplexy patients and from 12 age- and sex-matched healthy controls were pooled. The pooled samples were initially screened for candidate genes for narcolepsy by differential display analysis using annealing control primers (ACP). The second screening of the samples was carried out by semiquantitative PCR using gene-specific primers. Finally, the expression levels of the candidate genes were further confirmed by quantitative real-time PCR using a new set of samples (20 narcolepsy-cataplexy patients and 20 healthy controls). Results: The second screening revealed differential expression of 4 candidate genes. Among them, MX2 was confirmed as a significantly down-regulated gene in the white blood cells of narcoleptic patients by quantitative real-time PCR. Conclusion: We found the MX2 gene to be significantly less expressed in comparison with normal subjects in the white blood cells of narcoleptic patients. This gene is relevant to the immune system. Although differential display analysis using ACP technology has a limitation in that it does not help in determining the functional mechanism underlying sleep/wakefulness dysregulation, it is useful for identifying novel genetic factors related to narcolepsy, such as HLA molecules. Further studies are required to explore the functional relationship between the MX2 gene and narcolepsy pathophysiology. Citation: Tanaka S; Honda Y; Honda M. Identification of differentially expressed genes in blood cells of narcolepsy patients. SLEEP 2007;30(8):974-979. PMID:17702266

Evaluation and Design of Genome-Wide CRISPR/SpCas9 Knockout Screens

PubMed Central

Hart, Traver; Tong, Amy Hin Yan; Chan, Katie; Van Leeuwen, Jolanda; Seetharaman, Ashwin; Aregger, Michael; Chandrashekhar, Megha; Hustedt, Nicole; Seth, Sahil; Noonan, Avery; Habsid, Andrea; Sizova, Olga; Nedyalkova, Lyudmila; Climie, Ryan; Tworzyanski, Leanne; Lawson, Keith; Sartori, Maria Augusta; Alibeh, Sabriyeh; Tieu, David; Masud, Sanna; Mero, Patricia; Weiss, Alexander; Brown, Kevin R.; Usaj, Matej; Billmann, Maximilian; Rahman, Mahfuzur; Costanzo, Michael; Myers, Chad L.; Andrews, Brenda J.; Boone, Charles; Durocher, Daniel; Moffat, Jason

2017-01-01

The adaptation of CRISPR/SpCas9 technology to mammalian cell lines is transforming the study of human functional genomics. Pooled libraries of CRISPR guide RNAs (gRNAs) targeting human protein-coding genes and encoded in viral vectors have been used to systematically create gene knockouts in a variety of human cancer and immortalized cell lines, in an effort to identify whether these knockouts cause cellular fitness defects. Previous work has shown that CRISPR screens are more sensitive and specific than pooled-library shRNA screens in similar assays, but currently there exists significant variability across CRISPR library designs and experimental protocols. In this study, we reanalyze 17 genome-scale knockout screens in human cell lines from three research groups, using three different genome-scale gRNA libraries. Using the Bayesian Analysis of Gene Essentiality algorithm to identify essential genes, we refine and expand our previously defined set of human core essential genes from 360 to 684 genes. We use this expanded set of reference core essential genes, CEG2, plus empirical data from six CRISPR knockout screens to guide the design of a sequence-optimized gRNA library, the Toronto KnockOut version 3.0 (TKOv3) library. We then demonstrate the high effectiveness of the library relative to reference sets of essential and nonessential genes, as well as other screens using similar approaches. The optimized TKOv3 library, combined with the CEG2 reference set, provide an efficient, highly optimized platform for performing and assessing gene knockout screens in human cell lines. PMID:28655737

A genome-wide association study implicates diacylglycerol kinase eta (DGKH) and several other genes in the etiology of bipolar disorder

PubMed Central

Baum, AE; Akula, N; Cabanero, M; Cardona, I; Corona, W; Klemens, B; Schulze, TG; Cichon, S; Rietschel, M; Nöthen, MM; Georgi, A; Schumacher, J; Schwarz, M; Jamra, R Abou; Höfels, S; Propping, P; Satagopan, J; Detera-Wadleigh, SD; Hardy, J; McMahon, FJ

2008-01-01

The genetic basis of bipolar disorder has long been thought to be complex, with the potential involvement of multiple genes, but methods to analyze populations with respect to this complexity have only recently become available. We have carried out a genome-wide association study of bipolar disorder by genotyping over 550,000 SNPs in two independent case-control samples of European origin. The initial association screen was performed using pooled DNA; selected SNPs were confirmed by individual genotyping. While DNA pooling reduces power to detect genetic associations, there is a substantial cost savings and gain in efficiency. A total of 88 SNPs representing 80 different genes met the prior criteria for replication in both samples. Effect sizes were modest: no single SNP of large effect was detected. Of 37 SNPs selected for individual genotyping, the strongest association signal was detected at a marker within the first intron of DGKH (p = 1.5 × 10−8, experiment-wide p<0.01, OR= 1.59). This gene encodes diacylglycerol kinase eta, a key protein in the lithium-sensitive phosphatidyl inositol pathway. This first genome-wide association study of bipolar disorder shows that several genes, each of modest effect, reproducibly influence disease risk. Bipolar disorder may be a polygenic disease. PMID:17486107

The Diagnostic Performance of Stool DNA Testing for Colorectal Cancer: A Systematic Review and Meta-Analysis.

PubMed

Zhai, Rong-Lin; Xu, Fei; Zhang, Pei; Zhang, Wan-Li; Wang, Hui; Wang, Ji-Liang; Cai, Kai-Lin; Long, Yue-Ping; Lu, Xiao-Ming; Tao, Kai-Xiong; Wang, Guo-Bin

2016-02-01

This meta-analysis was designed to evaluate the diagnostic performance of stool DNA testing for colorectal cancer (CRC) and compare the performance between single-gene and multiple-gene tests.MEDLINE, Cochrane, EMBASE databases were searched using keywords colorectal cancers, stool/fecal, sensitivity, specificity, DNA, and screening. Sensitivity analysis, quality assessments, and performance bias were performed for the included studies.Fifty-three studies were included in the analysis with a total sample size of 7524 patients. The studies were heterogeneous with regard to the genes being analyzed for fecal genetic biomarkers of CRC, as well as the laboratory methods being used for each assay. The sensitivity of the different assays ranged from 2% to 100% and the specificity ranged from 81% to 100%. The meta-analysis found that the pooled sensitivities for single- and multigene assays were 48.0% and 77.8%, respectively, while the pooled specificities were 97.0% and 92.7%. Receiver operator curves and diagnostic odds ratios showed no significant difference between both tests with regard to sensitivity or specificity.This meta-analysis revealed that using assays that evaluated multiple genes compared with single-gene assays did not increase the sensitivity or specificity of stool DNA testing in detecting CRC.

Isolation of Novel CreERT2-Driver Lines in Zebrafish Using an Unbiased Gene Trap Approach

PubMed Central

Jungke, Peggy; Hammer, Juliane; Hans, Stefan; Brand, Michael

2015-01-01

Gene manipulation using the Cre/loxP-recombinase system has been successfully employed in zebrafish to study gene functions and lineage relationships. Recently, gene trapping approaches have been applied to produce large collections of transgenic fish expressing conditional alleles in various tissues. However, the limited number of available cell- and tissue-specific Cre/CreERT2-driver lines still constrains widespread application in this model organism. To enlarge the pool of existing CreERT2-driver lines, we performed a genome-wide gene trap screen using a Tol2-based mCherry-T2a-CreERT2 (mCT2aC) gene trap vector. This cassette consists of a splice acceptor and a mCherry-tagged variant of CreERT2 which enables simultaneous labeling of the trapping event, as well as CreERT2 expression from the endogenous promoter. Using this strategy, we generated 27 novel functional CreERT2-driver lines expressing in a cell- and tissue-specific manner during development and adulthood. This study summarizes the analysis of the generated CreERT2-driver lines with respect to functionality, expression, integration, as well as associated phenotypes. Our results significantly enlarge the existing pool of CreERT2-driver lines in zebrafish and combined with Cre–dependent effector lines, the new CreERT2-driver lines will be important tools to manipulate the zebrafish genome. PMID:26083735

Detection of Orientia tsutsugamushi (Rickettsiales: rickettsiaceae) in unengorged chiggers (Acari: Trombiculidae) from Oita Prefecture, Japan, by nested polymerase chain reaction.

PubMed

Pham, X D; Otsuka, Y; Suzuki, H; Takaoka, H

2001-03-01

The current study surveyed the 56-kDa type-specific antigen (TSA) gene DNAs of Orientia tsutsugamushi (Hayashi) in approximately 4.000 unengorged chiggers obtained from the soil or ground surface in an endemic and a nonendemic area of the Tsutsugamushi disease in Oita Prefecture, southwestern Japan, by nested polymerase chain reaction (PCR). Serotypes of O. tsutsugamushi were identified by restriction fragment-length polymorphism (RFLP) analysis. In the endemic area, 242 pools from five species [234 pools of Leptotrombidium scutellare (Nagayo, Miyagawa, Mitamura, Tamiya and Tenjin), two L. pallidum (Nagayo, Miyagawa, Mitamura and Tamiya), four L. kitasatoi (Fukuzumi & Obata), one L. fuji (Kuwata, Berge and Philip), and one Neotrombicula japonica (Tanaka, Kaiwa, Teramura and Kagaya)] were tested, and eight (seven pools of L. scutellare and one N. japonica) were positive for O. tsutsugamushi. Among the seven positive pools of L. scutellare, the distribution of serotypes was as follows: Kuroki (4), Gilliam (1), Karp (1), and Kawasaki (1). The first two serotypes (Kuroki and Gilliam) were identified for the first time in this species. In the nonendemic area, 128 pools from eight species were tested, and 13 were positive for O. tsutsugamushi. The positive rate was as follows: L. pallidum (4/41). L. kitasatoi (1/18), Gahrliepia saduski Womersley (2/10), L. fuji (4/50), L. himizu (Sasa, Kumada, Hayashi, Enomoto, Fukuzumi and Obata) (1/2), and Miyatrombicula kochiensis (Sasa, Kawashima and Egashira) (1/3). The latter three species were shown for the first time to harbor O. tsutsugamushi. All ofthe positive pools were Kuroki, except for two pools (one L. pallidum and one L. fuji), which were Gilliam (this serotype was also detected for the first time in L. pallidum). Further analysis revealed no differences in the nucleotide sequences (125 bp of variable domain 1 of TSA gene) of the same serotypes (i.e., Kuroki and Gilliam) among the positive samples. These data indicate that O. tsutsugamushi was widely distributed in various trombiculid species, even in the nonendemic area. The data are also suggestive of a possible horizontal transmission of O. tsutsugamushi among trombiculid species.

SNP discovery in common bean by restriction-associated DNA (RAD) sequencing for genetic diversity and population structure analysis.

PubMed

Valdisser, Paula Arielle M R; Pappas, Georgios J; de Menezes, Ivandilson P P; Müller, Bárbara S F; Pereira, Wendell J; Narciso, Marcelo G; Brondani, Claudio; Souza, Thiago L P O; Borba, Tereza C O; Vianello, Rosana P

2016-06-01

Researchers have made great advances into the development and application of genomic approaches for common beans, creating opportunities to driving more real and applicable strategies for sustainable management of the genetic resource towards plant breeding. This work provides useful polymorphic single-nucleotide polymorphisms (SNPs) for high-throughput common bean genotyping developed by RAD (restriction site-associated DNA) sequencing. The RAD tags were generated from DNA pooled from 12 common bean genotypes, including breeding lines of different gene pools and market classes. The aligned sequences identified 23,748 putative RAD-SNPs, of which 3357 were adequate for genotyping; 1032 RAD-SNPs with the highest ADT (assay design tool) score are presented in this article. The RAD-SNPs were structurally annotated in different coding (47.00 %) and non-coding (53.00 %) sequence components of genes. A subset of 384 RAD-SNPs with broad genome distribution was used to genotype a diverse panel of 95 common bean germplasms and revealed a successful amplification rate of 96.6 %, showing 73 % of polymorphic SNPs within the Andean group and 83 % in the Mesoamerican group. A slightly increased He (0.161, n = 21) value was estimated for the Andean gene pool, compared to the Mesoamerican group (0.156, n = 74). For the linkage disequilibrium (LD) analysis, from a group of 580 SNPs (289 RAD-SNPs and 291 BARC-SNPs) genotyped for the same set of genotypes, 70.2 % were in LD, decreasing to 0.10 %in the Andean group and 0.77 % in the Mesoamerican group. Haplotype patterns spanning 310 Mb of the genome (60 %) were characterized in samples from different origins. However, the haplotype frameworks were under-represented for the Andean (7.85 %) and Mesoamerican (5.55 %) gene pools separately. In conclusion, RAD sequencing allowed the discovery of hundreds of useful SNPs for broad genetic analysis of common bean germplasm. From now, this approach provides an excellent panel of molecular tools for whole genome analysis, allowing integrating and better exploring the common bean breeding practices.

Sequencing of a QTL-rich region of the Theobroma cacao genome using pooled BACs and the identification of trait specific candidate genes

PubMed Central

2011-01-01

Background BAC-based physical maps provide for sequencing across an entire genome or a selected sub-genomic region of biological interest. Such a region can be approached with next-generation whole-genome sequencing and assembly as if it were an independent small genome. Using the minimum tiling path as a guide, specific BAC clones representing the prioritized genomic interval are selected, pooled, and used to prepare a sequencing library. Results This pooled BAC approach was taken to sequence and assemble a QTL-rich region, of ~3 Mbp and represented by twenty-seven BACs, on linkage group 5 of the Theobroma cacao cv. Matina 1-6 genome. Using various mixtures of read coverages from paired-end and linear 454 libraries, multiple assemblies of varied quality were generated. Quality was assessed by comparing the assembly of 454 reads with a subset of ten BACs individually sequenced and assembled using Sanger reads. A mixture of reads optimal for assembly was identified. We found, furthermore, that a quality assembly suitable for serving as a reference genome template could be obtained even with a reduced depth of sequencing coverage. Annotation of the resulting assembly revealed several genes potentially responsible for three T. cacao traits: black pod disease resistance, bean shape index, and pod weight. Conclusions Our results, as with other pooled BAC sequencing reports, suggest that pooling portions of a minimum tiling path derived from a BAC-based physical map is an effective method to target sub-genomic regions for sequencing. While we focused on a single QTL region, other QTL regions of importance could be similarly sequenced allowing for biological discovery to take place before a high quality whole-genome assembly is completed. PMID:21794110

Sequencing of a QTL-rich region of the Theobroma cacao genome using pooled BACs and the identification of trait specific candidate genes.

PubMed

Feltus, Frank A; Saski, Christopher A; Mockaitis, Keithanne; Haiminen, Niina; Parida, Laxmi; Smith, Zachary; Ford, James; Staton, Margaret E; Ficklin, Stephen P; Blackmon, Barbara P; Cheng, Chun-Huai; Schnell, Raymond J; Kuhn, David N; Motamayor, Juan-Carlos

2011-07-27

BAC-based physical maps provide for sequencing across an entire genome or a selected sub-genomic region of biological interest. Such a region can be approached with next-generation whole-genome sequencing and assembly as if it were an independent small genome. Using the minimum tiling path as a guide, specific BAC clones representing the prioritized genomic interval are selected, pooled, and used to prepare a sequencing library. This pooled BAC approach was taken to sequence and assemble a QTL-rich region, of ~3 Mbp and represented by twenty-seven BACs, on linkage group 5 of the Theobroma cacao cv. Matina 1-6 genome. Using various mixtures of read coverages from paired-end and linear 454 libraries, multiple assemblies of varied quality were generated. Quality was assessed by comparing the assembly of 454 reads with a subset of ten BACs individually sequenced and assembled using Sanger reads. A mixture of reads optimal for assembly was identified. We found, furthermore, that a quality assembly suitable for serving as a reference genome template could be obtained even with a reduced depth of sequencing coverage. Annotation of the resulting assembly revealed several genes potentially responsible for three T. cacao traits: black pod disease resistance, bean shape index, and pod weight. Our results, as with other pooled BAC sequencing reports, suggest that pooling portions of a minimum tiling path derived from a BAC-based physical map is an effective method to target sub-genomic regions for sequencing. While we focused on a single QTL region, other QTL regions of importance could be similarly sequenced allowing for biological discovery to take place before a high quality whole-genome assembly is completed.

Novel quantitative real-time LCR for the sensitive detection of SNP frequencies in pooled DNA: method development, evaluation and application.

PubMed

Psifidi, Androniki; Dovas, Chrysostomos; Banos, Georgios

2011-01-19

Single nucleotide polymorphisms (SNP) have proven to be powerful genetic markers for genetic applications in medicine, life science and agriculture. A variety of methods exist for SNP detection but few can quantify SNP frequencies when the mutated DNA molecules correspond to a small fraction of the wild-type DNA. Furthermore, there is no generally accepted gold standard for SNP quantification, and, in general, currently applied methods give inconsistent results in selected cohorts. In the present study we sought to develop a novel method for accurate detection and quantification of SNP in DNA pooled samples. The development and evaluation of a novel Ligase Chain Reaction (LCR) protocol that uses a DNA-specific fluorescent dye to allow quantitative real-time analysis is described. Different reaction components and thermocycling parameters affecting the efficiency and specificity of LCR were examined. Several protocols, including gap-LCR modifications, were evaluated using plasmid standard and genomic DNA pools. A protocol of choice was identified and applied for the quantification of a polymorphism at codon 136 of the ovine PRNP gene that is associated with susceptibility to a transmissible spongiform encephalopathy in sheep. The real-time LCR protocol developed in the present study showed high sensitivity, accuracy, reproducibility and a wide dynamic range of SNP quantification in different DNA pools. The limits of detection and quantification of SNP frequencies were 0.085% and 0.35%, respectively. The proposed real-time LCR protocol is applicable when sensitive detection and accurate quantification of low copy number mutations in DNA pools is needed. Examples include oncogenes and tumour suppressor genes, infectious diseases, pathogenic bacteria, fungal species, viral mutants, drug resistance resulting from point mutations, and genetically modified organisms in food.

Novel Quantitative Real-Time LCR for the Sensitive Detection of SNP Frequencies in Pooled DNA: Method Development, Evaluation and Application

PubMed Central

Psifidi, Androniki; Dovas, Chrysostomos; Banos, Georgios

2011-01-01

Background Single nucleotide polymorphisms (SNP) have proven to be powerful genetic markers for genetic applications in medicine, life science and agriculture. A variety of methods exist for SNP detection but few can quantify SNP frequencies when the mutated DNA molecules correspond to a small fraction of the wild-type DNA. Furthermore, there is no generally accepted gold standard for SNP quantification, and, in general, currently applied methods give inconsistent results in selected cohorts. In the present study we sought to develop a novel method for accurate detection and quantification of SNP in DNA pooled samples. Methods The development and evaluation of a novel Ligase Chain Reaction (LCR) protocol that uses a DNA-specific fluorescent dye to allow quantitative real-time analysis is described. Different reaction components and thermocycling parameters affecting the efficiency and specificity of LCR were examined. Several protocols, including gap-LCR modifications, were evaluated using plasmid standard and genomic DNA pools. A protocol of choice was identified and applied for the quantification of a polymorphism at codon 136 of the ovine PRNP gene that is associated with susceptibility to a transmissible spongiform encephalopathy in sheep. Conclusions The real-time LCR protocol developed in the present study showed high sensitivity, accuracy, reproducibility and a wide dynamic range of SNP quantification in different DNA pools. The limits of detection and quantification of SNP frequencies were 0.085% and 0.35%, respectively. Significance The proposed real-time LCR protocol is applicable when sensitive detection and accurate quantification of low copy number mutations in DNA pools is needed. Examples include oncogenes and tumour suppressor genes, infectious diseases, pathogenic bacteria, fungal species, viral mutants, drug resistance resulting from point mutations, and genetically modified organisms in food. PMID:21283808

Metabolic traits of an uncultured archaeal lineage--MSBL1--from brine pools of the Red Sea.

PubMed

Mwirichia, Romano; Alam, Intikhab; Rashid, Mamoon; Vinu, Manikandan; Ba-Alawi, Wail; Anthony Kamau, Allan; Kamanda Ngugi, David; Göker, Markus; Klenk, Hans-Peter; Bajic, Vladimir; Stingl, Ulrich

2016-01-13

The candidate Division MSBL1 (Mediterranean Sea Brine Lakes 1) comprises a monophyletic group of uncultured archaea found in different hypersaline environments. Previous studies propose methanogenesis as the main metabolism. Here, we describe a metabolic reconstruction of MSBL1 based on 32 single-cell amplified genomes from Brine Pools of the Red Sea (Atlantis II, Discovery, Nereus, Erba and Kebrit). Phylogeny based on rRNA genes as well as conserved single copy genes delineates the group as a putative novel lineage of archaea. Our analysis shows that MSBL1 may ferment glucose via the Embden-Meyerhof-Parnas pathway. However, in the absence of organic carbon, carbon dioxide may be fixed via the ribulose bisphosphate carboxylase, Wood-Ljungdahl pathway or reductive TCA cycle. Therefore, based on the occurrence of genes for glycolysis, absence of the core genes found in genomes of all sequenced methanogens and the phylogenetic position, we hypothesize that the MSBL1 are not methanogens, but probably sugar-fermenting organisms capable of autotrophic growth. Such a mixotrophic lifestyle would confer survival advantage (or possibly provide a unique narrow niche) when glucose and other fermentable sugars are not available.

Molecular evidence of RNA polymerase II gene reveals the origin of worldwide cultivated barley

PubMed Central

Wang, Yonggang; Ren, Xifeng; Sun, Dongfa; Sun, Genlou

2016-01-01

The origin and domestication of cultivated barley have long been under debate. A population-based resequencing and phylogenetic analysis of the single copy of RPB2 gene was used to address barley domestication, to explore genetic differentiation of barley populations on the worldwide scale, and to understand gene-pool exchanges during the spread and subsequent development of barley cultivation. Our results revealed significant genetic differentiation among three geographically distinct wild barley populations. Differences in haplotype composition among populations from different geographical regions revealed that modern cultivated barley originated from two major wild barley populations: one from the Near East Fertile Crescent and the other from the Tibetan Plateau, supporting polyphyletic origin of cultivated barley. The results of haplotype frequencies supported multiple domestications coupled with widespread introgression events that generated genetic admixture between divergent barley gene pools. Our results not only provide important insight into the domestication and evolution of cultivated barley, but also enhance our understanding of introgression and distinct selection pressures in different environments on shaping the genetic diversity of worldwide barley populations, thus further facilitating the effective use of the wild barley germplasm. PMID:27786300

Genomics of high molecular weight plasmids isolated from an on-farm biopurification system.

PubMed

Martini, María C; Wibberg, Daniel; Lozano, Mauricio; Torres Tejerizo, Gonzalo; Albicoro, Francisco J; Jaenicke, Sebastian; van Elsas, Jan Dirk; Petroni, Alejandro; Garcillán-Barcia, M Pilar; de la Cruz, Fernando; Schlüter, Andreas; Pühler, Alfred; Pistorio, Mariano; Lagares, Antonio; Del Papa, María F

2016-06-20

The use of biopurification systems (BPS) constitutes an efficient strategy to eliminate pesticides from polluted wastewaters from farm activities. BPS environments contain a high microbial density and diversity facilitating the exchange of information among bacteria, mediated by mobile genetic elements (MGEs), which play a key role in bacterial adaptation and evolution in such environments. Here we sequenced and characterized high-molecular-weight plasmids from a bacterial collection of an on-farm BPS. The high-throughput-sequencing of the plasmid pool yielded a total of several Mb sequence information. Assembly of the sequence data resulted in six complete replicons. Using in silico analyses we identified plasmid replication genes whose encoding proteins represent 13 different Pfam families, as well as proteins involved in plasmid conjugation, indicating a large diversity of plasmid replicons and suggesting the occurrence of horizontal gene transfer (HGT) events within the habitat analyzed. In addition, genes conferring resistance to 10 classes of antimicrobial compounds and those encoding enzymes potentially involved in pesticide and aromatic hydrocarbon degradation were found. Global analysis of the plasmid pool suggest that the analyzed BPS represents a key environment for further studies addressing the dissemination of MGEs carrying catabolic genes and pathway assembly regarding degradation capabilities.

Identification of an ancestral resistance gene cluster involved in the coevolution process between Phaseolus vulgaris and its fungal pathogen Colletotrichum lindemuthianum.

PubMed

Geffroy, V; Sicard, D; de Oliveira, J C; Sévignac, M; Cohen, S; Gepts, P; Neema, C; Langin, T; Dron, M

1999-09-01

The recent cloning of plant resistance (R) genes and the sequencing of resistance gene clusters have shed light on the molecular evolution of R genes. However, up to now, no attempt has been made to correlate this molecular evolution with the host-pathogen coevolution process at the population level. Cross-inoculations were carried out between 26 strains of the fungal pathogen Colletotrichum lindemuthianum and 48 Phaseolus vulgaris plants collected in the three centers of diversity of the host species. A high level of diversity for resistance against the pathogen was revealed. Most of the resistance specificities were overcome in sympatric situations, indicating an adaptation of the pathogen to the local host. In contrast, plants were generally resistant to allopatric strains, suggesting that R genes that were efficient against exotic strains but had been overcome locally were maintained in the plant genome. These results indicated that coevolution processes between the two protagonists led to a differentiation for resistance in the three centers of diversity of the host. To improve our understanding of the molecular evolution of these different specificities, a recombinant inbred (RI) population derived from two representative genotypes of the Andean (JaloEEP558) and Mesoamerican (BAT93) gene pools was used to map anthracnose specificities. A gene cluster comprising both Andean (Co-y; Co-z) and Mesoamerican (Co-9) host resistance specificities was identified, suggesting that this locus existed prior to the separation of the two major gene pools of P. vulgaris. Molecular analysis revealed a high level of complexity at this locus. It harbors 11 restriction fragment length polymorphisms when R gene analog (RGA) clones are used. The relationship between the coevolution process and diversification of resistance specificities at resistance gene clusters is discussed.

Molecular detection of Rickettsia aeschlimannii in Hyalomma spp. ticks from camels (Camelus dromedarius) in Nigeria, West Africa.

PubMed

Kamani, J; Baneth, G; Apanaskevich, D A; Mumcuoglu, K Y; Harrus, S

2015-06-01

Several species of the spotted fever group rickettsiae have been identified as emerging pathogens throughout the world, including in Africa. In this study, 197 Hyalomma ticks (Ixodida: Ixodidae) collected from 51 camels (Camelus dromedarius) in Kano, northern Nigeria, were screened by amplification and sequencing of the citrate synthase (gltA), outer membrane protein A (ompA) and 17-kDa antigen gene fragments. Rickettsia sp. gltA fragments were detected in 43.3% (42/97) of the tick pools tested. Rickettsial ompA gene fragments (189 bp and 630 bp) were detected in 64.3% (n = 27) and 23.8% (n = 10) of the gltA-positive tick pools by real-time and conventional polymerase chain reaction (PCR), respectively. The amplicons were 99-100% identical to Rickettsia aeschlimannii TR/Orkun-H and R. aeschlimannii strain EgyRickHimp-El-Arish in GenBank. Furthermore, 17-kDa antigen gene fragments of 214 bp and 265 bp were detected in 59.5% (n = 25) and 38.1% (n = 16), respectively, of tick pools, and sequences were identical to one another and 99-100% identical to those of the R. aeschlimannii strain Ibadan A1 in GenBank. None of the Hyalomma impressum ticks collected were positive for Rickettsia sp. DNA. Rickettsia sp. gltA fragments (133 bp) were detected in 18.8% of camel blood samples, but all samples were negative for the other genes targeted. This is the first report to describe the molecular detection of R. aeschlimannii in Hyalomma spp. ticks from camels in Nigeria. © 2015 The Royal Entomological Society.

Coxiella burnetii and Rickettsia conorii: Two zoonotic pathogens in peridomestic rodents and their ectoparasites in Nigeria.

PubMed

Kamani, Joshua; Baneth, Gad; Gutiérrez, Ricardo; Nachum-Biala, Yaarit; Mumcuoglu, Kosta Y; Harrus, Shimon

2018-01-01

Rodents are hosts of numerous pathogenic agents of public health importance globally. Their ability to harbor these pathogens without showing overt clinical signs of disease has epidemiologic consequences. In some rural settings in Nigeria, humans and rodents do not only share feeds and abode, but the latter may end up on the table of the former as a source of protein, thereby increasing the risks of disease transmission. Molecular assays were used to detect and characterize two agents of zoonotic importance, Coxiella burnetii and Rickettsia spp. in 194 peridomestic rodents captured in a peri-urban setting in Nigeria, and 32 pools of ectoparasites removed from them, to determine their possible role in the epidemiology of these diseases in this country. Targeting and characterizing the insertion sequence IS1111, C. burnetii DNA was detected in 4 out of 194 (2.1%) rodents comprising 3 out of 121 (2.5%) Rattus norvegicus and 1 out of 48 (2.1%) Rattus rattus screened in this study. Rickettsia spp. DNA was detected in two Rhipicephalus sanginueus sensu lato pools (i.e. RT1 and RT4) using the citrate synthase (gltA) gene and further characterized by amplification and sequence analysis of six genes to determine their identity. The RT1 sample consistently gave 98-100% identity to Rickettsia conorii str. Malish 7 for the various genes and loci studied. However, the identity of RT4 could not be definitively determined due to variable identities to different Rickettsia spp. according to the gene or loci under consideration. Further isolation study to determine if the RT4 characterized is a new variant or a mixture of sequences of different rickettsiae within the pool will be worthwhile. Copyright © 2017 Elsevier GmbH. All rights reserved.

From genomes to vaccines: Leishmania as a model.

PubMed Central

Almeida, Renata; Norrish, Alan; Levick, Mark; Vetrie, David; Freeman, Tom; Vilo, Jaak; Ivens, Alasdair; Lange, Uta; Stober, Carmel; McCann, Sharon; Blackwell, Jenefer M

2002-01-01

The 35 Mb genome of Leishmania should be sequenced by late 2002. It contains approximately 8500 genes that will probably translate into more than 10 000 proteins. In the laboratory we have been piloting strategies to try to harness the power of the genome-proteome for rapid screening of new vaccine candidate. To this end, microarray analysis of 1094 unique genes identified using an EST analysis of 2091 cDNA clones from spliced leader libraries prepared from different developmental stages of Leishmania has been employed. The plan was to identify amastigote-expressed genes that could be used in high-throughput DNA-vaccine screens to identify potential new vaccine candidates. Despite the lack of transcriptional regulation that polycistronic transcription in Leishmania dictates, the data provide evidence for a high level of post-transcriptional regulation of RNA abundance during the developmental cycle of promastigotes in culture and in lesion-derived amastigotes of Leishmania major. This has provided 147 candidates from the 1094 unique genes that are specifically upregulated in amastigotes and are being used in vaccine studies. Using DNA vaccination, it was demonstrated that pooling strategies can work to identify protective vaccines, but it was found that some potentially protective antigens are masked by other disease-exacerbatory antigens in the pool. A total of 100 new vaccine candidates are currently being tested separately and in pools to extend this analysis, and to facilitate retrospective bioinformatic analysis to develop predictive algorithms for sequences that constitute potentially protective antigens. We are also working with other members of the Leishmania Genome Network to determine whether RNA expression determined by microarray analyses parallels expression at the protein level. We believe we are making good progress in developing strategies that will allow rapid translation of the sequence of Leishmania into potential interventions for disease control in humans. PMID:11839176

Core element characterization of Rhodococcus promoters and development of a promoter-RBS mini-pool with different activity levels for efficient gene expression.

PubMed

Jiao, Song; Yu, Huimin; Shen, Zhongyao

2018-09-25

To satisfy the urgent demand for promoter engineering that can accurately regulate the metabolic circuits and expression of specific genes in the Rhodococcus microbial platform, a promoter-ribosome binding site (RBS) coupled mini-pool with fine-tuning of different activity levels was successfully established. Transcriptome analyses of R. ruber TH revealed several representative promoters with different activity levels, e.g., Pami, Pcs, Pnh, P50sl36, PcbiM, PgroE and Pniami. β-Galactosidase (LacZ) reporter measurement demonstrated that different gene expression levels could be obtained with these natural promoters combined with an optimal RBS of ami. Further use of these promoters to overexpress the nitrile hydratase (NHase) gene with RBSami in R. ruber THdAdN produced different expression levels consistent with the transcription analyses. The -35 and -10 core elements of different promoters were further analyzed, and the conserved sequences were revealed to be TTGNNN and (T/C)GNNA(A/C)AAT. By mutating the core elements of the strong promoters, Pnh and Pami, into the above consensus sequence, two even stronger promoters, PnhM and PamiM, were obtained with 2.2-fold and 7.7-fold improvements in transcription, respectively. Integrating several strategies, including transcriptome promoter screening, -35 and -10 core element identification, core element point-mutation, RBS optimization and diverse reporter verification, a fine-tuning promoter-RBS combination mini-pool with different activity levels in Rhodococcus strains was successfully established. This development is significant for broad applications of the Rhodococcus genus as a microbial platform. Copyright © 2018 Elsevier B.V. All rights reserved.

Mining Gene Regulatory Networks by Neural Modeling of Expression Time-Series.

PubMed

Rubiolo, Mariano; Milone, Diego H; Stegmayer, Georgina

2015-01-01

Discovering gene regulatory networks from data is one of the most studied topics in recent years. Neural networks can be successfully used to infer an underlying gene network by modeling expression profiles as times series. This work proposes a novel method based on a pool of neural networks for obtaining a gene regulatory network from a gene expression dataset. They are used for modeling each possible interaction between pairs of genes in the dataset, and a set of mining rules is applied to accurately detect the subjacent relations among genes. The results obtained on artificial and real datasets confirm the method effectiveness for discovering regulatory networks from a proper modeling of the temporal dynamics of gene expression profiles.

Transcriptomic Analysis Using Olive Varieties and Breeding Progenies Identifies Candidate Genes Involved in Plant Architecture.

PubMed

González-Plaza, Juan J; Ortiz-Martín, Inmaculada; Muñoz-Mérida, Antonio; García-López, Carmen; Sánchez-Sevilla, José F; Luque, Francisco; Trelles, Oswaldo; Bejarano, Eduardo R; De La Rosa, Raúl; Valpuesta, Victoriano; Beuzón, Carmen R

2016-01-01

Plant architecture is a critical trait in fruit crops that can significantly influence yield, pruning, planting density and harvesting. Little is known about how plant architecture is genetically determined in olive, were most of the existing varieties are traditional with an architecture poorly suited for modern growing and harvesting systems. In the present study, we have carried out microarray analysis of meristematic tissue to compare expression profiles of olive varieties displaying differences in architecture, as well as seedlings from their cross pooled on the basis of their sharing architecture-related phenotypes. The microarray used, previously developed by our group has already been applied to identify candidates genes involved in regulating juvenile to adult transition in the shoot apex of seedlings. Varieties with distinct architecture phenotypes and individuals from segregating progenies displaying opposite architecture features were used to link phenotype to expression. Here, we identify 2252 differentially expressed genes (DEGs) associated to differences in plant architecture. Microarray results were validated by quantitative RT-PCR carried out on genes with functional annotation likely related to plant architecture. Twelve of these genes were further analyzed in individual seedlings of the corresponding pool. We also examined Arabidopsis mutants in putative orthologs of these targeted candidate genes, finding altered architecture for most of them. This supports a functional conservation between species and potential biological relevance of the candidate genes identified. This study is the first to identify genes associated to plant architecture in olive, and the results obtained could be of great help in future programs aimed at selecting phenotypes adapted to modern cultivation practices in this species.

Identification and verification of differentially expressed genes in the caprine hypothalamic-pituitary-gonadal axis that are associated with litter size.

PubMed

Feng, Tao; Cao, Gui-Ling; Chu, Ming-Xing; Di, Ran; Huang, Dong-Wei; Liu, Qiu-Yue; Pan, Zhang-Yuan; Jin, Mei; Zhang, Ying-Jie; Li, Ning

2015-02-01

Litter size is a favorable economic trait for the goat industry, but remains a complex trait controlled by multiple genes in multiple organs. Several genes have been identified that may affect embryo survival, follicular development, and the health of fetuses during pregnancy. Jining Grey goats demonstrate the largest litter size among goat breeds indigenous to China. In order to better understand the genetic basis of this trait, six suppression subtractive hybridization (SSH) cDNA libraries were constructed using pooled mRNAs from hypothalamuses, pituitaries, and ovaries of sexually mature and adult polytocous Jining Grey goats, as testers, versus the pooled corresponding mRNAs of monotocous Liaoning Cashmere goats, as drivers. A total of 1,458 true-positive clones--including 955 known genes and 481 known and 22 unknown expressed sequence tags--were obtained from the SSH libraries by sequencing and alignment. The known genes were categorized into cellular processes and signaling information storage and processing, and metabolism. Three genes (FTH1, GH, and SAA) were selected to validate the SSH results by quantitative real-time PCR; all three were up-regulated in the corresponding tissues in the tester group indicating that these are candidate genes associated with the large litter size of Jining Grey goats. Several other identified genes may affect embryo survival, follicular development, and health during pregnancy. This study provides insights into the mechanistic basis by which the caprine hypothalamic-pituitary-gonadal axis affects reproductive traits and provides a theoretical basis for goat production and breeding. © 2015 Wiley Periodicals, Inc.

High frequency of phylogenetically diverse reductive dehalogenase-homologous genes in deep subseafloor sedimentary metagenomes

PubMed Central

Kawai, Mikihiko; Futagami, Taiki; Toyoda, Atsushi; Takaki, Yoshihiro; Nishi, Shinro; Hori, Sayaka; Arai, Wataru; Tsubouchi, Taishi; Morono, Yuki; Uchiyama, Ikuo; Ito, Takehiko; Fujiyama, Asao; Inagaki, Fumio; Takami, Hideto

2014-01-01

Marine subsurface sediments on the Pacific margin harbor diverse microbial communities even at depths of several hundreds meters below the seafloor (mbsf) or more. Previous PCR-based molecular analysis showed the presence of diverse reductive dehalogenase gene (rdhA) homologs in marine subsurface sediment, suggesting that anaerobic respiration of organohalides is one of the possible energy-yielding pathways in the organic-rich sedimentary habitat. However, primer-independent molecular characterization of rdhA has remained to be demonstrated. Here, we studied the diversity and frequency of rdhA homologs by metagenomic analysis of five different depth horizons (0.8, 5.1, 18.6, 48.5, and 107.0 mbsf) at Site C9001 off the Shimokita Peninsula of Japan. From all metagenomic pools, remarkably diverse rdhA-homologous sequences, some of which are affiliated with novel clusters, were observed with high frequency. As a comparison, we also examined frequency of dissimilatory sulfite reductase genes (dsrAB), key functional genes for microbial sulfate reduction. The dsrAB were also widely observed in the metagenomic pools whereas the frequency of dsrAB genes was generally smaller than that of rdhA-homologous genes. The phylogenetic composition of rdhA-homologous genes was similar among the five depth horizons. Our metagenomic data revealed that subseafloor rdhA homologs are more diverse than previously identified from PCR-based molecular studies. Spatial distribution of similar rdhA homologs across wide depositional ages indicates that the heterotrophic metabolic processes mediated by the genes can be ecologically important, functioning in the organic-rich subseafloor sedimentary biosphere. PMID:24624126

High frequency of phylogenetically diverse reductive dehalogenase-homologous genes in deep subseafloor sedimentary metagenomes.

PubMed

Kawai, Mikihiko; Futagami, Taiki; Toyoda, Atsushi; Takaki, Yoshihiro; Nishi, Shinro; Hori, Sayaka; Arai, Wataru; Tsubouchi, Taishi; Morono, Yuki; Uchiyama, Ikuo; Ito, Takehiko; Fujiyama, Asao; Inagaki, Fumio; Takami, Hideto

2014-01-01

Marine subsurface sediments on the Pacific margin harbor diverse microbial communities even at depths of several hundreds meters below the seafloor (mbsf) or more. Previous PCR-based molecular analysis showed the presence of diverse reductive dehalogenase gene (rdhA) homologs in marine subsurface sediment, suggesting that anaerobic respiration of organohalides is one of the possible energy-yielding pathways in the organic-rich sedimentary habitat. However, primer-independent molecular characterization of rdhA has remained to be demonstrated. Here, we studied the diversity and frequency of rdhA homologs by metagenomic analysis of five different depth horizons (0.8, 5.1, 18.6, 48.5, and 107.0 mbsf) at Site C9001 off the Shimokita Peninsula of Japan. From all metagenomic pools, remarkably diverse rdhA-homologous sequences, some of which are affiliated with novel clusters, were observed with high frequency. As a comparison, we also examined frequency of dissimilatory sulfite reductase genes (dsrAB), key functional genes for microbial sulfate reduction. The dsrAB were also widely observed in the metagenomic pools whereas the frequency of dsrAB genes was generally smaller than that of rdhA-homologous genes. The phylogenetic composition of rdhA-homologous genes was similar among the five depth horizons. Our metagenomic data revealed that subseafloor rdhA homologs are more diverse than previously identified from PCR-based molecular studies. Spatial distribution of similar rdhA homologs across wide depositional ages indicates that the heterotrophic metabolic processes mediated by the genes can be ecologically important, functioning in the organic-rich subseafloor sedimentary biosphere.

Multiplexed droplet single-cell RNA-sequencing using natural genetic variation.

PubMed

Kang, Hyun Min; Subramaniam, Meena; Targ, Sasha; Nguyen, Michelle; Maliskova, Lenka; McCarthy, Elizabeth; Wan, Eunice; Wong, Simon; Byrnes, Lauren; Lanata, Cristina M; Gate, Rachel E; Mostafavi, Sara; Marson, Alexander; Zaitlen, Noah; Criswell, Lindsey A; Ye, Chun Jimmie

2018-01-01

Droplet single-cell RNA-sequencing (dscRNA-seq) has enabled rapid, massively parallel profiling of transcriptomes. However, assessing differential expression across multiple individuals has been hampered by inefficient sample processing and technical batch effects. Here we describe a computational tool, demuxlet, that harnesses natural genetic variation to determine the sample identity of each droplet containing a single cell (singlet) and detect droplets containing two cells (doublets). These capabilities enable multiplexed dscRNA-seq experiments in which cells from unrelated individuals are pooled and captured at higher throughput than in standard workflows. Using simulated data, we show that 50 single-nucleotide polymorphisms (SNPs) per cell are sufficient to assign 97% of singlets and identify 92% of doublets in pools of up to 64 individuals. Given genotyping data for each of eight pooled samples, demuxlet correctly recovers the sample identity of >99% of singlets and identifies doublets at rates consistent with previous estimates. We apply demuxlet to assess cell-type-specific changes in gene expression in 8 pooled lupus patient samples treated with interferon (IFN)-β and perform eQTL analysis on 23 pooled samples.

Nitrate Reduction Functional Genes and Nitrate Reduction Potentials Persist in Deeper Estuarine Sediments. Why?

PubMed Central

Papaspyrou, Sokratis; Smith, Cindy J.; Dong, Liang F.; Whitby, Corinne; Dumbrell, Alex J.; Nedwell, David B.

2014-01-01

Denitrification and dissimilatory nitrate reduction to ammonium (DNRA) are processes occurring simultaneously under oxygen-limited or anaerobic conditions, where both compete for nitrate and organic carbon. Despite their ecological importance, there has been little investigation of how denitrification and DNRA potentials and related functional genes vary vertically with sediment depth. Nitrate reduction potentials measured in sediment depth profiles along the Colne estuary were in the upper range of nitrate reduction rates reported from other sediments and showed the existence of strong decreasing trends both with increasing depth and along the estuary. Denitrification potential decreased along the estuary, decreasing more rapidly with depth towards the estuary mouth. In contrast, DNRA potential increased along the estuary. Significant decreases in copy numbers of 16S rRNA and nitrate reducing genes were observed along the estuary and from surface to deeper sediments. Both metabolic potentials and functional genes persisted at sediment depths where porewater nitrate was absent. Transport of nitrate by bioturbation, based on macrofauna distributions, could only account for the upper 10 cm depth of sediment. A several fold higher combined freeze-lysable KCl-extractable nitrate pool compared to porewater nitrate was detected. We hypothesised that his could be attributed to intracellular nitrate pools from nitrate accumulating microorganisms like Thioploca or Beggiatoa. However, pyrosequencing analysis did not detect any such organisms, leaving other bacteria, microbenthic algae, or foraminiferans which have also been shown to accumulate nitrate, as possible candidates. The importance and bioavailability of a KCl-extractable nitrate sediment pool remains to be tested. The significant variation in the vertical pattern and abundance of the various nitrate reducing genes phylotypes reasonably suggests differences in their activity throughout the sediment column. This raises interesting questions as to what the alternative metabolic roles for the various nitrate reductases could be, analogous to the alternative metabolic roles found for nitrite reductases. PMID:24728381

Evolution and ecology of influenza A viruses.

PubMed Central

Webster, R G; Bean, W J; Gorman, O T; Chambers, T M; Kawaoka, Y

1992-01-01

In this review we examine the hypothesis that aquatic birds are the primordial source of all influenza viruses in other species and study the ecological features that permit the perpetuation of influenza viruses in aquatic avian species. Phylogenetic analysis of the nucleotide sequence of influenza A virus RNA segments coding for the spike proteins (HA, NA, and M2) and the internal proteins (PB2, PB1, PA, NP, M, and NS) from a wide range of hosts, geographical regions, and influenza A virus subtypes support the following conclusions. (i) Two partly overlapping reservoirs of influenza A viruses exist in migrating waterfowl and shorebirds throughout the world. These species harbor influenza viruses of all the known HA and NA subtypes. (ii) Influenza viruses have evolved into a number of host-specific lineages that are exemplified by the NP gene and include equine Prague/56, recent equine strains, classical swine and human strains, H13 gull strains, and all other avian strains. Other genes show similar patterns, but with extensive evidence of genetic reassortment. Geographical as well as host-specific lineages are evident. (iii) All of the influenza A viruses of mammalian sources originated from the avian gene pool, and it is possible that influenza B viruses also arose from the same source. (iv) The different virus lineages are predominantly host specific, but there are periodic exchanges of influenza virus genes or whole viruses between species, giving rise to pandemics of disease in humans, lower animals, and birds. (v) The influenza viruses currently circulating in humans and pigs in North America originated by transmission of all genes from the avian reservoir prior to the 1918 Spanish influenza pandemic; some of the genes have subsequently been replaced by others from the influenza gene pool in birds. (vi) The influenza virus gene pool in aquatic birds of the world is probably perpetuated by low-level transmission within that species throughout the year. (vii) There is evidence that most new human pandemic strains and variants have originated in southern China. (viii) There is speculation that pigs may serve as the intermediate host in genetic exchange between influenza viruses in avian and humans, but experimental evidence is lacking. (ix) Once the ecological properties of influenza viruses are understood, it may be possible to interdict the introduction of new influenza viruses into humans. Images PMID:1579108

Recombinant protein production from stable mammalian cell lines and pools.

PubMed

Hacker, David L; Balasubramanian, Sowmya

2016-06-01

We highlight recent developments for the production of recombinant proteins from suspension-adapted mammalian cell lines. We discuss the generation of stable cell lines using transposons and lentivirus vectors (non-targeted transgene integration) and site-specific recombinases (targeted transgene integration). Each of these methods results in the generation of cell lines with protein yields that are generally superior to those achievable through classical plasmid transfection that depends on the integration of the transfected DNA by non-homologous DNA end-joining. This is the main reason why these techniques can also be used for the generation of stable cell pools, heterogenous populations of recombinant cells generated by gene delivery and genetic selection without resorting to single cell cloning. This allows the time line from gene transfer to protein production to be reduced. Copyright © 2016 Elsevier Ltd. All rights reserved.

The Polynesian gene pool: an early contribution by Amerindians to Easter Island.

PubMed

Thorsby, Erik

2012-03-19

It is now generally accepted that Polynesia was first settled by peoples from southeast Asia. An alternative that eastern parts of Polynesia were first inhabited by Amerindians has found little support. There are, however, many indications of a 'prehistoric' (i.e. before Polynesia was discovered by Europeans) contact between Polynesia and the Americas, but genetic evidence of a prehistoric Amerindian contribution to the Polynesian gene pool has been lacking. We recently carried out genomic HLA (human leucocyte antigen) typing as well as typing for mitochondrial DNA (mtDNA) and Y chromosome markers of blood samples collected in 1971 and 2008 from reputedly non-admixed Easter Islanders. All individuals carried HLA alleles and mtDNA types previously found in Polynesia, and most of the males carried Y chromosome markers of Polynesian origin (a few had European Y chromosome markers), further supporting an initial Polynesian population on Easter Island. The HLA investigations revealed, however, that some individuals also carried HLA alleles which have previously almost only been found in Amerindians. We could trace the introduction of these Amerindian alleles to before the Peruvian slave trades, i.e. before the 1860s, and provide suggestive evidence that they were introduced already in prehistoric time. Our results demonstrate an early Amerindian contribution to the Polynesian gene pool on Easter Island, and illustrate the usefulness of typing for immunogenetic markers such as HLA to complement mtDNA and Y chromosome analyses in anthropological investigations.

Microsatellite diversity and genetic structure among common bean (Phaseolus vulgaris L.) landraces in Brazil, a secondary center of diversity

PubMed Central

Burle, Marília Lobo; Fonseca, Jaime Roberto; Kami, James A.

2010-01-01

Brazil is the largest producer and consumer of common bean (Phaseolus vulgaris L.), which is the most important source of human dietary protein in that country. This study assessed the genetic diversity and the structure of a sample of 279 geo-referenced common bean landraces from Brazil, using molecular markers. Sixty-seven microsatellite markers spread over the 11 linkage groups of the common bean genome, as well as Phaseolin, PvTFL1y, APA and four SCAR markers were used. As expected, the sample showed lower genetic diversity compared to the diversity in the primary center of diversification. Andean and Mesoamerican gene pools were both present but the latter gene pool was four times more frequent than the former. The two gene pools could be clearly distinguished; limited admixture was observed between these groups. The Mesoamerican group consisted of two sub-populations, with a high level of admixture between them leading to a large proportion of stabilized hybrids not observed in the centers of domestication. Thus, Brazil can be considered a secondary center of diversification of common bean. A high degree of genome-wide multilocus associations even among unlinked loci was observed, confirming the high level of structure in the sample and suggesting that association mapping should be conducted in separate Andean and Mesoamerican Brazilian samples. Electronic supplementary material The online version of this article (doi:10.1007/s00122-010-1350-5) contains supplementary material, which is available to authorized users. PMID:20502861

Comparison of microsatellite variations between Red Junglefowl and a commercial chicken gene pool.

PubMed

Tadano, R; Kinoshita, K; Mizutani, M; Tsudzuki, M

2014-02-01

It is assumed that Red Junglefowl (Gallus gallus) is one of the main ancestors of domestic chickens (Gallus gallus domesticus). Differences in microsatellite polymorphisms between Red Junglefowl and modern commercial chickens, which are used for egg and meat production, have not been fully reported. A total of 361 individuals from 1 Red Junglefowl population that has been maintained as a closed flock, 5 final cross-bred commercial layer populations (white-, tinted-, and brown-egg layers), and 2 final cross-bred commercial broiler populations were genotyped for 40 autosomal microsatellite loci. We compared microsatellite variations in Red Junglefowl with those in a commercial chicken gene pool. The contribution of each population to the genetic diversity was also estimated based on the molecular coancestry. In total, 302 distinct alleles were detected in 1 Red Junglefowl and 7 commercial chicken populations, of which 31 alleles (10.3%) were unique to Red Junglefowl, most of which occurred at a high frequency. The genetic differentiation between Red Junglefowl and commercial chickens (pairwise FST) ranged from 0.32 to 0.47. According to the neighbor-joining tree based on the modified Cavalli-Sforza chord distances and the Bayesian clustering analysis, Red Junglefowl was genetically distant from the commercial chicken gene pool tested. In all of the populations analyzed, Red Junglefowl made the highest contribution to genetic diversity. These results suggest that Red Junglefowl has a distinct distribution of microsatellite alleles and that there is a high level of genetic divergence between Red Junglefowl and commercial chickens.

Genome sequence of Thalassospira xiamenensis type strain M-5.

PubMed

Lai, Qiliang; Shao, Zongze

2012-12-01

Thalassospira xiamenensis M-5(T) was isolated from the surface water of a waste oil pool at the oil storage dock in the city of Xiamen, Fujian Province, China. Here, we present the draft genome of strain M-5(T), which contains 4,705,237 bp with a G+C content of 54.65% and contains 4,343 protein-coding genes and 46 tRNA genes.

Saturation of an Intra-Gene Pool Linkage Map: Towards a Unified Consensus Linkage Map for Fine Mapping and Synteny Analysis in Common Bean

PubMed Central

Galeano, Carlos H.; Fernandez, Andrea C.; Franco-Herrera, Natalia; Cichy, Karen A.; McClean, Phillip E.; Vanderleyden, Jos; Blair, Matthew W.

2011-01-01

Map-based cloning and fine mapping to find genes of interest and marker assisted selection (MAS) requires good genetic maps with reproducible markers. In this study, we saturated the linkage map of the intra-gene pool population of common bean DOR364×BAT477 (DB) by evaluating 2,706 molecular markers including SSR, SNP, and gene-based markers. On average the polymorphism rate was 7.7% due to the narrow genetic base between the parents. The DB linkage map consisted of 291 markers with a total map length of 1,788 cM. A consensus map was built using the core mapping populations derived from inter-gene pool crosses: DOR364×G19833 (DG) and BAT93×JALO EEP558 (BJ). The consensus map consisted of a total of 1,010 markers mapped, with a total map length of 2,041 cM across 11 linkage groups. On average, each linkage group on the consensus map contained 91 markers of which 83% were single copy markers. Finally, a synteny analysis was carried out using our highly saturated consensus maps compared with the soybean pseudo-chromosome assembly. A total of 772 marker sequences were compared with the soybean genome. A total of 44 syntenic blocks were identified. The linkage group Pv6 presented the most diverse pattern of synteny with seven syntenic blocks, and Pv9 showed the most consistent relations with soybean with just two syntenic blocks. Additionally, a co-linear analysis using common bean transcript map information against soybean coding sequences (CDS) revealed the relationship with 787 soybean genes. The common bean consensus map has allowed us to map a larger number of markers, to obtain a more complete coverage of the common bean genome. Our results, combined with synteny relationships provide tools to increase marker density in selected genomic regions to identify closely linked polymorphic markers for indirect selection, fine mapping or for positional cloning. PMID:22174773

Teaching the Hardy-Weinberg Law

ERIC Educational Resources Information Center

Dudley, B. A. C.

1972-01-01

Describes an approach to teaching the Hardy-Weinberg Law in high school genetics class. Instructional procedures used help in developing this concept in broad generalization form rather than merely a mathematical model of a gene pool. (PS)

Differential gene regulation of GHSR signaling pathway in the arcuate nucleus and NPY neurons by fasting, diet-induced obesity, and 17β-estradiol

PubMed Central

Yasrebi, Ali; Hsieh, Anna; Mamounis, Kyle J.; Krumm, Elizabeth A.; Yang, Jennifer A.; Magby, Jason; Hu, Pu; Roepke, Troy A.

2015-01-01

Ghrelin’s receptor, growth hormone secretagogue receptor (GHSR), is highly expressed in the arcuate nucleus (ARC) and in neuropeptide Y (NPY) neurons. Fasting, diet-induced obesity (DIO), and 17β-estradiol (E2) influence ARC Ghsr expression. It is unknown if these effects occur in NPY neurons. Therefore, we examined the expression of Npy, Agrp, and GHSR signaling pathway genes after fasting, DIO, and E2 replacement in ARC and pools of NPY neurons. In males, fasting increased ARC Ghsr and NPY Foxo1 but decreased NPY Ucp2. In males, DIO decreased ARC and NPY Ghsr and Cpt1c. In fed females, E2 increased Agrp, Ghsr, Cpt1c, and Foxo1 in ARC. In NPY pools, E2 decreased Foxo1 in fed females but increased Foxo1 in fasted females. DIO in females suppressed Agrp and augmented Cpt1c in NPY neurons. In summary, genes involved in GHSR signaling are differentially regulated between the ARC and NPY neurons in a sex-dependent manner. PMID:26577678

Functional Analysis With a Barcoder Yeast Gene Overexpression System

PubMed Central

Douglas, Alison C.; Smith, Andrew M.; Sharifpoor, Sara; Yan, Zhun; Durbic, Tanja; Heisler, Lawrence E.; Lee, Anna Y.; Ryan, Owen; Göttert, Hendrikje; Surendra, Anu; van Dyk, Dewald; Giaever, Guri; Boone, Charles; Nislow, Corey; Andrews, Brenda J.

2012-01-01

Systematic analysis of gene overexpression phenotypes provides an insight into gene function, enzyme targets, and biological pathways. Here, we describe a novel functional genomics platform that enables a highly parallel and systematic assessment of overexpression phenotypes in pooled cultures. First, we constructed a genome-level collection of ~5100 yeast barcoder strains, each of which carries a unique barcode, enabling pooled fitness assays with a barcode microarray or sequencing readout. Second, we constructed a yeast open reading frame (ORF) galactose-induced overexpression array by generating a genome-wide set of yeast transformants, each of which carries an individual plasmid-born and sequence-verified ORF derived from the Saccharomyces cerevisiae full-length EXpression-ready (FLEX) collection. We combined these collections genetically using synthetic genetic array methodology, generating ~5100 strains, each of which is barcoded and overexpresses a specific ORF, a set we termed “barFLEX.” Additional synthetic genetic array allows the barFLEX collection to be moved into different genetic backgrounds. As a proof-of-principle, we describe the properties of the barFLEX overexpression collection and its application in synthetic dosage lethality studies under different environmental conditions. PMID:23050238

Polymorphisms in Genes of Relevance for Oestrogen and Oxytocin Pathways and Risk of Barrett's Oesophagus and Oesophageal Adenocarcinoma: A Pooled Analysis from the BEACON Consortium.

PubMed

Lagergren, Katarina; Ek, Weronica E; Levine, David; Chow, Wong-Ho; Bernstein, Leslie; Casson, Alan G; Risch, Harvey A; Shaheen, Nicholas J; Bird, Nigel C; Reid, Brian J; Corley, Douglas A; Hardie, Laura J; Wu, Anna H; Fitzgerald, Rebecca C; Pharoah, Paul; Caldas, Carlos; Romero, Yvonne; Vaughan, Thomas L; MacGregor, Stuart; Whiteman, David; Westberg, Lars; Nyren, Olof; Lagergren, Jesper

2015-01-01

The strong male predominance in oesophageal adenocarcinoma (OAC) and Barrett's oesophagus (BO) continues to puzzle. Hormonal influence, e.g. oestrogen or oxytocin, might contribute. This genetic-epidemiological study pooled 14 studies from three continents, Australia, Europe, and North America. Polymorphisms in 3 key genes coding for the oestrogen pathway (receptor alpha (ESR1), receptor beta (ESR2), and aromatase (CYP19A1)), and 3 key genes of the oxytocin pathway (the oxytocin receptor (OXTR), oxytocin protein (OXT), and cyclic ADP ribose hydrolase glycoprotein (CD38)), were analysed using a gene-based approach, versatile gene-based test association study (VEGAS). Among 1508 OAC patients, 2383 BO patients, and 2170 controls, genetic variants within ESR1 were associated with BO in males (p = 0.0058) and an increased risk of OAC and BO combined in males (p = 0.0023). Genetic variants within OXTR were associated with an increased risk of BO in both sexes combined (p = 0.0035) and in males (p = 0.0012). We followed up these suggestive findings in a further smaller data set, but found no replication. There were no significant associations between the other 4 genes studied and risk of OAC, BO, separately on in combination, in males and females combined or in males only. Genetic variants in the oestrogen receptor alpha and the oxytocin receptor may be associated with an increased risk of BO or OAC, but replication in other large samples are needed.

Understanding genetic control of root system architecture in soybean: Insights into the genetic basis of lateral root number.

PubMed

Prince, Silvas J; Valliyodan, Babu; Ye, Heng; Yang, Ming; Tai, Shuaishuai; Hu, Wushu; Murphy, Mackensie; Durnell, Lorellin A; Song, Li; Joshi, Trupti; Liu, Yang; Van de Velde, Jan; Vandepoele, Klaas; Grover Shannon, J; Nguyen, Henry T

2018-05-10

Developing crops with better root systems is a promising strategy to ensure productivity in both optimum and stress environments. Root system architectural (RSA) traits in 397 soybean accessions were characterized and a high-density single nucleotide polymorphisms (SNP) based genome-wide association study was performed to identify the underlying genes associated with root structure. SNPs associated with root architectural traits specific to landraces and elite germplasm pools were detected. Four loci were detected in landraces for lateral root number (LRN) and distribution of root thickness in diameter class I with a major locus on chromosome 16. This major loci was detected in the coding region of unknown protein, and subsequent analyses demonstrated that root traits are affected with mutated haplotypes of the gene. In elite germplasm pool, three significant SNPs in alanine-glyoxalate aminotransferase, Leucine-Rich Repeat receptor/No apical meristem and unknown functional genes were found to govern multiple traits including root surface area and volume. However, no major loci were detected for LRN in elite germplasm. Nucleotide diversity analysis found evidence of selective sweeps around the landraces LRN gene. Soybean accessions with minor and mutated allelic variants of LRN gene were found to perform better in both water-limited and optimal field conditions. This article is protected by copyright. All rights reserved.

Lack of association between TPH2 gene polymorphisms with major depressive disorder in multiethnic Malaysian population.

PubMed

Nazree, Nur Elia; Loke, Ai Chin; Zainal, Nor Zuraida; Mohamed, Zahurin

2015-03-01

Numerous association studies of candidate genes studies with major depressive disorder (MDD) have been conducted for many years; however, the evidence of association between genes and the risk of developing MDD still remains inconclusive. In this study, we aimed to investigate the association between the tryptophan hydroxylase 2 (TPH2) gene and MDD in three ethnic groups (Malay, Chinese and Indian) within the Malaysian population. Two hundred and sixty five MDD patients who fulfilled the Diagnostic and Statistical Manual of Mental Disorders-IV criteria for MDD and 332 healthy controls were recruited for the study. All cases and controls were then genotyped for TPH2 polymorphisms rs1386494, rs1386495 and rs7305115. Single locus analysis in pooled and ethnically stratified subjects revealed no association between each of the three variants of the TPH2 gene with susceptibility to MDD. Strong linkage disequilibrium was detected between rs1386495 and rs1386494 in pooled subjects; however, no significant association was found in the haplotype analysis. In this study, we suggest that in both the Chinese and Indian populations, gender distribution differ significantly between cases and controls, showing that women are more at risk of developing MDD compared with men. Therefore, we suggest that the occurrence of MDD in both Chinese and Indians in the Malaysian population may be influenced by gender. Copyright © 2013 Wiley Publishing Asia Pty Ltd.

Identification of susceptibility genes and genetic modifiers of human diseases

NASA Astrophysics Data System (ADS)

Abel, Kenneth; Kammerer, Stefan; Hoyal, Carolyn; Reneland, Rikard; Marnellos, George; Nelson, Matthew R.; Braun, Andreas

2005-03-01

The completion of the human genome sequence enables the discovery of genes involved in common human disorders. The successful identification of these genes is dependent on the availability of informative sample sets, validated marker panels, a high-throughput scoring technology, and a strategy for combining these resources. We have developed a universal platform technology based on mass spectrometry (MassARRAY) for analyzing nucleic acids with high precision and accuracy. To fuel this technology, we generated more than 100,000 validated assays for single nucleotide polymorphisms (SNPs) covering virtually all known and predicted human genes. We also established a large DNA sample bank comprised of more than 50,000 consented healthy and diseased individuals. This combination of reagents and technology allows the execution of large-scale genome-wide association studies. Taking advantage of MassARRAY"s capability for quantitative analysis of nucleic acids, allele frequencies are estimated in sample pools containing large numbers of individual DNAs. To compare pools as a first-pass "filtering" step is a tremendous advantage in throughput and cost over individual genotyping. We employed this approach in numerous genome-wide, hypothesis-free searches to identify genes associated with common complex diseases, such as breast cancer, osteoporosis, and osteoarthritis, and genes involved in quantitative traits like high density lipoproteins cholesterol (HDL-c) levels and central fat. Access to additional well-characterized patient samples through collaborations allows us to conduct replication studies that validate true disease genes. These discoveries will expand our understanding of genetic disease predisposition, and our ability for early diagnosis and determination of specific disease subtype or progression stage.

miRNA-mediated 'tug-of-war' model reveals ceRNA propensity of genes in cancers.

PubMed

Swain, Arpit Chandan; Mallick, Bibekanand

2018-06-01

Competing endogenous RNA (ceRNA) are transcripts that cross-regulate each other at the post-transcriptional level by competing for shared microRNA response elements (MREs). These have been implicated in various biological processes impacting cell-fate decisions and diseases including cancer. There are several studies that predict possible ceRNA pairs by adopting various machine-learning and mathematical approaches; however, there is no method that enables us to gauge as well as compare the propensity of the ceRNA of a gene and precisely envisages which among a pair exerts a stronger pull on the shared miRNA pool. In this study, we developed a method that uses the 'tug of war of genes' concept to predict and quantify ceRNA potential of a gene for the shared miRNA pool in cancers based on a score represented by SoCeR (score of competing endogenous RNA). The method was executed on the RNA-Seq transcriptional profiles of genes and miRNA available at TCGA along with CLIP-supported miRNA-target sites to predict ceRNA in 32 cancer types which were validated with already reported cases. The proposed method can be used to determine the sequestering capability of the gene of interest as well as in ranking the probable ceRNA candidates of a gene. Finally, we developed standalone applications (SoCeR tool) to aid researchers in easier implementation of the method in analysing different data sets or diseases. © 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.

Upregulated Genes In Sporadic, Idiopathic Pulmonary Arterial Hypertension

PubMed Central

Edgar, Alasdair J; Chacón, Matilde R; Bishop, Anne E; Yacoub, Magdi H; Polak, Julia M

2006-01-01

Background To elucidate further the pathogenesis of sporadic, idiopathic pulmonary arterial hypertension (IPAH) and identify potential therapeutic avenues, differential gene expression in IPAH was examined by suppression subtractive hybridisation (SSH). Methods Peripheral lung samples were obtained immediately after removal from patients undergoing lung transplant for IPAH without familial disease, and control tissues consisted of similarly sampled pieces of donor lungs not utilised during transplantation. Pools of lung mRNA from IPAH cases containing plexiform lesions and normal donor lungs were used to generate the tester and driver cDNA libraries, respectively. A subtracted IPAH cDNA library was made by SSH. Clones isolated from this subtracted library were examined for up regulated expression in IPAH using dot blot arrays of positive colony PCR products using both pooled cDNA libraries as probes. Clones verified as being upregulated were sequenced. For two genes the increase in expression was verified by northern blotting and data analysed using Student's unpaired two-tailed t-test. Results We present preliminary findings concerning candidate genes upregulated in IPAH. Twenty-seven upregulated genes were identified out of 192 clones examined. Upregulation in individual cases of IPAH was shown by northern blot for tissue inhibitor of metalloproteinase-3 and decorin (P < 0.01) compared with the housekeeping gene glyceraldehydes-3-phosphate dehydrogenase. Conclusion Four of the up regulated genes, magic roundabout, hevin, thrombomodulin and sucrose non-fermenting protein-related kinase-1 are expressed specifically by endothelial cells and one, muscleblind-1, by muscle cells, suggesting that they may be associated with plexiform lesions and hypertrophic arterial wall remodelling, respectively. PMID:16390543

New enzymes from environmental cassette arrays: Functional attributes of a phosphotransferase and an RNA-methyltransferase

PubMed Central

Nield, Blair S.; Willows, Robert D.; Torda, Andrew E.; Gillings, Michael R.; Holmes, Andrew J.; Nevalainen, K.M. Helena; Stokes, H.W.; Mabbutt, Bridget C.

2004-01-01

By targeting gene cassettes by polymerase chain reaction (PCR) directly from environmentally derived DNA, we are able to amplify entire open reading frames (ORFs) independently of prior sequence knowledge. Approximately 10% of the mobile genes recovered by these means can be attributed to known protein families. Here we describe the characterization of two ORFs which show moderate homology to known proteins: (1) an aminoglycoside phosphotransferase displaying 25% sequence identity with APH(7″) from Streptomyces hygroscopicus, and (2) an RNA methyltransferase sharing 25%–28% identity with a group of recently defined bacterial RNA methyltransferases distinct from the SpoU enzyme family. Our novel genes were expressed as recombinant products and assayed for appropriate enzyme activity. The aminoglycoside phosphotransferase displayed ATPase activity, consistent with the presence of characteristic Mg2+-binding residues. Unlike related APH(4) or APH(7″) enzymes, however, this activity was not enhanced by hygromycin B or kanamycin, suggesting the normal substrate to be a different aminoglycoside. The RNA methyltransferase contains sequence motifs of the RNA methyltransferase superfamily, and our recombinant version showed methyltransferase activity with RNA. Our data confirm that gene cassettes present in the environment encode folded enzymes with novel sequence variation and demonstrable catalytic activity. Our PCR approach (cassette PCR) may be used to identify a diverse range of ORFs from any environmental sample, as well as to directly access the gene pool found in mobile gene cassettes commonly associated with integrons. This gene pool can be accessed from both cultured and uncultured microbial samples as a source of new enzymes and proteins. PMID:15152095

New enzymes from environmental cassette arrays: functional attributes of a phosphotransferase and an RNA-methyltransferase.

PubMed

Nield, Blair S; Willows, Robert D; Torda, Andrew E; Gillings, Michael R; Holmes, Andrew J; Nevalainen, K M Helena; Stokes, H W; Mabbutt, Bridget C

2004-06-01

By targeting gene cassettes by polymerase chain reaction (PCR) directly from environmentally derived DNA, we are able to amplify entire open reading frames (ORFs) independently of prior sequence knowledge. Approximately 10% of the mobile genes recovered by these means can be attributed to known protein families. Here we describe the characterization of two ORFs which show moderate homology to known proteins: (1) an aminoglycoside phosphotransferase displaying 25% sequence identity with APH(7") from Streptomyces hygroscopicus, and (2) an RNA methyltransferase sharing 25%-28% identity with a group of recently defined bacterial RNA methyltransferases distinct from the SpoU enzyme family. Our novel genes were expressed as recombinant products and assayed for appropriate enzyme activity. The aminoglycoside phosphotransferase displayed ATPase activity, consistent with the presence of characteristic Mg(2+)-binding residues. Unlike related APH(4) or APH(7") enzymes, however, this activity was not enhanced by hygromycin B or kanamycin, suggesting the normal substrate to be a different aminoglycoside. The RNA methyltransferase contains sequence motifs of the RNA methyltransferase superfamily, and our recombinant version showed methyltransferase activity with RNA. Our data confirm that gene cassettes present in the environment encode folded enzymes with novel sequence variation and demonstrable catalytic activity. Our PCR approach (cassette PCR) may be used to identify a diverse range of ORFs from any environmental sample, as well as to directly access the gene pool found in mobile gene cassettes commonly associated with integrons. This gene pool can be accessed from both cultured and uncultured microbial samples as a source of new enzymes and proteins.

ABC-B transporter genes in Dirofilaria immitis.

PubMed

Bourguinat, Catherine; Che, Hua; Mani, Thangadurai; Keller, Kathy; Prichard, Roger K

2016-08-01

Dirofilaria immitis is a filarial nematode causing infection and heartworm disease in dogs and other canids, cats, and occasionally in humans. Prevention with macrocyclic lactones (ML) is recommended during the mosquito transmission season. Recently, ML resistance has been reported. ABC-B transporter genes are thought to be involved in the mechanism of ML resistance in other nematodes. This study aimed to identify all the ABC-B transporter genes in D. immitis using as a reference the nDi.2.2 D. immitis whole genome, which is not completely annotated. Using bioinformatic tools and PCR amplification on pooled D. immitis genomic DNA and on pooled cDNA, nine ABC transporter genes including one pseudogene were characterized. Bioinformatic and phylogenetic analyses allowed identification of three P-glycoproteins (Pgps) (Dim-pgp-3 Dim-pgp-10, Dim-pgp-11), of two ABC-B half transporter genes (one ortholog of Cel-haf-4 and Cel-haf-9; and one ortholog of Cel-haf-1 and Cel-haf-3), of one ABC half transporter gene (ortholog of Cel-haf-5) that contained an ABC-C motif, and of one additional half transporter that would require functional study for characterization. The number of ABC-B transporter genes identified was lower than in Caenorhabditis elegans and Haemonchus contortus. Further studies are needed to understand their possible role in ML resistance in D. immitis. These ABC transporters constitute a base for ML resistance investigation in D. immitis and advance our understanding of the molecular biology of this parasite. Copyright © 2016. Published by Elsevier Ltd.

Recombination walking: genetic selection of clones from pooled libraries of yeast artificial chromosomes by homologous recombination.

PubMed Central

Miller, A M; Savinelli, E A; Couture, S M; Hannigan, G M; Han, Z; Selden, R F; Treco, D A

1993-01-01

Recombination walking is based on the genetic selection of specific human clones from a yeast artificial chromosome (YAC) library by homologous recombination. The desired clone is selected from a pooled (unordered) YAC library, eliminating labor-intensive steps typically used in organizing and maintaining ordered YAC libraries. Recombination walking represents an efficient approach to library screening and is well suited for chromosome-walking approaches to the isolation of genes associated with common diseases. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8367472

CRISPR-UMI: single-cell lineage tracing of pooled CRISPR-Cas9 screens.

PubMed

Michlits, Georg; Hubmann, Maria; Wu, Szu-Hsien; Vainorius, Gintautas; Budusan, Elena; Zhuk, Sergei; Burkard, Thomas R; Novatchkova, Maria; Aichinger, Martin; Lu, Yiqing; Reece-Hoyes, John; Nitsch, Roberto; Schramek, Daniel; Hoepfner, Dominic; Elling, Ulrich

2017-12-01

Pooled CRISPR screens are a powerful tool for assessments of gene function. However, conventional analysis is based exclusively on the relative abundance of integrated single guide RNAs (sgRNAs) between populations, which does not discern distinct phenotypes and editing outcomes generated by identical sgRNAs. Here we present CRISPR-UMI, a single-cell lineage-tracing methodology for pooled screening to account for cell heterogeneity. We generated complex sgRNA libraries with unique molecular identifiers (UMIs) that allowed for screening of clonally expanded, individually tagged cells. A proof-of-principle CRISPR-UMI negative-selection screen provided increased sensitivity and robustness compared with conventional analysis by accounting for underlying cellular and editing-outcome heterogeneity and detection of outlier clones. Furthermore, a CRISPR-UMI positive-selection screen uncovered new roadblocks in reprogramming mouse embryonic fibroblasts as pluripotent stem cells, distinguishing reprogramming frequency and speed (i.e., effect size and probability). CRISPR-UMI boosts the predictive power, sensitivity, and information content of pooled CRISPR screens.

Microorganism genomics, compositions and methods related thereto

DOEpatents

Handelsman, Jo; Goodman, Robert M.; Rondon, Michelle R.

2001-01-01

The present invention provides methods and compositions for accessing, in a generally unbaised manner, a diverse genetic pool for genes involved in biosynthetic pathways. The invention also provides compounds which can be identified by cloning biosynthetic pathways.

Admixture Aberration Analysis: Application to Mapping in Admixed Population Using Pooled DNA

NASA Astrophysics Data System (ADS)

Bercovici, Sivan; Geiger, Dan

Admixture mapping is a gene mapping approach used for the identification of genomic regions harboring disease susceptibility genes in the case of recently admixed populations such as African Americans. We present a novel method for admixture mapping, called admixture aberration analysis (AAA), that uses a DNA pool of affected admixed individuals. We demonstrate through simulations that AAA is a powerful and economical mapping method under a range of scenarios, capturing complex human diseases such as hypertension and end stage kidney disease. The method has a low false-positive rate and is robust to deviation from model assumptions. Finally, we apply AAA on 600 prostate cancer-affected African Americans, replicating a known risk locus. Simulation results indicate that the method can yield over 96% reduction in genotyping. Our method is implemented as a Java program called AAAmap and is freely available.

Molecular characterization of primary gene pool of chickpea based on ISSR markers.

PubMed

Choudhary, Pooja; Khanna, Suruchi M; Jain, Pradeep K; Bharadwaj, Chellapilla; Kumar, Jitendra; Lakhera, Pramesh C; Srinivasan, Ramamurthy

2013-04-01

Genetic diversity and relationships within and among members of the primary gene pool of chickpea, including 38 accessions of Cicer arietinum, six of C. reticulatum,, and four of C. echinospermum, were investigated using 31 ISSR markers. The study revealed moderate diversity, detecting 141 fragments, of which 79 (56%) were polymorphic. Averages were 0.125 for polymorphic information content, 0.350 for marker index, and 0.715 for resolving power. The UPGMA dendrogram and the principal coordinate analysis revealed a clear differentiation between wild and cultivated accessions. The clustering pattern did not strictly follow the grouping of accessions by geographic origin but was in good agreement with the pedigree data and the seed type. The study demonstrates that ISSRs provide promising marker tools in revealing genetic diversity and relationships in chickpea and can contribute to efficient identification, conservation, and utilization of germplasm for plant improvement through conventional as well as molecular breeding approaches.

Donor specificity of YjiC glycosyltransferase determines the conjugation of cytosolic NDP-sugar in in vivo glycosylation reactions.

PubMed

Pandey, Ramesh Prasad; Parajuli, Prakash; Gurung, Rit Bahadur; Sohng, Jae Kyung

2016-09-01

Escherichia coli BL21 (DE3) was engineered by blocking glucose-1-phosphate utilizing glucose phosphate isomerase (pgi), glucose-6-phosphate dehydrogenase (zwf) and uridylyltransferase (galU) genes to produce pool of four different rare dTDP-sugars. The cytosolic pool of dTDP-l-rhamnose, dTDP-d-viosamine, dTDP-4-amino 4,6-dideoxy-d-galactose, and dTDP-3-amino 3,6-dideoxy-d-galactose was generated by overexpressing respective dTDP-sugars biosynthesis genes from various microbial sources. A flexible glycosyltransferase YjiC, from Bacillus licheniformis DSM 13 was also overexpressed to transfer sugar moieties to 3-hydroxyl group of 3-hydroxyflavone, a core unit of flavonoids. Among four rare dTDP-sugars generated in cytosol of engineered strains, YjiC solely transferred l-rhamnose from dTDP-l-rhamnose and tuned to rhamnosyltransferase. Copyright © 2016. Published by Elsevier Inc.

Identification of presumed ancestral DNA sequences of phaseolin in Phaseolus vulgaris.

PubMed Central

Kami, J; Velásquez, V B; Debouck, D G; Gepts, P

1995-01-01

Common bean (Phaseolus vulgaris) consists of two major geographic gene pools, one distributed in Mexico, Central America, and Colombia and the other in the southern Andes (southern Peru, Bolivia, and Argentina). Amplification and sequencing of members of the multigene family coding for phaseolin, the major seed storage protein of the common bean, provide evidence for accumulation of tandem direct repeats in both introns and exons during evolution of the multigene family in this species. The presumed ancestral phaseolin sequences, without tandem repeats, were found in recently discovered but nearly extinct wild common bean populations of Ecuador and northern Peru that are intermediate between the two major gene pools of the species based on geographical and molecular arguments. Our results illustrate the usefulness of tandem direct repeats in establishing the polarity of DNA sequence divergence and therefore in proposing phylogenies. Images Fig. 1 Fig. 3 PMID:7862642

Transcriptomic Analysis Using Olive Varieties and Breeding Progenies Identifies Candidate Genes Involved in Plant Architecture

PubMed Central

González-Plaza, Juan J.; Ortiz-Martín, Inmaculada; Muñoz-Mérida, Antonio; García-López, Carmen; Sánchez-Sevilla, José F.; Luque, Francisco; Trelles, Oswaldo; Bejarano, Eduardo R.; De La Rosa, Raúl; Valpuesta, Victoriano; Beuzón, Carmen R.

2016-01-01

Plant architecture is a critical trait in fruit crops that can significantly influence yield, pruning, planting density and harvesting. Little is known about how plant architecture is genetically determined in olive, were most of the existing varieties are traditional with an architecture poorly suited for modern growing and harvesting systems. In the present study, we have carried out microarray analysis of meristematic tissue to compare expression profiles of olive varieties displaying differences in architecture, as well as seedlings from their cross pooled on the basis of their sharing architecture-related phenotypes. The microarray used, previously developed by our group has already been applied to identify candidates genes involved in regulating juvenile to adult transition in the shoot apex of seedlings. Varieties with distinct architecture phenotypes and individuals from segregating progenies displaying opposite architecture features were used to link phenotype to expression. Here, we identify 2252 differentially expressed genes (DEGs) associated to differences in plant architecture. Microarray results were validated by quantitative RT-PCR carried out on genes with functional annotation likely related to plant architecture. Twelve of these genes were further analyzed in individual seedlings of the corresponding pool. We also examined Arabidopsis mutants in putative orthologs of these targeted candidate genes, finding altered architecture for most of them. This supports a functional conservation between species and potential biological relevance of the candidate genes identified. This study is the first to identify genes associated to plant architecture in olive, and the results obtained could be of great help in future programs aimed at selecting phenotypes adapted to modern cultivation practices in this species. PMID:26973682

Molecular detection of Rickettsia species in ticks collected from the southwestern provinces of the Republic of Korea.

PubMed

Noh, Yoontae; Lee, Yeong Seon; Kim, Heung-Chul; Chong, Sung-Tae; Klein, Terry A; Jiang, Ju; Richards, Allen L; Lee, Hae Kyeong; Kim, Su Yeon

2017-01-10

Rickettsiae constitute a group of arthropod-borne, Gram-negative, obligate intracellular bacteria that are the causative agents of diseases ranging from mild to life threatening that impact on medical and veterinary health worldwide. A total of 6,484 ticks were collected by tick drag from June-October 2013 in the southwestern provinces of the Republic of Korea (ROK) (Jeollanam, n = 3,995; Jeollabuk, n = 680; Chungcheongnam, n = 1,478; and Chungcheongbuk, n = 331). Ticks were sorted into 311 pools according to species, collection site, and stage of development. DNA preparations of tick pools were assayed for rickettsiae by 17 kDa antigen gene and ompA nested PCR (nPCR) assays and the resulting amplicons sequenced to determine the identity and prevalence of spotted fever group rickettsiae (SFGR). Haemaphysalis longicornis (4,471; 52 adults, 123 nymphs and 4,296 larvae) were the most commonly collected ticks, followed by Haemaphysalis flava (1,582; 28 adults, 263 nymphs and 1,291 larvae), and Ixodes nipponensis (431; 25 adults, 5 nymphs and 401 larvae). The minimum field infection rate/100 ticks (assuming 1 positive tick/pool) was 0.93% for the 17 kDa antigen gene and 0.82% for the ompA nPCR assays. The partial 17 kDa antigen and ompA gene sequences from positive pools of H. longicornis were similar to: Rickettsia sp. HI550 (99.4-100%), Rickettsia sp. FUJ98 (99.3-100%), Rickettsia sp. HIR/D91 (99.3-100%), and R. japonica (99.7%). One sequence of the partial 17 kDa antigen gene for H. flava was similar to Rickettsia sp. 17kd-005 (99.7%), while seven sequences of the 17 kDa antigen gene obtained from I. nipponensis ticks were similar to R. monacensis IrR/Munich (98.7-100%) and Rickettsia sp. IRS3 (98.9%). SFG rickettsiae were detected in three species of ixodid ticks collected in the southwestern provinces of the ROK during 2013. A number of rickettsiae have been recently reported from ticks in Korea, some of which were identified as medically important. Results from this study and previous reports demonstrate the need to conduct longitudinal investigations to identify tick-borne rickettsiae and better understand their geographical distributions and potential impact on medical and veterinary health, in addition to risk communication and development of rickettsial disease prevention strategies.

Assessing pooled BAC and whole genome shotgun strategies for assembly of complex genomes.

PubMed

Haiminen, Niina; Feltus, F Alex; Parida, Laxmi

2011-04-15

We investigate if pooling BAC clones and sequencing the pools can provide for more accurate assembly of genome sequences than the "whole genome shotgun" (WGS) approach. Furthermore, we quantify this accuracy increase. We compare the pooled BAC and WGS approaches using in silico simulations. Standard measures of assembly quality focus on assembly size and fragmentation, which are desirable for large whole genome assemblies. We propose additional measures enabling easy and visual comparison of assembly quality, such as rearrangements and redundant sequence content, relative to the known target sequence. The best assembly quality scores were obtained using 454 coverage of 15× linear and 5× paired (3kb insert size) reads (15L-5P) on Arabidopsis. This regime gave similarly good results on four additional plant genomes of very different GC and repeat contents. BAC pooling improved assembly scores over WGS assembly, coverage and redundancy scores improving the most. BAC pooling works better than WGS, however, both require a physical map to order the scaffolds. Pool sizes up to 12Mbp work well, suggesting this pooling density to be effective in medium-scale re-sequencing applications such as targeted sequencing of QTL intervals for candidate gene discovery. Assuming the current Roche/454 Titanium sequencing limitations, a 12 Mbp region could be re-sequenced with a full plate of linear reads and a half plate of paired-end reads, yielding 15L-5P coverage after read pre-processing. Our simulation suggests that massively over-sequencing may not improve accuracy. Our scoring measures can be used generally to evaluate and compare results of simulated genome assemblies.

Genome Sequence of Thalassospira xiamenensis Type Strain M-5

PubMed Central

Lai, Qiliang

2012-01-01

Thalassospira xiamenensis M-5T was isolated from the surface water of a waste oil pool at the oil storage dock in the city of Xiamen, Fujian Province, China. Here, we present the draft genome of strain M-5T, which contains 4,705,237 bp with a G+C content of 54.65% and contains 4,343 protein-coding genes and 46 tRNA genes. PMID:23209216

Gene Therapy for Fracture Repair

DTIC Science & Technology

2007-05-01

Methods: We have adopted the Agilent rat oligomer chip to analyze our fracture RNA in our microarray analysis. This chip has 20,046 unique gene...signal during fluorescent labeling of the cDNA. This approach is highly advantageous for reducing the RNA input into the system, minimizing the numbers...perform the analysis on these extremely limited samples without pooling the RNA from multiple individuals. We are therefore able to analyze the

High-density SNP genotyping array for hexaploid wheat and its secondary and tertiary gene pool.

PubMed

Winfield, Mark O; Allen, Alexandra M; Burridge, Amanda J; Barker, Gary L A; Benbow, Harriet R; Wilkinson, Paul A; Coghill, Jane; Waterfall, Christy; Davassi, Alessandro; Scopes, Geoff; Pirani, Ali; Webster, Teresa; Brew, Fiona; Bloor, Claire; King, Julie; West, Claire; Griffiths, Simon; King, Ian; Bentley, Alison R; Edwards, Keith J

2016-05-01

In wheat, a lack of genetic diversity between breeding lines has been recognized as a significant block to future yield increases. Species belonging to bread wheat's secondary and tertiary gene pools harbour a much greater level of genetic variability, and are an important source of genes to broaden its genetic base. Introgression of novel genes from progenitors and related species has been widely employed to improve the agronomic characteristics of hexaploid wheat, but this approach has been hampered by a lack of markers that can be used to track introduced chromosome segments. Here, we describe the identification of a large number of single nucleotide polymorphisms that can be used to genotype hexaploid wheat and to identify and track introgressions from a variety of sources. We have validated these markers using an ultra-high-density Axiom(®) genotyping array to characterize a range of diploid, tetraploid and hexaploid wheat accessions and wheat relatives. To facilitate the use of these, both the markers and the associated sequence and genotype information have been made available through an interactive web site. © 2015 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

Associating quantitative behavioral traits with gene expression in the brain: searching for diamonds in the hay.

PubMed

Reiner-Benaim, Anat; Yekutieli, Daniel; Letwin, Noah E; Elmer, Gregory I; Lee, Norman H; Kafkafi, Neri; Benjamini, Yoav

2007-09-01

Gene expression and phenotypic functionality can best be associated when they are measured quantitatively within the same experiment. The analysis of such a complex experiment is presented, searching for associations between measures of exploratory behavior in mice and gene expression in brain regions. The analysis of such experiments raises several methodological problems. First and foremost, the size of the pool of potential discoveries being screened is enormous yet only few biologically relevant findings are expected, making the problem of multiple testing especially severe. We present solutions based on screening by testing related hypotheses, then testing the hypotheses of interest. In one variant the subset is selected directly, in the other one a tree of hypotheses is tested hierarchical; both variants control the False Discovery Rate (FDR). Other problems in such experiments are in the fact that the level of data aggregation may be different for the quantitative traits (one per animal) and gene expression measurements (pooled across animals); in that the association may not be linear; and in the resolution of interest only few replications exist. We offer solutions to these problems as well. The hierarchical FDR testing strategies presented here can serve beyond the structure of our motivating example study to any complex microarray study. Supplementary data are available at Bioinformatics online.

Stimulation of autophagy by the p53 target gene Sestrin2.

PubMed

Maiuri, Maria Chiara; Malik, Shoaib Ahmad; Morselli, Eugenia; Kepp, Oliver; Criollo, Alfredo; Mouchel, Pierre-Luc; Carnuccio, Rosa; Kroemer, Guido

2009-05-15

The oncosuppressor protein p53 regulates autophagy in a dual fashion. The pool of cytoplasmic p53 protein represses autophagy in a transcription-independent fashion, while the pool of nuclear p53 stimulates autophagy through the transactivation of specific genes. Here we report the discovery that Sestrin2, a novel p53 target gene, is involved in the induction of autophagy. Depletion of Sestrin2 by RNA interference reduced the level of autophagy in a panel of p53-sufficient human cancer cell lines responding to distinct autophagy inducers. In quantitative terms, Sestrin2 depletion was as efficient in preventing autophagy induction as was the depletion of Dram, another p53 target gene. Knockout of either Sestrin2 or Dram reduced autophagy elicited by nutrient depletion, rapamycin, lithium or thapsigargin. Moreover, autophagy induction by nutrient depletion or pharmacological stimuli led to an increase in Sestrin2 expression levels in p53-proficient cells. In strict contrast, the depletion of Sestrin2 or Dram failed to affect autophagy in p53-deficient cells and did not modulate the inhibition of baseline autophagy by a cytoplasmic p53 mutant that was reintroduced into p53-deficient cells. We conclude that Sestrin2 acts as a positive regulator of autophagy in p53-proficient cells.

Annotating ebony on the fly.

PubMed

Kohn, Michael H; Wittkopp, Patricia J

2007-07-01

The distinctive black phenotype of ebony mutants has made it one of the most widely used phenotypic markers in Drosophila genetics. Without doubt, ebony showcases the fruits of the fly community's labours to annotate gene function. As of this writing, FlyBase lists 142 references, 1277 fly stocks, 15 phenotypes and 44 alleles. In addition to its namesake pigmentation phenotype, ebony mutants affect other traits, including phototaxis and courtship. With phenotypic consequences of ebony variants readily apparent in the laboratory, does natural selection also see them in the wild? In this issue of Molecular Ecology, Pool & Aquadro investigate this question and found signs of natural selection on the ebony gene that appear to have resulted from selection for darker pigmentation at higher elevations in sub-Saharan populations of Drosophila melanogaster. Such findings from population genomic analysis of wild-derived strains should be included in gene annotations to provide a more holistic view of a gene's function. The evolutionary annotation of ebony added by Pool & Aquadro substantiates that pigmentation can be adaptive and implicates elevation as an important selective factor. This is important progress because the selective factors seem to differ between populations and species. In addition, the study raises issues to consider when extrapolating from selection at the molecular level to selection at the phenotypic level.

[Genetic diversity and relationships of northern eurasia populations for polymorphic Alu-insertions].

PubMed

Khitrinskaia, I Iu; Khar'kov, V N; Voevoda, M I; Stepanova, V A

2014-01-01

We for the first time have examined the autosomal gene pool of the Siberia, Central Asian and the Far East populations (27 populations of 12 ethnic groups) using a set of polymorphic Alu insertions in the human genome. The results of the analysis testify (i) to a significant level of genetic diversity in the Northern Eurasian populations and (ii) to a considerable differentiation of gene pool in the population of this region. It has been shown that at the CD4 locus, the frequency of Alu (-) is inversely related to the Mongoloid component of the population, the lowest and highest frequencies of the Alu deletion at locus CD4 were recorded respectively in Eskimo (0.012) and Russian and Ukrainian (0.35). The analysis of gene flow proved Caucasoid populations (Russian, Tajik and Uzbek), as well as those of Turkic ethnic groups from the Southern Siberia (Altaians and Tuvinians) and Khanty and Mansy populations to be the recipients of a considerable gene flow from the outside of the concerned population system, as compared with the East Siberian and the Far East ethnic groups. The results of the correlation analysis received with use polymorphic Alu insertion testify to the greatest correlation of genetic distances with anthropological characteristics of populations.

SNPGenie: estimating evolutionary parameters to detect natural selection using pooled next-generation sequencing data.

PubMed

Nelson, Chase W; Moncla, Louise H; Hughes, Austin L

2015-11-15

New applications of next-generation sequencing technologies use pools of DNA from multiple individuals to estimate population genetic parameters. However, no publicly available tools exist to analyse single-nucleotide polymorphism (SNP) calling results directly for evolutionary parameters important in detecting natural selection, including nucleotide diversity and gene diversity. We have developed SNPGenie to fill this gap. The user submits a FASTA reference sequence(s), a Gene Transfer Format (.GTF) file with CDS information and a SNP report(s) in an increasing selection of formats. The program estimates nucleotide diversity, distance from the reference and gene diversity. Sites are flagged for multiple overlapping reading frames, and are categorized by polymorphism type: nonsynonymous, synonymous, or ambiguous. The results allow single nucleotide, single codon, sliding window, whole gene and whole genome/population analyses that aid in the detection of positive and purifying natural selection in the source population. SNPGenie version 1.2 is a Perl program with no additional dependencies. It is free, open-source, and available for download at https://github.com/hugheslab/snpgenie. nelsoncw@email.sc.edu or austin@biol.sc.edu Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Whole exome sequencing in recurrent early pregnancy loss.

PubMed

Qiao, Ying; Wen, Jiadi; Tang, Flamingo; Martell, Sally; Shomer, Naomi; Leung, Peter C K; Stephenson, Mary D; Rajcan-Separovic, Evica

2016-05-01

Exome sequencing can identify genetic causes of idiopathic recurrent pregnancy loss (RPL). We identified compound heterozygous deleterious mutations affecting DYNC2H1 and ALOX15 in two out of four families with RPL. Both genes have a role in early development. Bioinformatics analysis of all genes with rare and putatively pathogenic mutations in miscarriages and couples showed enrichment in pathways relevant to pregnancy loss, including the complement and coagulation cascades pathways. Next generation sequencing (NGS) is increasingly being used to identify known and novel gene mutations in children with developmental delay and in fetuses with ultrasound-detected anomalies. In contrast, NGS is rarely used to study pregnancy loss. Chromosome microarray analysis detects putatively causative DNA copy number variants (CNVs) in ∼2% of miscarriages and CNVs of unknown significance (predominantly parental in origin) in up to 40% of miscarriages. Therefore, a large number of miscarriages still have an unknown cause. Whole exome sequencing (WES) was performed using Illumina HiSeq 2000 platform on seven euploid miscarriages from four families with RPL. Golden Helix SVS v8.1.5 was used for data assessment and inheritance analysis for deleterious DNA variants predicted to severely disrupt protein-coding genes by introducing a frameshift, loss of the stop codon, gain of the stop codon, changes in splicing or the initial codon. Webgestalt (http://bioinfo.vanderbilt.edu/webgestalt/) was used for pathway and disease association enrichment analysis of a gene pool containing putatively pathogenic variants in miscarriages and couples in comparison to control gene pools. Compound heterozygous mutations in DYNC2H1 and ALOX15 were identified in miscarriages from two families with RPL. DYNC2H1 is involved in cilia biogenesis and has been associated with fetal lethality in humans. ALOX15 is expressed in placenta and its dysregulation has been associated with inflammation, placental, dysfunction, abnormal oxidative stress response and angiogenesis. The pool of putatively pathogenic single nucleotide variants (SNVs) and small insertions and deletions (indels) detected in the miscarriages showed enrichment in 'complement and coagulation cascades pathway', and 'ciliary motility disorders'. We conclude that CNVs, individual SNVs and pool of deleterious gene mutations identified by exome sequencing could contribute to RPL. The size of our sample cohort is small. The functional effect of candidate mutations should be evaluated to determine whether the mutations are causative. This is the first study to assess whether SNVs may contribute to the pathogenesis of miscarriage. Furthermore, our findings suggest that collective effect of mutations in relevant biological pathways could be implicated in RPL. The study was funded by Canadian Institutes of Health Research (grant MOP 106467) and Michael Smith Foundation of Health Research Career Scholar salary award to ERS. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Whole exome sequencing in recurrent early pregnancy loss

PubMed Central

Qiao, Ying; Wen, Jiadi; Tang, Flamingo; Martell, Sally; Shomer, Naomi; Leung, Peter C.K.; Stephenson, Mary D.; Rajcan-Separovic, Evica

2016-01-01

STUDY HYPOTHESIS Exome sequencing can identify genetic causes of idiopathic recurrent pregnancy loss (RPL). STUDY FINDING We identified compound heterozygous deleterious mutations affecting DYNC2H1 and ALOX15 in two out of four families with RPL. Both genes have a role in early development. Bioinformatics analysis of all genes with rare and putatively pathogenic mutations in miscarriages and couples showed enrichment in pathways relevant to pregnancy loss, including the complement and coagulation cascades pathways. WHAT IS KNOWN ALREADY Next generation sequencing (NGS) is increasingly being used to identify known and novel gene mutations in children with developmental delay and in fetuses with ultrasound-detected anomalies. In contrast, NGS is rarely used to study pregnancy loss. Chromosome microarray analysis detects putatively causative DNA copy number variants (CNVs) in ∼2% of miscarriages and CNVs of unknown significance (predominantly parental in origin) in up to 40% of miscarriages. Therefore, a large number of miscarriages still have an unknown cause. STUDY DESIGN, SAMPLES/MATERIALS, METHODS Whole exome sequencing (WES) was performed using Illumina HiSeq 2000 platform on seven euploid miscarriages from four families with RPL. Golden Helix SVS v8.1.5 was used for data assessment and inheritance analysis for deleterious DNA variants predicted to severely disrupt protein-coding genes by introducing a frameshift, loss of the stop codon, gain of the stop codon, changes in splicing or the initial codon. Webgestalt (http://bioinfo.vanderbilt.edu/webgestalt/) was used for pathway and disease association enrichment analysis of a gene pool containing putatively pathogenic variants in miscarriages and couples in comparison to control gene pools. MAIN RESULTS AND THE ROLE OF CHANCE Compound heterozygous mutations in DYNC2H1 and ALOX15 were identified in miscarriages from two families with RPL. DYNC2H1 is involved in cilia biogenesis and has been associated with fetal lethality in humans. ALOX15 is expressed in placenta and its dysregulation has been associated with inflammation, placental, dysfunction, abnormal oxidative stress response and angiogenesis. The pool of putatively pathogenic single nucleotide variants (SNVs) and small insertions and deletions (indels) detected in the miscarriages showed enrichment in ‘complement and coagulation cascades pathway’, and ‘ciliary motility disorders’. We conclude that CNVs, individual SNVs and pool of deleterious gene mutations identified by exome sequencing could contribute to RPL. LIMITATIONS, REASONS FOR CAUTION The size of our sample cohort is small. The functional effect of candidate mutations should be evaluated to determine whether the mutations are causative. WIDER IMPLICATIONS OF THE FINDINGS This is the first study to assess whether SNVs may contribute to the pathogenesis of miscarriage. Furthermore, our findings suggest that collective effect of mutations in relevant biological pathways could be implicated in RPL. STUDY FUNDING AND COMPETING INTEREST(S) The study was funded by Canadian Institutes of Health Research (grant MOP 106467) and Michael Smith Foundation of Health Research Career Scholar salary award to ERS. PMID:26826164

Genomics of crop wild relatives: expanding the gene pool for crop improvement.

PubMed

Brozynska, Marta; Furtado, Agnelo; Henry, Robert J

2016-04-01

Plant breeders require access to new genetic diversity to satisfy the demands of a growing human population for more food that can be produced in a variable or changing climate and to deliver the high-quality food with nutritional and health benefits demanded by consumers. The close relatives of domesticated plants, crop wild relatives (CWRs), represent a practical gene pool for use by plant breeders. Genomics of CWR generates data that support the use of CWR to expand the genetic diversity of crop plants. Advances in DNA sequencing technology are enabling the efficient sequencing of CWR and their increased use in crop improvement. As the sequencing of genomes of major crop species is completed, attention has shifted to analysis of the wider gene pool of major crops including CWR. A combination of de novo sequencing and resequencing is required to efficiently explore useful genetic variation in CWR. Analysis of the nuclear genome, transcriptome and maternal (chloroplast and mitochondrial) genome of CWR is facilitating their use in crop improvement. Genome analysis results in discovery of useful alleles in CWR and identification of regions of the genome in which diversity has been lost in domestication bottlenecks. Targeting of high priority CWR for sequencing will maximize the contribution of genome sequencing of CWR. Coordination of global efforts to apply genomics has the potential to accelerate access to and conservation of the biodiversity essential to the sustainability of agriculture and food production. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

Lake-specific variation in growth, migration timing and survival of juvenile sockeye salmon Oncorhynchus nerka: separating environmental from genetic influences.

PubMed

Reed, T E; Martinek, G; Quinn, T P

2010-08-01

Time series on juvenile life-history traits obtained from sockeye salmon Oncorhynchus nerka were analysed to assess lake-specific environmental influences on juvenile migration timing, size and survival of fish from a common gene pool. Every year for the past two decades, O. nerka have been spawned at a hatchery facility, and the progeny released into two lakes that differ in average summer temperatures, limnological attributes and growth opportunities. Juveniles reared in the warmer, more productive Crosswind Lake were larger and heavier as smolts compared to those from the cooler, less productive Summit Lake and had higher in-lake and subsequent marine survival. Crosswind Lake smolts migrated from the lake to sea slightly earlier in the season but the migration timing distributions overlapped considerably across years. Fry stocking density had a negative effect on smolt length for both lakes, and a negative effect on in-lake survival in Summit Lake. Taken together, the results revealed a strong effect of lake-rearing environment on the expression of life-history variation in O. nerka. The stocking of these lakes each year with juveniles from a single mixed-source population provided a large-scale reverse common-garden experiment, where the same gene pool was exposed to different environments, rather than the different gene pools in the same environment approach typical of evolutionary ecology studies. Other researchers are encouraged to seek and exploit similar serendipitous situations, which might allow environmental and genetic influences on ecologically important traits to be distinguished in natural or semi-natural settings.

Genetic linkage analysis using pooled DNA and infrared detection of tailed STRP primer patterns

NASA Astrophysics Data System (ADS)

Oetting, William S.; Wildenberg, Scott C.; King, Richard A.

1996-04-01

The mapping of a disease locus to a specific chromosomal region is an important step in the eventual isolation and analysis of a disease causing gene. Conventional mapping methods analyze large multiplex families and/or smaller nuclear families to find linkage between the disease and a chromosome marker that maps to a known chromosomal region. This analysis is time consuming and tedious, typically requiring the determination of 30,000 genotypes or more. For appropriate populations, we have instead utilized pooled DNA samples for gene mapping which greatly reduces the amount of time necessary for an initial chromosomal screen. This technique assumes a common founder for the disease locus of interest and searches for a region of a chromosome shared between affected individuals. Our analysis involves the PCR amplification of short tandem repeat polymorphisms (STRP) to detect these shared regions. In order to reduce the cost of genotyping, we have designed unlabeled tailed PCR primers which, when combined with a labeled universal primer, provides for an alternative to synthesizing custom labeled primers. The STRP pattern is visualized with an infrared fluorescence based automated DNA sequencer and the patterns quantitated by densitometric analysis of the allele pattern. Differences in the distribution of alleles between pools of affected and unaffected individuals, including a reduction in the number of alleles in the affected pool, indicate the sharing of a region of a chromosome. We have found this method effective for markers 10 - 15 cM away from the disease locus for a recessive genetic disease.

BAC-pool sequencing and analysis confirms growth-associated QTLs in the Asian seabass genome.

PubMed

Shen, Xueyan; Ngoh, Si Yan; Thevasagayam, Natascha May; Prakki, Sai Rama Sridatta; Bhandare, Pranjali; Tan, Andy Wee Kiat; Tan, Gui Quan; Singh, Siddharth; Phua, Norman Chun Han; Vij, Shubha; Orbán, László

2016-11-08

The Asian seabass is an important marine food fish that has been cultured for several decades in Asia Pacific. However, the lack of a high quality reference genome has hampered efforts to improve its selective breeding. A 3D BAC pool set generated in this study was screened using 22 SSR markers located on linkage group 2 which contains a growth-related QTL region. Seventy-two clones corresponding to 22 FPC contigs were sequenced by Illumina MiSeq technology. We co-assembled the MiSeq-derived scaffolds from each FPC contig with error-corrected PacBio reads, resulting in 187 sequences covering 9.7 Mb. Eleven genes annotated within this region were found to be potentially associated with growth and their tissue-specific expression was investigated. Correlation analysis demonstrated that SNPs in ctsb, skp1 and ppp2ca can be potentially used as markers for selecting fast-growing fingerlings. Conserved syntenies between seabass LG2 and five other teleosts were identified. This study i) provided a 10 Mb targeted genome assembly; ii) demonstrated NGS of BAC pools as a potential approach for mining candidates underlying QTLs of this species; iii) detected eleven genes potentially responsible for growth in the QTL region; and iv) identified useful SNP markers for selective breeding programs of Asian seabass.

Prevalence and diversity of H9N2 avian influenza in chickens of Northern Vietnam, 2014.

PubMed

Thuy, Duong Mai; Peacock, Thomas P; Bich, Vu Thi Ngoc; Fabrizio, Thomas; Hoang, Dang Nguyen; Tho, Nguyen Dang; Diep, Nguyen Thi; Nguyen, Minh; Hoa, Le Nguyen Minh; Trang, Hau Thi Thu; Choisy, Marc; Inui, Ken; Newman, Scott; Trung, Nguyen Vu; van Doorn, Rogier; To, Thanh Long; Iqbal, Munir; Bryant, Juliet E

2016-10-01

Despite their classification as low pathogenicity avian influenza viruses (LPAIV), A/H9N2 viruses cause significant losses in poultry in many countries throughout Asia, the Middle East and North Africa. To date, poultry surveillance in Vietnam has focused on detection of influenza H5 viruses, and there is limited understanding of influenza H9 epidemiology and transmission dynamics. We determined prevalence and diversity of influenza A viruses in chickens from live bird markets (LBM) of 7 northern Vietnamese provinces, using pooled oropharyngeal swabs collected from October to December 2014. Screening by real time RT-PCR revealed 1207/4900 (24.6%) of pooled swabs to be influenza A virus positive; overall prevalence estimates after accounting for pooling (5 swabs/pools) were 5.8% (CI 5.4-6.0). Subtyping was performed on 468 pooled swabs with M gene Ct<26. No influenza H7 was detected; 422 (90.1%) were H9 positive; and 22 (4.7%) were H5 positive. There was no evidence was of interaction between H9 and H5 virus detection rates. We sequenced 17 whole genomes of A/H9N2, 2 of A/H5N6, and 11 partial genomes. All H9N2 viruses had internal genes that clustered with genotype 57 and were closely related to Chinese human isolates of A/H7N9 and A/H10N8. Using a nucleotide divergence cutoff of 98%, we identified 9 distinct H9 genotypes. Phylogenetic analysis suggested multiple introductions of H9 viruses to northern Vietnam rather than in-situ transmission. Further investigations of H9 prevalence and diversity in other regions of Vietnam are warranted to assess H9 endemicity elsewhere in the country. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Historical biome distribution and recent human disturbance shape the diversity of arbuscular mycorrhizal fungi.

PubMed

Pärtel, Meelis; Öpik, Maarja; Moora, Mari; Tedersoo, Leho; Szava-Kovats, Robert; Rosendahl, Søren; Rillig, Matthias C; Lekberg, Ylva; Kreft, Holger; Helgason, Thorunn; Eriksson, Ove; Davison, John; de Bello, Francesco; Caruso, Tancredi; Zobel, Martin

2017-10-01

The availability of global microbial diversity data, collected using standardized metabarcoding techniques, makes microorganisms promising models for investigating the role of regional and local factors in driving biodiversity. Here we modelled the global diversity of symbiotic arbuscular mycorrhizal (AM) fungi using currently available data on AM fungal molecular diversity (small subunit (SSU) ribosomal RNA (rRNA) gene sequences) in field samples. To differentiate between regional and local effects, we estimated species pools (sets of potentially suitable taxa) for each site, which are expected to reflect regional processes. We then calculated community completeness, an index showing the fraction of the species pool present, which is expected to reflect local processes. We found significant spatial variation, globally in species pool size, as well as in local and dark diversity (absent members of the species pool). Species pool size was larger close to areas containing tropical grasslands during the last glacial maximum, which are possible centres of diversification. Community completeness was greater in regions of high wilderness (remoteness from human disturbance). Local diversity was correlated with wilderness and current connectivity to mountain grasslands. Applying the species pool concept to symbiotic fungi facilitated a better understanding of how biodiversity can be jointly shaped by large-scale historical processes and recent human disturbance. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

Fine Mapping of a Clubroot Resistance Gene in Chinese Cabbage Using SNP Markers Identified from Bulked Segregant RNA Sequencing

PubMed Central

Huang, Zhen; Peng, Gary; Liu, Xunjia; Deora, Abhinandan; Falk, Kevin C.; Gossen, Bruce D.; McDonald, Mary R.; Yu, Fengqun

2017-01-01

Clubroot, caused by Plasmodiophora brassicae, is an important disease of canola (Brassica napus) in western Canada and worldwide. In this study, a clubroot resistance gene (Rcr2) was identified and fine mapped in Chinese cabbage cv. “Jazz” using single-nucleotide polymorphisms (SNP) markers identified from bulked segregant RNA sequencing (BSR-Seq) and molecular markers were developed for use in marker assisted selection. In total, 203.9 million raw reads were generated from one pooled resistant (R) and one pooled susceptible (S) sample, and >173,000 polymorphic SNP sites were identified between the R and S samples. One significant peak was observed between 22 and 26 Mb of chromosome A03, which had been predicted by BSR-Seq to contain the causal gene Rcr2. There were 490 polymorphic SNP sites identified in the region. A segregating population consisting of 675 plants was analyzed with 15 SNP sites in the region using the Kompetitive Allele Specific PCR method, and Rcr2 was fine mapped between two SNP markers, SNP_A03_32 and SNP_A03_67 with 0.1 and 0.3 cM from Rcr2, respectively. Five SNP markers co-segregated with Rcr2 in this region. Variants were identified in 14 of 36 genes annotated in the Rcr2 target region. The numbers of poly variants differed among the genes. Four genes encode TIR-NBS-LRR proteins and two of them Bra019410 and Bra019413, had high numbers of polymorphic variants and so are the most likely candidates of Rcr2. PMID:28894454

Messenger RNA profiling of human platelets by microarray hybridization.

PubMed

Bugert, Peter; Dugrillon, Alex; Günaydin, Ayse; Eichler, Hermann; Klüter, Harald

2003-10-01

Platelets are generally believed to be inactive in terms of de novo protein synthesis. On the other hand, the presence of ribosomes and mRNA molecules is well established. Many studies have used reverse transcriptase (RT) -PCR for detection of gene transcripts in platelets. As RT-PCR is a very sensitive method, any leukocyte contamination of platelet preparations can lead to false results. We performed three filtration procedures to minimize leukocyte contamination of pooled buffy-coat platelet concentrates prior to RNA isolation. Furthermore, by applying a genomic PCR approach with 50 amplification cycles we demonstrated that nucleated cells were not detectable. Microarray hybridization was used to analyze 9,850 individual human genes in RNA from purified platelets. In total we identified 1,526 (15.5%) positive genes. The data were confirmed in six individual experiments each performed on a PC pooled from four individual blood donations. Genes specific for nucleated blood cells such as CD4, CD83 and others were negative and verified the purity of PC. Overrepresentation of positive genes was found in the functional categories of glycoproteins/integrins (22.6% vs. 15.5%, p=0.029) and receptors (20.7% vs. 15.5%, p<0.001). Gene transcripts encoding RANTES, GRO-alpha, MIP-1alpha, MIP-1beta, and others were found at high levels of signal intensity and confirmed literature data. This work provides a mRNA profile of human platelets and a complete list of results can be downloaded from the website of our institute www.ma.uni-heidelberg.de/inst/iti/plt_array.xls. The knowledge about gene transcripts may have an impact on the characterization of novel proteins and their functions in platelets.

Systematic Integration of Brain eQTL and GWAS Identifies ZNF323 as a Novel Schizophrenia Risk Gene and Suggests Recent Positive Selection Based on Compensatory Advantage on Pulmonary Function

PubMed Central

Luo, Xiong-Jian; Mattheisen, Manuel; Li, Ming; Huang, Liang; Rietschel, Marcella; Børglum, Anders D.; Als, Thomas D.; van den Oord, Edwin J.; Aberg, Karolina A.; Mors, Ole; Mortensen, Preben Bo; Luo, Zhenwu; Degenhardt, Franziska; Cichon, Sven; Schulze, Thomas G.; Nöthen, Markus M.; Su, Bing; Zhao, Zhongming; Gan, Lin; Yao, Yong-Gang

2015-01-01

Genome-wide association studies have identified multiple risk variants and loci that show robust association with schizophrenia. Nevertheless, it remains unclear how these variants confer risk to schizophrenia. In addition, the driving force that maintains the schizophrenia risk variants in human gene pool is poorly understood. To investigate whether expression-associated genetic variants contribute to schizophrenia susceptibility, we systematically integrated brain expression quantitative trait loci and genome-wide association data of schizophrenia using Sherlock, a Bayesian statistical framework. Our analyses identified ZNF323 as a schizophrenia risk gene (P = 2.22×10–6). Subsequent analyses confirmed the association of the ZNF323 and its expression-associated single nucleotide polymorphism rs1150711 in independent samples (gene-expression: P = 1.40×10–6; single-marker meta-analysis in the combined discovery and replication sample comprising 44123 individuals: P = 6.85×10−10). We found that the ZNF323 was significantly downregulated in hippocampus and frontal cortex of schizophrenia patients (P = .0038 and P = .0233, respectively). Evidence for pleiotropic effects was detected (association of rs1150711 with lung function and gene expression of ZNF323 in lung: P = 6.62×10–5 and P = 9.00×10–5, respectively) with the risk allele (T allele) for schizophrenia acting as protective allele for lung function. Subsequent population genetics analyses suggest that the risk allele (T) of rs1150711 might have undergone recent positive selection in human population. Our findings suggest that the ZNF323 is a schizophrenia susceptibility gene whose expression may influence schizophrenia risk. Our study also illustrates a possible mechanism for maintaining schizophrenia risk variants in the human gene pool. PMID:25759474

Combinatorial Pooling Enables Selective Sequencing of the Barley Gene Space

PubMed Central

Lonardi, Stefano; Duma, Denisa; Alpert, Matthew; Cordero, Francesca; Beccuti, Marco; Bhat, Prasanna R.; Wu, Yonghui; Ciardo, Gianfranco; Alsaihati, Burair; Ma, Yaqin; Wanamaker, Steve; Resnik, Josh; Bozdag, Serdar; Luo, Ming-Cheng; Close, Timothy J.

2013-01-01

For the vast majority of species – including many economically or ecologically important organisms, progress in biological research is hampered due to the lack of a reference genome sequence. Despite recent advances in sequencing technologies, several factors still limit the availability of such a critical resource. At the same time, many research groups and international consortia have already produced BAC libraries and physical maps and now are in a position to proceed with the development of whole-genome sequences organized around a physical map anchored to a genetic map. We propose a BAC-by-BAC sequencing protocol that combines combinatorial pooling design and second-generation sequencing technology to efficiently approach denovo selective genome sequencing. We show that combinatorial pooling is a cost-effective and practical alternative to exhaustive DNA barcoding when preparing sequencing libraries for hundreds or thousands of DNA samples, such as in this case gene-bearing minimum-tiling-path BAC clones. The novelty of the protocol hinges on the computational ability to efficiently compare hundred millions of short reads and assign them to the correct BAC clones (deconvolution) so that the assembly can be carried out clone-by-clone. Experimental results on simulated data for the rice genome show that the deconvolution is very accurate, and the resulting BAC assemblies have high quality. Results on real data for a gene-rich subset of the barley genome confirm that the deconvolution is accurate and the BAC assemblies have good quality. While our method cannot provide the level of completeness that one would achieve with a comprehensive whole-genome sequencing project, we show that it is quite successful in reconstructing the gene sequences within BACs. In the case of plants such as barley, this level of sequence knowledge is sufficient to support critical end-point objectives such as map-based cloning and marker-assisted breeding. PMID:23592960

Combinatorial pooling enables selective sequencing of the barley gene space.

PubMed

Lonardi, Stefano; Duma, Denisa; Alpert, Matthew; Cordero, Francesca; Beccuti, Marco; Bhat, Prasanna R; Wu, Yonghui; Ciardo, Gianfranco; Alsaihati, Burair; Ma, Yaqin; Wanamaker, Steve; Resnik, Josh; Bozdag, Serdar; Luo, Ming-Cheng; Close, Timothy J

2013-04-01

For the vast majority of species - including many economically or ecologically important organisms, progress in biological research is hampered due to the lack of a reference genome sequence. Despite recent advances in sequencing technologies, several factors still limit the availability of such a critical resource. At the same time, many research groups and international consortia have already produced BAC libraries and physical maps and now are in a position to proceed with the development of whole-genome sequences organized around a physical map anchored to a genetic map. We propose a BAC-by-BAC sequencing protocol that combines combinatorial pooling design and second-generation sequencing technology to efficiently approach denovo selective genome sequencing. We show that combinatorial pooling is a cost-effective and practical alternative to exhaustive DNA barcoding when preparing sequencing libraries for hundreds or thousands of DNA samples, such as in this case gene-bearing minimum-tiling-path BAC clones. The novelty of the protocol hinges on the computational ability to efficiently compare hundred millions of short reads and assign them to the correct BAC clones (deconvolution) so that the assembly can be carried out clone-by-clone. Experimental results on simulated data for the rice genome show that the deconvolution is very accurate, and the resulting BAC assemblies have high quality. Results on real data for a gene-rich subset of the barley genome confirm that the deconvolution is accurate and the BAC assemblies have good quality. While our method cannot provide the level of completeness that one would achieve with a comprehensive whole-genome sequencing project, we show that it is quite successful in reconstructing the gene sequences within BACs. In the case of plants such as barley, this level of sequence knowledge is sufficient to support critical end-point objectives such as map-based cloning and marker-assisted breeding.

Mucopolysaccharidosis VI in cats - clarification regarding genetic testing.

PubMed

Lyons, Leslie A; Grahn, Robert A; Genova, Francesca; Beccaglia, Michela; Hopwood, John J; Longeri, Maria

2016-07-02

The release of new DNA-based diagnostic tools has increased tremendously in companion animals. Over 70 different DNA variants are now known for the cat, including DNA variants in disease-associated genes and genes causing aesthetically interesting traits. The impact genetic tests have on animal breeding and health management is significant because of the ability to control the breeding of domestic cats, especially breed cats. If used properly, genetic testing can prevent the production of diseased animals, causing the reduction of the frequency of the causal variant in the population, and, potentially, the eventual eradication of the disease. However, testing of some identified DNA variants may be unwarranted and cause undo strife within the cat breeding community and unnecessary reduction of gene pools and availability of breeding animals. Testing for mucopolysaccharidosis Type VI (MPS VI) in cats, specifically the genetic testing of the L476P (c.1427T>C) and the D520N (c.1558G>A) variants in arylsulfatase B (ARSB), has come under scrutiny. No health problems are associated with the D520N (c.1558G>A) variant, however, breeders that obtain positive results for this variant are speculating as to possible correlation with health concerns. Birman cats already have a markedly reduced gene pool and have a high frequency of the MPS VI D520N variant. Further reduction of the gene pool by eliminating cats that are heterozygous or homozygous for only the MPS VI D520N variant could lead to more inbreeding depression effects on the breed population. Herein is debated the genetic testing of the MPS VI D520N variant in cats. Surveys from different laboratories suggest the L476P (c.1427T>C) disease-associated variant should be monitored in the cat breed populations, particularly breeds with Siamese derivations and outcrosses. However, the D520N has no evidence of association with disease in cats and testing is not recommended in the absence of L476P genotyping. Selection against the D520N is not warranted in cat populations. More rigorous guidelines may be required to support the genetic testing of DNA variants in all animal species.

Technique for quantitative RT-PCR analysis directly from single muscle fibers.

PubMed

Wacker, Michael J; Tehel, Michelle M; Gallagher, Philip M

2008-07-01

The use of single-cell quantitative RT-PCR has greatly aided the study of gene expression in fields such as muscle physiology. For this study, we hypothesized that single muscle fibers from a biopsy can be placed directly into the reverse transcription buffer and that gene expression data can be obtained without having to first extract the RNA. To test this hypothesis, biopsies were taken from the vastus lateralis of five male subjects. Single muscle fibers were isolated and underwent RNA isolation (technique 1) or placed directly into reverse transcription buffer (technique 2). After cDNA conversion, individual fiber cDNA was pooled and quantitative PCR was performed using primer-probes for beta(2)-microglobulin, glyceraldehyde-3-phosphate dehydrogenase, insulin-like growth factor I receptor, and glucose transporter subtype 4. The no RNA extraction method provided similar quantitative PCR data as that of the RNA extraction method. A third technique was also tested in which we used one-quarter of an individual fiber's cDNA for PCR (not pooled) and the average coefficient of variation between fibers was <8% (cycle threshold value) for all genes studied. The no RNA extraction technique was tested on isolated muscle fibers using a gene known to increase after exercise (pyruvate dehydrogenase kinase 4). We observed a 13.9-fold change in expression after resistance exercise, which is consistent with what has been previously observed. These results demonstrate a successful method for gene expression analysis directly from single muscle fibers.

Genetic Linkage of Soil Carbon Pools and Microbial Functions in Subtropical Freshwater Wetlands in Response to Experimental Warming

PubMed Central

Wang, Hang; He, Zhili; Lu, Zhenmei; Zhou, Jizhong; Van Nostrand, Joy D.; Xu, Xinhua

2012-01-01

Rising climate temperatures in the future are predicted to accelerate the microbial decomposition of soil organic matter. A field microcosm experiment was carried out to examine the impact of soil warming in freshwater wetlands on different organic carbon (C) pools and associated microbial functional responses. GeoChip 4.0, a functional gene microarray, was used to determine microbial gene diversity and functional potential for C degradation. Experimental warming significantly increased soil pore water dissolved organic C and phosphorus (P) concentrations, leading to a higher potential for C emission and P export. Such losses of total organic C stored in soil could be traced back to the decomposition of recalcitrant organic C. Warming preferentially stimulated genes for degrading recalcitrant C over labile C. This was especially true for genes encoding cellobiase and mnp for cellulose and lignin degradation, respectively. We confirmed this with warming-enhanced polyphenol oxidase and peroxidase activities for recalcitrant C acquisition and greater increases in recalcitrant C use efficiency than in labile C use efficiency (average percentage increases of 48% versus 28%, respectively). The relative abundance of lignin-degrading genes increased by 15% under warming; meanwhile, soil fungi, as the primary decomposers of lignin, were greater in abundance by 27%. This work suggests that future warming may enhance the potential for accelerated fungal decomposition of lignin-like compounds, leading to greater microbially mediated C losses than previously estimated in freshwater wetlands. PMID:22923398

High-resolution phylogenetic analysis of southeastern Europe traces major episodes of paternal gene flow among Slavic populations.

PubMed

Pericić, Marijana; Lauc, Lovorka Barać; Klarić, Irena Martinović; Rootsi, Siiri; Janićijevic, Branka; Rudan, Igor; Terzić, Rifet; Colak, Ivanka; Kvesić, Ante; Popović, Dan; Sijacki, Ana; Behluli, Ibrahim; Dordevic, Dobrivoje; Efremovska, Ljudmila; Bajec, Dorde D; Stefanović, Branislav D; Villems, Richard; Rudan, Pavao

2005-10-01

The extent and nature of southeastern Europe (SEE) paternal genetic contribution to the European genetic landscape were explored based on a high-resolution Y chromosome analysis involving 681 males from seven populations in the region. Paternal lineages present in SEE were compared with previously published data from 81 western Eurasian populations and 5,017 Y chromosome samples. The finding that five major haplogroups (E3b1, I1b* (xM26), J2, R1a, and R1b) comprise more than 70% of SEE total genetic variation is consistent with the typical European Y chromosome gene pool. However, distribution of major Y chromosomal lineages and estimated expansion signals clarify the specific role of this region in structuring of European, and particularly Slavic, paternal genetic heritage. Contemporary Slavic paternal gene pool, mostly characterized by the predominance of R1a and I1b* (xM26) and scarcity of E3b1 lineages, is a result of two major prehistoric gene flows with opposite directions: the post-Last Glacial Maximum R1a expansion from east to west, the Younger Dryas-Holocene I1b* (xM26) diffusion out of SEE in addition to subsequent R1a and I1b* (xM26) putative gene flows between eastern Europe and SEE, and a rather weak extent of E3b1 diffusion toward regions nowadays occupied by Slavic-speaking populations.

Assessing pooled BAC and whole genome shotgun strategies for assembly of complex genomes

PubMed Central

2011-01-01

Background We investigate if pooling BAC clones and sequencing the pools can provide for more accurate assembly of genome sequences than the "whole genome shotgun" (WGS) approach. Furthermore, we quantify this accuracy increase. We compare the pooled BAC and WGS approaches using in silico simulations. Standard measures of assembly quality focus on assembly size and fragmentation, which are desirable for large whole genome assemblies. We propose additional measures enabling easy and visual comparison of assembly quality, such as rearrangements and redundant sequence content, relative to the known target sequence. Results The best assembly quality scores were obtained using 454 coverage of 15× linear and 5× paired (3kb insert size) reads (15L-5P) on Arabidopsis. This regime gave similarly good results on four additional plant genomes of very different GC and repeat contents. BAC pooling improved assembly scores over WGS assembly, coverage and redundancy scores improving the most. Conclusions BAC pooling works better than WGS, however, both require a physical map to order the scaffolds. Pool sizes up to 12Mbp work well, suggesting this pooling density to be effective in medium-scale re-sequencing applications such as targeted sequencing of QTL intervals for candidate gene discovery. Assuming the current Roche/454 Titanium sequencing limitations, a 12 Mbp region could be re-sequenced with a full plate of linear reads and a half plate of paired-end reads, yielding 15L-5P coverage after read pre-processing. Our simulation suggests that massively over-sequencing may not improve accuracy. Our scoring measures can be used generally to evaluate and compare results of simulated genome assemblies. PMID:21496274

Clinicopathological Characteristics of Patients with Non-Small-Cell Lung Cancer Who Harbor EML4-ALK Fusion Gene: A Meta-Analysis

PubMed Central

Zhao, Fengzhi; Xu, Meng; Lei, Honcho; Zhou, Ziqi; Wang, Liang; Li, Ping; Zhao, Jianfu; Hu, Penghui

2015-01-01

Background A novel fusion gene of echinoderm microtubule-associated protein-like 4 (EML4) and anaplastic lymphoma kinase (ALK) has been recently identified in non-small-cell lung cancers (NSCLCs). Patients with the EML4-ALK fusion gene demonstrate unique clinicopathological and physiological characteristics. Here we present a meta-analysis of large-scale studies to evaluate the clinicopathological characteristics of NSCLC patients harboring the EML4-ALK fusion gene. Methods Both English and Chinese databases were systematically used to search the materials of the clinicopathological characteristics of patients with NSCLC harboring the EML4-ALK fusion gene. Pooled relative risk (RR) estimates and the 95% confidence intervals (95% CI) were calculated with the fixed or random effect model. Publication bias and chi-square test were also calculated. Results 27 retrospective studies were included in our meta-analysis. These studies included a total of 6950 patients. The incidence rate of EML4-ALK fusion in NSCLC patients was found to be 6.8% (472/6950). The correlation of the EML4-ALK fusion gene and clinicopathological characteristics of NSCLC patients demonstrated a significant difference in smoking status, histological types, stage, and ethnic characteristics. The positive rate of the EML4-ALK fusion gene expression in females were slightly higher than that in males, but not significantly (P = 0.52). In addition, the EML4-ALK fusion gene was mutually exclusive of the EGFR and KRAS mutation genes (P = 0.00). Conclusion Our pooled analysis revealed that the EML4-ALK fusion gene was observed predominantly in adenocarcinoma, non-smoking and NSCLC patients, especially those diagnosed in the advanced clinical stage of NSCLC. Additionally, the EML4-ALK fusion gene was exclusive of the EGFR and KRAS mutation genes. We surmise that IHC assay is a valuable tool for the prescreening of patients with ALK fusion gene in clinical practice, and FISH assay can be performed as a confirmation method. These insights might be helpful in guiding the appropriate molecular target therapy for NSCLC. PMID:25706305

Clinicopathological characteristics of patients with non-small-cell lung cancer who harbor EML4-ALK fusion gene: a meta-analysis.

PubMed

Zhao, Fengzhi; Xu, Meng; Lei, Honcho; Zhou, Ziqi; Wang, Liang; Li, Ping; Zhao, Jianfu; Hu, Penghui

2015-01-01

A novel fusion gene of echinoderm microtubule-associated protein-like 4 (EML4) and anaplastic lymphoma kinase (ALK) has been recently identified in non-small-cell lung cancers (NSCLCs). Patients with the EML4-ALK fusion gene demonstrate unique clinicopathological and physiological characteristics. Here we present a meta-analysis of large-scale studies to evaluate the clinicopathological characteristics of NSCLC patients harboring the EML4-ALK fusion gene. Both English and Chinese databases were systematically used to search the materials of the clinicopathological characteristics of patients with NSCLC harboring the EML4-ALK fusion gene. Pooled relative risk (RR) estimates and the 95% confidence intervals (95% CI) were calculated with the fixed or random effect model. Publication bias and chi-square test were also calculated. 27 retrospective studies were included in our meta-analysis. These studies included a total of 6950 patients. The incidence rate of EML4-ALK fusion in NSCLC patients was found to be 6.8% (472/6950). The correlation of the EML4-ALK fusion gene and clinicopathological characteristics of NSCLC patients demonstrated a significant difference in smoking status, histological types, stage, and ethnic characteristics. The positive rate of the EML4-ALK fusion gene expression in females were slightly higher than that in males, but not significantly (P = 0.52). In addition, the EML4-ALK fusion gene was mutually exclusive of the EGFR and KRAS mutation genes (P = 0.00). Our pooled analysis revealed that the EML4-ALK fusion gene was observed predominantly in adenocarcinoma, non-smoking and NSCLC patients, especially those diagnosed in the advanced clinical stage of NSCLC. Additionally, the EML4-ALK fusion gene was exclusive of the EGFR and KRAS mutation genes. We surmise that IHC assay is a valuable tool for the prescreening of patients with ALK fusion gene in clinical practice, and FISH assay can be performed as a confirmation method. These insights might be helpful in guiding the appropriate molecular target therapy for NSCLC.

In-vivo-induced antigenic determinants of Fusobacterium nucleatum subsp. nucleatum.

PubMed

Lee, H-R; Rhyu, I-C; Kim, H-D; Jun, H-K; Min, B-M; Lee, S-H; Choi, B-K

2011-04-01

Fusobacterium nucleatum plays a pivotal role in dental plaque biofilm formation and is known to be involved in chronic inflammatory systemic disease. However, limited knowledge of F. nucleatum genes expressed in vivo interferes with our understanding of pathogenesis. In this study, we identified F. nucleatum genes induced in vivo using in-vivo-induced antigen technology (IVIAT). Among 30,000 recombinant clones screened, 87 reacted reproducibly with pooled sera from 10 patients with periodontitis. The clones encoded for 32 different proteins, of which 28 could be assigned to their functions, which were categorized in translation, transcription, transport, energy metabolism, cell envelope, cellular process, fatty acid and phospholipid metabolism, transposition, cofactor biosynthesis, amino acid biosynthesis, and DNA replication. Putative virulence factors detected were ABC transporter, butyrate-acetoacetate CoA-transferase, hemin receptor, hemolysin, hemolysin-related protein, LysR family transcriptional regulator, serine protease, and transposase. Analysis of immune responses to the in-vivo-induced (ivi) antigens in five patients demonstrated that most were reactive to these proteins, confirming results with pooled sera. IVIAT-identified F. nucleatum genes in this study may accelerate the elucidation of F. nucleatum-mediated molecular pathogenesis. © 2011 John Wiley & Sons A/S.

Identification of Genes from the Fungal Pathogen Cryptococcus neoformans Related to Transmigration into the Central Nervous System

PubMed Central

Tseng, Hsiang-Kuang; Liu, Chang-Pan; Price, Michael S.; Jong, Ambrose Y.; Chang, Jui-Chih; Toffaletti, Dena L.; Betancourt-Quiroz, Marisol; Frazzitta, Aubrey E.; Cho, Wen-Long; Perfect, John R.

2012-01-01

Background A mouse brain transmigration assessment (MBTA) was created to investigate the central nervous system (CNS) pathogenesis of cryptococcal meningoencephalitis. Methodology/Principal Findings Two cryptococcal mutants were identified from a pool of 109 pre-selected mutants that were signature-tagged with the nourseothricin acetyltransferase (NAT) resistance cassette. These two mutants displayed abnormal transmigration into the central nervous system. One mutant displaying decreased transmigration contains a null mutation in the putative FNX1 gene, whereas the other mutant possessing a null mutation in the putative RUB1 gene exhibited increased transmigration into the brain. Two macrophage adhesion-defective mutants in the pool, 12F1 and 3C9, showed reduced phagocytosis by macrophages, but displayed no defects in CNS entry suggesting that transit within macrophages (the “Trojan horse” model of CNS entry) is not the primary mechanism for C. neoformans migration into the CNS in this MBTA. Conclusions/Significance This research design provides a new strategy for genetic impact studies on how Cryptococcus passes through the blood-brain barrier (BBB), and the specific isolated mutants in this assay support a transcellular mechanism of CNS entry. PMID:23028773

Differential gene regulation of GHSR signaling pathway in the arcuate nucleus and NPY neurons by fasting, diet-induced obesity, and 17β-estradiol.

PubMed

Yasrebi, Ali; Hsieh, Anna; Mamounis, Kyle J; Krumm, Elizabeth A; Yang, Jennifer A; Magby, Jason; Hu, Pu; Roepke, Troy A

2016-02-15

Ghrelin's receptor, growth hormone secretagogue receptor (GHSR), is highly expressed in the arcuate nucleus (ARC) and in neuropeptide Y (NPY) neurons. Fasting, diet-induced obesity (DIO), and 17β-estradiol (E2) influence ARC Ghsr expression. It is unknown if these effects occur in NPY neurons. Therefore, we examined the expression of Npy, Agrp, and GHSR signaling pathway genes after fasting, DIO, and E2 replacement in ARC and pools of NPY neurons. In males, fasting increased ARC Ghsr and NPY Foxo1 but decreased NPY Ucp2. In males, DIO decreased ARC and NPY Ghsr and Cpt1c. In fed females, E2 increased Agrp, Ghsr, Cpt1c, and Foxo1 in ARC. In NPY pools, E2 decreased Foxo1 in fed females but increased Foxo1 in fasted females. DIO in females suppressed Agrp and augmented Cpt1c in NPY neurons. In summary, genes involved in GHSR signaling are differentially regulated between the ARC and NPY neurons in a sex-dependent manner. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Aspen phenylpropanoid genes' expression levels correlate with genets' tannin richness and vary both in responses to soil nitrogen and associations with phenolic profiles.

PubMed

Decker, Vicki H G; Bandau, Franziska; Gundale, Michael J; Cole, Christopher T; Albrectsen, Benedicte R

2017-02-01

Condensed tannin (CT) contents of European aspen (Populus tremula L.) vary among genotypes, and increases in nitrogen (N) availability generally reduce plants' tannin production in favor of growth, through poorly understood mechanisms. We hypothesized that intrinsic tannin production rates may co-vary with gene expression responses to soil N and resource allocation within the phenylpropanoid pathway (PPP). Thus, we examined correlations between soil N levels and both expression patterns of eight PPP genes (measured by quantitative-reverse transcription PCR) and foliar phenolic compounds (measured by liquid chromatography-mass spectrometry) in young aspen genets with intrinsically extreme CT levels. Monitored phenolics included salicinoids, lignins, flavones, flavonols, CT precursors and CTs. The PPP genes were consistently expressed more strongly in high-CT trees. Low N supplements reduced expression of genes throughout the PPP in all genets, while high N doses restored expression of genes at the beginning and end of the pathway. These PPP changes were not reflected in pools of tannin precursors, but varying correlations between gene expression and foliar phenolic pools were detected in young and mature leaves, suggesting that processes linking gene expression and the resulting phenolics vary spatially and temporally. Precursor fluxes suggested that CT-related metabolic rate or sink controls are linked to intrinsic carbon allocation strategies associated with N responses. Overall, we found more negative correlations (indicative of allocation trade-offs) between PPP gene expression and phenolic products following N additions in low-CT plants than in high-CT plants. The tannin-related expression dynamics suggest that, in addition to defense, relative tannin levels may also be indicative of intraspecific variations in the way aspen genets respond to soil fertility. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Microdissection of Shoot Meristem Functional Domains

USDA-ARS?s Scientific Manuscript database

The shoot apical meristem (SAM) maintains a pool of indeterminate cells within the SAM proper, while lateral organs are initiated from the SAM periphery. Laser microdissection–microarray technology was used to compare transcriptional profiles within these SAM domains to identify novel maize genes th...

Genetic diversity in Gossypium genus

USDA-ARS?s Scientific Manuscript database

The overall objectives of this paper are to report on cotton germplasm resources, morphobiological and agronomic diversity of Gossypium genus and review efforts on molecular genetic diversity of cotton gene pools as well as on the challenges and perspectives of exploiting genetic diversity in cotton...

Genomic analyses of tropical beef cattle fertility based on genotyping pools of Brahman cows with unknown pedigree.

PubMed

Reverter, A; Porto-Neto, L R; Fortes, M R S; McCulloch, R; Lyons, R E; Moore, S; Nicol, D; Henshall, J; Lehnert, S A

2016-10-01

We introduce an innovative approach to lowering the overall cost of obtaining genomic EBV (GEBV) and encourage their use in commercial extensive herds of Brahman beef cattle. In our approach, the DNA genotyping of cow herds from 2 independent properties was performed using a high-density bovine SNP chip on DNA from pooled blood samples, grouped according to the result of a pregnancy test following their first and second joining opportunities. For the DNA pooling strategy, 15 to 28 blood samples from the same phenotype and contemporary group were allocated to pools. Across the 2 properties, a total of 183 pools were created representing 4,164 cows. In addition, blood samples from 309 bulls from the same properties were also taken. After genotyping and quality control, 74,584 remaining SNP were used for analyses. Pools and individual DNA samples were related by means of a "hybrid" genomic relationship matrix. The pooled genotyping analysis of 2 large and independent commercial populations of tropical beef cattle was able to recover significant and plausible associations between SNP and pregnancy test outcome. We discuss 24 SNP with significant association ( < 1.0 × 10) and mapped within 40 kb of an annotated gene. We have established a method to estimate the GEBV in young herd bulls for a trait that is currently unable to be predicted at all. In summary, our novel approach allowed us to conduct genomic analyses of fertility in 2 large commercial Brahman herds managed under extensive pastoral conditions.

Systematic Functional Interrogation of Rare Cancer Variants Identifies Oncogenic Alleles | Office of Cancer Genomics

Cancer.gov

Cancer genome characterization efforts now provide an initial view of the somatic alterations in primary tumors. However, most point mutations occur at low frequency, and the function of these alleles remains undefined. We have developed a scalable systematic approach to interrogate the function of cancer-associated gene variants. We subjected 474 mutant alleles curated from 5,338 tumors to pooled in vivo tumor formation assays and gene expression profiling. We identified 12 transforming alleles, including two in genes (PIK3CB, POT1) that have not been shown to be tumorigenic.

Endogenous miRNA and Target Concentrations Determine Susceptibility to Potential ceRNA Competition

PubMed Central

Bosson, Andrew D.; Zamudio, Jesse R.; Sharp, Phillip A.

2016-01-01

SUMMARY Target competition (ceRNA crosstalk) within miRNA-regulated gene networks has been proposed to influence biological systems. To assess target competition, we characterize and quantitate miRNA networks in two cell types. Argonaute iCLIP reveals that hierarchical binding of high- to low-affinity miRNA targets is a key characteristic of in vivo activity. Quantification of cellular miRNA and mRNA/ncRNA target pool levels indicates that miRNA:target pool ratios and an affinity partitioned target pool accurately predict in vivo Ago binding profiles and miRNA susceptibility to target competition. Using single-cell reporters, we directly test predictions and estimate that ~3,000 additional high-affinity target sites can affect active miRNA families with low endogenous miRNA:target ratios, such as miR-92/25. In contrast, the highly expressed miR-294 and let-7 families are not susceptible to increases of nearly 10,000 sites. These results show differential susceptibility based on endogenous miRNA:target pool ratios and provide a physiological context for ceRNA competition in vivo. PMID:25449132

Sample pooling for real-time PCR detection and virulence determination of the footrot pathogen Dichelobacter nodosus.

PubMed

Frosth, Sara; König, Ulrika; Nyman, Ann-Kristin; Aspán, Anna

2017-09-01

Dichelobacter nodosus is the principal cause of ovine footrot and strain virulence is an important factor in disease severity. Therefore, detection and virulence determination of D. nodosus is important for proper diagnosis of the disease. Today this is possible by real-time PCR analysis. Analysis of large numbers of samples is costly and laborious; therefore, pooling of individual samples is common in surveillance programs. However, pooling can reduce the sensitivity of the method. The aim of this study was to develop a pooling method for real-time PCR analysis that would allow sensitive detection and simultaneous virulence determination of D. nodosus. A total of 225 sheep from 17 flocks were sampled using ESwabs within the Swedish Footrot Control Program in 2014. Samples were first analysed individually and then in pools of five by real-time PCR assays targeting the 16S rRNA and aprV2/B2 genes of D. nodosus. Each pool consisted of four negative and one positive D. nodosus samples with varying amounts of the bacterium. In the individual analysis, 61 (27.1%) samples were positive in the 16S rRNA and the aprV2/B2 PCR assays and 164 (72.9%) samples were negative. All samples positive in the aprV2/B2 PCR-assay were of aprB2 variant. The pooled analysis showed that all 41 pools were also positive for D. nodosus 16S rRNA and the aprB2 variant. The diagnostic sensitivity for pooled and individual samples was therefore similar. Our method includes concentration of the bacteria before DNA-extraction. This may account for the maintenance of diagnostic sensitivity. Diagnostic sensitivity in the real-time PCR assays of the pooled samples were comparable to the sensitivity obtained for individually analysed samples. Even sub-clinical infections were able to be detected in the pooled PCR samples which is important for control of the disease. This method may therefore be implemented in footrot control programs where it can replace analysis of individual samples.

The Role of the Horizontal Gene Pool and Lateral Gene Transfer in Enhancing Microbial Activities in Marine Sediments

DTIC Science & Technology

2006-05-10

nifH encoding plasmids of diazotrophic bacteria isolated from roots of a salt marsh grass. Meeting Abstract, 105th General Meeting of the American...When the method was applied to 100 endogenous plasmids isolated from cultivated marine diazotrophs from salt marsh grass rhizoplane niches remarkably...Beeson, K.E., D.L. Erdner, C.E. Bagwell, C.R. Lovell, and P.A. Sobecky. 2002. Differentiation of plasmids in marine diazotroph assemblages

Natural Allelic Diversity, Genetic Structure and Linkage Disequilibrium Pattern in Wild Chickpea

PubMed Central

Kujur, Alice; Das, Shouvik; Badoni, Saurabh; Kumar, Vinod; Singh, Mohar; Bansal, Kailash C.; Tyagi, Akhilesh K.; Parida, Swarup K.

2014-01-01

Characterization of natural allelic diversity and understanding the genetic structure and linkage disequilibrium (LD) pattern in wild germplasm accessions by large-scale genotyping of informative microsatellite and single nucleotide polymorphism (SNP) markers is requisite to facilitate chickpea genetic improvement. Large-scale validation and high-throughput genotyping of genome-wide physically mapped 478 genic and genomic microsatellite markers and 380 transcription factor gene-derived SNP markers using gel-based assay, fluorescent dye-labelled automated fragment analyser and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass array have been performed. Outcome revealed their high genotyping success rate (97.5%) and existence of a high level of natural allelic diversity among 94 wild and cultivated Cicer accessions. High intra- and inter-specific polymorphic potential and wider molecular diversity (11–94%) along with a broader genetic base (13–78%) specifically in the functional genic regions of wild accessions was assayed by mapped markers. It suggested their utility in monitoring introgression and transferring target trait-specific genomic (gene) regions from wild to cultivated gene pool for the genetic enhancement. Distinct species/gene pool-wise differentiation, admixed domestication pattern, and differential genome-wide recombination and LD estimates/decay observed in a six structured population of wild and cultivated accessions using mapped markers further signifies their usefulness in chickpea genetics, genomics and breeding. PMID:25222488

Investigating the genetics of Bti resistance using mRNA tag sequencing: application on laboratory strains and natural populations of the dengue vector Aedes aegypti

PubMed Central

Paris, Margot; Marcombe, Sebastien; Coissac, Eric; Corbel, Vincent; David, Jean-Philippe; Després, Laurence

2013-01-01

Mosquito control is often the main method used to reduce mosquito-transmitted diseases. In order to investigate the genetic basis of resistance to the bio-insecticide Bacillus thuringiensis subsp. israelensis (Bti), we used information on polymorphism obtained from cDNA tag sequences from pooled larvae of laboratory Bti-resistant and susceptible Aedes aegypti mosquito strains to identify and analyse 1520 single nucleotide polymorphisms (SNPs). Of the 372 SNPs tested, 99.2% were validated using DNA Illumina GoldenGate® array, with a strong correlation between the allelic frequencies inferred from the pooled and individual data (r = 0.85). A total of 11 genomic regions and five candidate genes were detected using a genome scan approach. One of these candidate genes showed significant departures from neutrality in the resistant strain at sequence level. Six natural populations from Martinique Island were sequenced for the 372 tested SNPs with a high transferability (87%), and association mapping analyses detected 14 loci associated with Bti resistance, including one located in a putative receptor for Cry11 toxins. Three of these loci were also significantly differentiated between the laboratory strains, suggesting that most of the genes associated with resistance might differ between the two environments. It also suggests that common selected regions might harbour key genes for Bti resistance. PMID:24187584

Possible basis for the emergence of H1N1 viruses with pandemic potential from avian hosts.

PubMed

Koçer, Zeynep A; Krauss, Scott; Zanin, Mark; Danner, Angela; Gulati, Shelly; Jones, Jeremy C; Friedman, Kimberly; Graham, Allison; Forrest, Heather; Seiler, Jon; Air, Gillian M; Webster, Robert G

2015-07-01

Influenza A viruses of the H1N1 subtype have emerged from the avian influenza gene pool in aquatic birds and caused human pandemics at least twice during the past century. Despite this fact, surprisingly little is known about the H1N1 gene pool in the aquatic bird reservoir. A preliminary study showed that an H1N1 virus from a shorebird of the Charadriiformes order was transmitted between animals through the airborne route of infection, whereas an H1N1 virus from a bird of the Anseriformes order was not. Here we show that two of the three H1N1 viruses isolated from Charadriiformes species in 2009 were transmitted between animals through the airborne route of infection, and five H1N1 isolates from Anseriformes species were not. The one H1N1 virus from a Charadriiformes species that failed to transmit through the airborne route was a reassortant possessing multiple internal gene segments from Anseriformes species. The molecular differences between the airborne-transmissible and non-airborne-transmissible H1N1 viruses were multigenic, involving the selection of virus with human-like receptor-binding specificity (α2-6 sialic acid) and multiple differences in the polymerase complex, mainly in the PB2, PB1-F2, and nonstructural genes.

Possible basis for the emergence of H1N1 viruses with pandemic potential from avian hosts

PubMed Central

Koçer, Zeynep A; Krauss, Scott; Zanin, Mark; Danner, Angela; Gulati, Shelly; Jones, Jeremy C; Friedman, Kimberly; Graham, Allison; Forrest, Heather; Seiler, Jon; Air, Gillian M; Webster, Robert G

2015-01-01

Influenza A viruses of the H1N1 subtype have emerged from the avian influenza gene pool in aquatic birds and caused human pandemics at least twice during the past century. Despite this fact, surprisingly little is known about the H1N1 gene pool in the aquatic bird reservoir. A preliminary study showed that an H1N1 virus from a shorebird of the Charadriiformes order was transmitted between animals through the airborne route of infection, whereas an H1N1 virus from a bird of the Anseriformes order was not. Here we show that two of the three H1N1 viruses isolated from Charadriiformes species in 2009 were transmitted between animals through the airborne route of infection, and five H1N1 isolates from Anseriformes species were not. The one H1N1 virus from a Charadriiformes species that failed to transmit through the airborne route was a reassortant possessing multiple internal gene segments from Anseriformes species. The molecular differences between the airborne-transmissible and non-airborne-transmissible H1N1 viruses were multigenic, involving the selection of virus with human-like receptor-binding specificity (α2-6 sialic acid) and multiple differences in the polymerase complex, mainly in the PB2, PB1-F2, and nonstructural genes. PMID:26251829

Origins of domestic dog in southern East Asia is supported by analysis of Y-chromosome DNA.

PubMed

Ding, Z-L; Oskarsson, M; Ardalan, A; Angleby, H; Dahlgren, L-G; Tepeli, C; Kirkness, E; Savolainen, P; Zhang, Y-P

2012-05-01

Global mitochondrial DNA (mtDNA) data indicates that the dog originates from domestication of wolf in Asia South of Yangtze River (ASY), with minor genetic contributions from dog-wolf hybridisation elsewhere. Archaeological data and autosomal single nucleotide polymorphism data have instead suggested that dogs originate from Europe and/or South West Asia but, because these datasets lack data from ASY, evidence pointing to ASY may have been overlooked. Analyses of additional markers for global datasets, including ASY, are therefore necessary to test if mtDNA phylogeography reflects the actual dog history and not merely stochastic events or selection. Here, we analyse 14,437 bp of Y-chromosome DNA sequence in 151 dogs sampled worldwide. We found 28 haplotypes distributed in five haplogroups. Two haplogroups were universally shared and included three haplotypes carried by 46% of all dogs, but two other haplogroups were primarily restricted to East Asia. Highest genetic diversity and virtually complete phylogenetic coverage was found within ASY. The 151 dogs were estimated to originate from 13-24 wolf founders, but there was no indication of post-domestication dog-wolf hybridisations. Thus, Y-chromosome and mtDNA data give strikingly similar pictures of dog phylogeography, most importantly that roughly 50% of the gene pools are shared universally but only ASY has nearly the full range of genetic diversity, such that the gene pools in all other regions may derive from ASY. This corroborates that ASY was the principal, and possibly sole region of wolf domestication, that a large number of wolves were domesticated, and that subsequent dog-wolf hybridisation contributed modestly to the dog gene pool.

Origins of domestic dog in Southern East Asia is supported by analysis of Y-chromosome DNA

PubMed Central

Ding, Z-L; Oskarsson, M; Ardalan, A; Angleby, H; Dahlgren, L-G; Tepeli, C; Kirkness, E; Savolainen, P; Zhang, Y-P

2012-01-01

Global mitochondrial DNA (mtDNA) data indicates that the dog originates from domestication of wolf in Asia South of Yangtze River (ASY), with minor genetic contributions from dog–wolf hybridisation elsewhere. Archaeological data and autosomal single nucleotide polymorphism data have instead suggested that dogs originate from Europe and/or South West Asia but, because these datasets lack data from ASY, evidence pointing to ASY may have been overlooked. Analyses of additional markers for global datasets, including ASY, are therefore necessary to test if mtDNA phylogeography reflects the actual dog history and not merely stochastic events or selection. Here, we analyse 14 437 bp of Y-chromosome DNA sequence in 151 dogs sampled worldwide. We found 28 haplotypes distributed in five haplogroups. Two haplogroups were universally shared and included three haplotypes carried by 46% of all dogs, but two other haplogroups were primarily restricted to East Asia. Highest genetic diversity and virtually complete phylogenetic coverage was found within ASY. The 151 dogs were estimated to originate from 13–24 wolf founders, but there was no indication of post-domestication dog–wolf hybridisations. Thus, Y-chromosome and mtDNA data give strikingly similar pictures of dog phylogeography, most importantly that roughly 50% of the gene pools are shared universally but only ASY has nearly the full range of genetic diversity, such that the gene pools in all other regions may derive from ASY. This corroborates that ASY was the principal, and possibly sole region of wolf domestication, that a large number of wolves were domesticated, and that subsequent dog–wolf hybridisation contributed modestly to the dog gene pool. PMID:22108628

Projected climate changes threaten ancient refugia of kelp forests in the North Atlantic.

PubMed

Assis, Jorge; Araújo, Miguel B; Serrão, Ester A

2018-01-01

Intraspecific genetic variability is critical for species adaptation and evolution and yet it is generally overlooked in projections of the biological consequences of climate change. We ask whether ongoing climate changes can cause the loss of important gene pools from North Atlantic relict kelp forests that persisted over glacial-interglacial cycles. We use ecological niche modelling to predict genetic diversity hotspots for eight species of large brown algae with different thermal tolerances (Arctic to warm temperate), estimated as regions of persistence throughout the Last Glacial Maximum (20,000 YBP), the warmer Mid-Holocene (6,000 YBP), and the present. Changes in the genetic diversity within ancient refugia were projected for the future (year 2100) under two contrasting climate change scenarios (RCP2.6 and RCP8.5). Models predicted distributions that matched empirical distributions in cross-validation, and identified distinct refugia at the low latitude ranges, which largely coincide among species with similar ecological niches. Transferred models into the future projected polewards expansions and substantial range losses in lower latitudes, where richer gene pools are expected (in Nova Scotia and Iberia for cold affinity species and Gibraltar, Alboran, and Morocco for warm-temperate species). These effects were projected for both scenarios but were intensified under the extreme RCP8.5 scenario, with the complete borealization (circum-Arctic colonization) of kelp forests, the redistribution of the biogeographical transitional zones of the North Atlantic, and the erosion of global gene pools across all species. As the geographic distribution of genetic variability is unknown for most marine species, our results represent a baseline for identification of locations potentially rich in unique phylogeographic lineages that are also climatic relics in threat of disappearing. © 2017 John Wiley & Sons Ltd.

A novel rhabdovirus, related to Merida virus, in field-collected mosquitoes from Anatolia and Thrace.

PubMed

Ergünay, Koray; Brinkmann, Annika; Litzba, Nadine; Günay, Filiz; Kar, Sırrı; Öter, Kerem; Örsten, Serra; Sarıkaya, Yasemen; Alten, Bülent; Nitsche, Andreas; Linton, Yvonne-Marie

2017-07-01

Next-generation sequencing technologies have significantly facilitated the discovery of novel viruses, and metagenomic surveillance of arthropods has enabled exploration of the diversity of novel or known viral agents. We have identified a novel rhabdovirus that is genetically related to the recently described Merida virus via next-generation sequencing in a mosquito pool from Thrace. The complete viral genome contains 11,798 nucleotides with 83% genome-wide nucleotide sequence similarity to Merida virus. Five major putative open reading frames that follow the canonical rhabdovirus genome organization were identified. A total of 1380 mosquitoes comprising 13 species, collected from Thrace and the Mediterranean and Aegean regions of Anatolia were screened for the novel virus using primers based on the N and L genes of the prototype genome. Eight positive pools (6.2%) exclusively comprised Culex pipiens sensu lato specimens originating from all study regions. Infections were observed in pools with female as well as male or mixed-sex individuals. The overall and Cx. pipiens-specific minimal infection rates were calculated to be 5.7 and 14.8, respectively. Sequencing of the PCR products revealed marked diversity within a portion of the N gene, with up to 4% divergence and distinct amino acid substitutions that were unrelated to the collection site. Phylogenetic analysis of the complete and partial viral polymerase (L gene) amino acid sequences placed the novel virus and Merida virus in a distinct group, indicating that these strains are closely related. The strain is tentatively named "Merida-like virus Turkey". Studies are underway to isolate and further explore the host range and distribution of this new strain.

Dose-response assessment for influenza A virus based on data sets of infection with its live attenuated reassortants.

PubMed

Watanabe, Toru; Bartrand, Timothy A; Omura, Tatsuo; Haas, Charles N

2012-03-01

Reported data sets on infection of volunteers challenged with wild-type influenza A virus at graded doses are few. Alternatively, we aimed at developing a dose-response assessment for this virus based on the data sets for its live attenuated reassortants. Eleven data sets for live attenuated reassortants that were fit to beta-Poisson and exponential dose-response models. Dose-response relationships for those reassortants were characterized by pooling analysis of the data sets with respect to virus subtype (H1N1 or H3N2), attenuation method (cold-adapted or avian-human gene reassortment), and human age (adults or children). Furthermore, by comparing the above data sets to a limited number of reported data sets for wild-type virus, we quantified the degree of attenuation of wild-type virus with gene reassortment and estimated its infectivity. As a result, dose-response relationships of all reassortants were best described by a beta-Poisson model. Virus subtype and human age were significant factors determining the dose-response relationship, whereas attenuation method affected only the relationship of H1N1 virus infection to adults. The data sets for H3N2 wild-type virus could be pooled with those for its reassortants on the assumption that the gene reassortment attenuates wild-type virus by at least 63 times and most likely 1,070 times. Considering this most likely degree of attenuation, 10% infectious dose of H3N2 wild-type virus for adults was estimated at 18 TCID50 (95% CI = 8.8-35 TCID50). The infectivity of wild-type H1N1 virus remains unknown as the data set pooling was unsuccessful. © 2011 Society for Risk Analysis.

Novel Rhizosphere Soil Alleles for the Enzyme 1-Aminocyclopropane-1-Carboxylate Deaminase Queried for Function with an In Vivo Competition Assay

PubMed Central

Jin, Zhao; Di Rienzi, Sara C.; Janzon, Anders; Werner, Jeff J.; Angenent, Largus T.; Dangl, Jeffrey L.; Fowler, Douglas M.

2015-01-01

Metagenomes derived from environmental microbiota encode a vast diversity of protein homologs. How this diversity impacts protein function can be explored through selection assays aimed to optimize function. While artificially generated gene sequence pools are typically used in selection assays, their usage may be limited because of technical or ethical reasons. Here, we investigate an alternative strategy, the use of soil microbial DNA as a starting point. We demonstrate this approach by optimizing the function of a widely occurring soil bacterial enzyme, 1-aminocyclopropane-1-carboxylate (ACC) deaminase. We identified a specific ACC deaminase domain region (ACCD-DR) that, when PCR amplified from the soil, produced a variant pool that we could swap into functional plasmids carrying ACC deaminase-encoding genes. Functional clones of ACC deaminase were selected for in a competition assay based on their capacity to provide nitrogen to Escherichia coli in vitro. The most successful ACCD-DR variants were identified after multiple rounds of selection by sequence analysis. We observed that previously identified essential active-site residues were fixed in the original unselected library and that additional residues went to fixation after selection. We identified a divergent essential residue whose presence hints at the possible use of alternative substrates and a cluster of neutral residues that did not influence ACCD performance. Using an artificial ACCD-DR variant library generated by DNA oligomer synthesis, we validated the same fixation patterns. Our study demonstrates that soil metagenomes are useful starting pools of protein-coding-gene diversity that can be utilized for protein optimization and functional characterization when synthetic libraries are not appropriate. PMID:26637602

[Structure of the gene pool of eastern Ukrainians from Y-chromosome haplogroups].

PubMed

Khar'kov, V N; Stepanov, V A; Borinskaia, S A; Kozhekbaeva, Zh M; Gusar, V A; Grechanina, E Ia; Puzyrev, V P; Khusnutdinova, E K; Iankovskiĭ, N K

2004-03-01

Y chromosomes from representative sample of Eastern Ukrainians (94 individuals) were analyzed for composition and frequencies of haplogroups, defined by 11 biallelic loci located in non-recombining part of the chromosome (SRY1532, YAP, 92R7, DYF155S2, 12f2, Tat, M9, M17, M25, M89, and M56). In the Ukrainian gene, pool six haplogroups were revealed: E, F (including G and I), J, N3, P, and R1a1. These haplogroups were earlier detected in a study of Y-chromosome diversity on the territory of Europe as a whole. The major haplogroup in the Ukrainian gene pool, haplogroup R1a1 (earlier designated HG3), accounted for about 44% of all Y chromosomes in the sample examined. This haplogroup is thought to mark the migration patterns of the early Indo-Europeans and is associated with the distribution of the Kurgan archaeological culture. The second major haplogroup is haplogroup F (21.3%), which is a combination of the lineages differing by the time of appearance. Haplogroup P found with the frequency of 9.6%, represents the genetic contribution of the population originating from the ancient autochthonous population of Europe. Haplogroups J and E (11.7 and 4.2%, respectively) mark the migration patterns of the Middle-Eastern agriculturists during the Neolithic. The presence of the N3 lineage (9.6%) is likely explained by a contribution of the assimilated Finno-Ugric tribes. The data on the composition and frequencies of Y-chromosome haplogroups in the sample studied substantially supplement the existing picture of the male lineage distribution in the Eastern Slav population.

Heterologous Expression of sahH Reveals That Biofilm Formation Is Autoinducer-2-independent in Streptococcus sanguinis but Is Associated with an Intact Activated Methionine Cycle*

PubMed Central

Redanz, Sylvio; Standar, Kerstin; Podbielski, Andreas; Kreikemeyer, Bernd

2012-01-01

Numerous studies have claimed deleterious effects of LuxS mutation on many bacterial phenotypes, including bacterial biofilm formation. Genetic complementation mostly restored the observed mutant phenotypes to WT levels, leading to the postulation that quorum sensing via a family of molecules generically termed autoinducer-2 (AI-2) is essential for many phenotypes. Because LuxS mutation has dual effects, this hypothesis needs to be investigated into the details for each bacterial species. In this study we used S. sanguinis SK36 as a model biofilm bacterium and employed physiological characterization and transcriptome approaches on WT and luxS-deficient strains, in combination with chemical, luxS, and sahH complementation experiments. SahH enables a direct conversion of SAH to homocysteine and thereby restores the activated methionine cycle in a luxS-negative background without formation of the AI-2 precursor 4,5-dihydroxy-2,3-pentanedione. With this strategy we were able to dissect the individual contribution of LuxS and AI-2 activity in detail. Our data revealed that S. sanguinis biofilm formation is independent from AI-2 substance pools and is rather supported by an intact activated methyl cycle. Of 216 differentially transcribed genes in the luxS mutant, 209 were restored by complementation with a gene encoding the S-adenosylhomocysteine hydrolase. Only nine genes, mainly involved in natural competence, were directly affected by the AI-2 quorum-sensing substance pool. Cumulatively, this suggested that biofilm formation in S. sanguinis is not under control of AI-2. Our study suggests that previously evaluated LuxS mutants in other species need to be revisited to resolve the precise contribution of AI-2 substance pools and the methionine pathways. PMID:22942290

Detection of rickettsiae in fleas and ticks from areas of Costa Rica with history of spotted fever group rickettsioses.

PubMed

Troyo, Adriana; Moreira-Soto, Rolando D; Calderon-Arguedas, Ólger; Mata-Somarribas, Carlos; Ortiz-Tello, Jusara; Barbieri, Amália R M; Avendaño, Adrián; Vargas-Castro, Luis E; Labruna, Marcelo B; Hun, Laya; Taylor, Lizeth

2016-10-01

Outbreaks of spotted fevers have been reported in Costa Rica since the 1950s, although vectors responsible for transmission to humans have not been directly identified. In this study, species of Rickettsia were detected in ectoparasites from Costa Rica, mostly from five study sites where cases of spotted fevers have been reported. Ticks and fleas were collected using drag cloths or directly from domestic and wild animals and pooled according to species, host, and location. Pools were analyzed initially by PCR to detect a fragment of Rickettsia spp. specific gltA gene, and those positive were confirmed by detection of htrA and/or ompA gene fragments. Partial sequences of the gltA gene were obtained, as well as at least one ompA and/or ompB partial sequence of each species. Rickettsia spp. were confirmed in 119 of 497 (23.9%) pools of ticks and fleas analyzed. Rickettsia rickettsii was identified in one nymph of Amblyomma mixtum and one nymph of Amblyomma varium. Other rickettsiae present were 'Candidatus Rickettsia amblyommii' in A. mixtum, Amblyomma ovale, Dermacentor nitens, and Rhipicephalus sanguineus s. l.; Rickettsia bellii in Amblyomma sabanerae; Rickettsia felis in Ctenocephalides felis; and Rickettsia sp. similar to 'Candidatus R. asemboensis' in C. felis, Pulex simulans, A. ovale, and Rhipicephalus microplus. Results show the presence of rickettsiae in vectors that may be responsible for transmission to humans in Costa Rica, and evidence suggests exposure to rickettsial organisms in the human environment may be common. This is the first study to report R. rickettsii in A. varium and in A. mixtum in Costa Rica. Copyright © 2016 Elsevier GmbH. All rights reserved.

From big data to diagnosis and prognosis: gene expression signatures in liver hepatocellular carcinoma.

PubMed

Yang, Hong; Zhang, Xin; Cai, Xiao-Yong; Wen, Dong-Yue; Ye, Zhi-Hua; Liang, Liang; Zhang, Lu; Wang, Han-Lin; Chen, Gang; Feng, Zhen-Bo

2017-01-01

Liver hepatocellular carcinoma accounts for the overwhelming majority of primary liver cancers and its belated diagnosis and poor prognosis call for novel biomarkers to be discovered, which, in the era of big data, innovative bioinformatics and computational techniques can prove to be highly helpful in. Big data aggregated from The Cancer Genome Atlas and Natural Language Processing were integrated to generate differentially expressed genes. Relevant signaling pathways of differentially expressed genes went through Gene Ontology enrichment analysis, Kyoto Encyclopedia of Genes and Genomes and Panther pathway enrichment analysis and protein-protein interaction network. The pathway ranked high in the enrichment analysis was further investigated, and selected genes with top priority were evaluated and assessed in terms of their diagnostic and prognostic values. A list of 389 genes was generated by overlapping genes from The Cancer Genome Atlas and Natural Language Processing. Three pathways demonstrated top priorities, and the one with specific associations with cancers, 'pathways in cancer,' was analyzed with its four highlighted genes, namely, BIRC5, E2F1, CCNE1, and CDKN2A, which were validated using Oncomine. The detection pool composed of the four genes presented satisfactory diagnostic power with an outstanding integrated AUC of 0.990 (95% CI [0.982-0.998], P  < 0.001, sensitivity: 96.0%, specificity: 96.5%). BIRC5 ( P  = 0.021) and CCNE1 ( P  = 0.027) were associated with poor prognosis, while CDKN2A ( P  = 0.066) and E2F1 ( P  = 0.088) demonstrated no statistically significant differences. The study illustrates liver hepatocellular carcinoma gene signatures, related pathways and networks from the perspective of big data, featuring the cancer-specific pathway with priority, 'pathways in cancer.' The detection pool of the four highlighted genes, namely BIRC5, E2F1, CCNE1 and CDKN2A, should be further investigated given its high evidence level of diagnosis, whereas the prognostic powers of BIRC5 and CCNE1 are equally attractive and worthy of attention.

A Self-Directed Method for Cell-Type Identification and Separation of Gene Expression Microarrays

PubMed Central

Zuckerman, Neta S.; Noam, Yair; Goldsmith, Andrea J.; Lee, Peter P.

2013-01-01

Gene expression analysis is generally performed on heterogeneous tissue samples consisting of multiple cell types. Current methods developed to separate heterogeneous gene expression rely on prior knowledge of the cell-type composition and/or signatures - these are not available in most public datasets. We present a novel method to identify the cell-type composition, signatures and proportions per sample without need for a-priori information. The method was successfully tested on controlled and semi-controlled datasets and performed as accurately as current methods that do require additional information. As such, this method enables the analysis of cell-type specific gene expression using existing large pools of publically available microarray datasets. PMID:23990767

Genes for psychosis and creativity: a promoter polymorphism of the neuregulin 1 gene is related to creativity in people with high intellectual achievement.

PubMed

Kéri, Szabolcs

2009-09-01

Why are genetic polymorphisms related to severe mental disorders retained in the gene pool of a population? A possible answer is that these genetic variations may have a positive impact on psychological functions. Here, I show that a biologically relevant polymorphism of the promoter region of the neuregulin 1 gene (SNP8NRG243177/rs6994992) is associated with creativity in people with high intellectual and academic performance. Intriguingly, the highest creative achievements and creative-thinking scores were found in people who carried the T/T genotype, which was previously shown to be related to psychosis risk and altered prefrontal activation.

Metagenomics and novel gene discovery

PubMed Central

Culligan, Eamonn P; Sleator, Roy D; Marchesi, Julian R; Hill, Colin

2014-01-01

Metagenomics provides a means of assessing the total genetic pool of all the microbes in a particular environment, in a culture-independent manner. It has revealed unprecedented diversity in microbial community composition, which is further reflected in the encoded functional diversity of the genomes, a large proportion of which consists of novel genes. Herein, we review both sequence-based and functional metagenomic methods to uncover novel genes and outline some of the associated problems of each type of approach, as well as potential solutions. Furthermore, we discuss the potential for metagenomic biotherapeutic discovery, with a particular focus on the human gut microbiome and finally, we outline how the discovery of novel genes may be used to create bioengineered probiotics. PMID:24317337

A strategy for rapid production and screening of yeast artificial chromosome libraries.

PubMed

Strauss, W M; Jaenisch, E; Jaenisch, R

1992-01-01

We describe methods for rapid production and screening of yeast artificial chromosome (YAC) libraries. Utilizing complete restriction digests of mouse genomic DNA for ligations in agarose, a 32,000-clone library was produced and screened in seven weeks. Screening was accomplished by subdividing primary transformation plates into pools of approximately 100 clones which were transferred into a master glycerol stock. These master stocks were used to inoculate liquid cultures to produce culture "pools," and ten pools of 100 clones were then combined to yield superpools of 1,000 clones. Both pool and superpool DNA was screened by polymerase chain reaction (PCR) and positive pools representing 100 clones were then plated on selective medium and screened by in situ hybridization. Screening by the two tiered PCR assay and by in situ hybridization was completed in 4-5 days. Utilizing this methodology we have isolated a 150 kb clone spanning the alpha 1(I) collagen (Col1a1) gene as well as 40 kb clones from the Hox-2 locus. To characterize the representation of the YAC library, the size distribution of genomic Sal I fragments was compared to that of clones picked at random from the library. The results demonstrate significant biasing of the cloned fragment distribution, resulting in a loss of representation for larger fragments.

[Investigation of toxigenic microcystis and microcystin pollution in Huayuankou Conservation Pool of Yellow River].

PubMed

Ban, Haiqun; Ba, Yue; Cheng, Xuemin; Wang, Guangzhou

2007-09-01

To investigate the contaminative, condition of planktonic algae, cyanobacteria, toxigenic microcystis and microcystin in Huayuankou Conservation Pool of Yellow River. From March 2005 to January 2006, water samples were taken 15 times by 2. 5L plastic sampler from Huayuankou Conservation Pool. The density of algae were counted by using blood cell counter. Phycocyanin intergenic spacer region (PC-IGS) and microcystin synthetase gene B (mcyB) of toxigenic microcystis was identified by the whole cell PCR. The concentration of microcystin was determined by ELISA kit. The positive results of PCR and ELISA were compared. Bacillariophyta, chlorophyta, cyanophyta (cyanobacteria) and euglenophyta were main algaes in Huayuankou conservation pool, and the dominant algae and cell density changed seasonally. Algae cell density and cyanobacteria cell density were higher in summer and autumn than in spring and winter. From July to November, 2005, PC-IGS and mcyB were detected positively by whole cell PCR. Microcystin was positively detected from July, the concentration of microcystin changed from 0 to 0.25microg/L, it was more higher in summer than other seasons. Toxigenic microcystis and microcystin could be detected in Huayuankou Conservation Pool of Yellow River. Whole cell PCR could be used to identify toxigenic microcystis.

DNA pooling: a comprehensive, multi-stage association analysis of ACSL6 and SIRT5 polymorphisms in schizophrenia.

PubMed

Chowdari, K V; Northup, A; Pless, L; Wood, J; Joo, Y H; Mirnics, K; Lewis, D A; Levitt, P R; Bacanu, S-A; Nimgaonkar, V L

2007-04-01

Many candidate gene association studies have evaluated incomplete, unrepresentative sets of single nucleotide polymorphisms (SNPs), producing non-significant results that are difficult to interpret. Using a rapid, efficient strategy designed to investigate all common SNPs, we tested associations between schizophrenia and two positional candidate genes: ACSL6 (Acyl-Coenzyme A synthetase long-chain family member 6) and SIRT5 (silent mating type information regulation 2 homologue 5). We initially evaluated the utility of DNA sequencing traces to estimate SNP allele frequencies in pooled DNA samples. The mean variances for the DNA sequencing estimates were acceptable and were comparable to other published methods (mean variance: 0.0008, range 0-0.0119). Using pooled DNA samples from cases with schizophrenia/schizoaffective disorder (Diagnostic and Statistical Manual of Mental Disorders edition IV criteria) and controls (n=200, each group), we next sequenced all exons, introns and flanking upstream/downstream sequences for ACSL6 and SIRT5. Among 69 identified SNPs, case-control allele frequency comparisons revealed nine suggestive associations (P<0.2). Each of these SNPs was next genotyped in the individual samples composing the pools. A suggestive association with rs 11743803 at ACSL6 remained (allele-wise P=0.02), with diminished evidence in an extended sample (448 cases, 554 controls, P=0.062). In conclusion, we propose a multi-stage method for comprehensive, rapid, efficient and economical genetic association analysis that enables simultaneous SNP detection and allele frequency estimation in large samples. This strategy may be particularly useful for research groups lacking access to high throughput genotyping facilities. Our analyses did not yield convincing evidence for associations of schizophrenia with ACSL6 or SIRT5.

Differential Gene Expression at Coral Settlement and Metamorphosis - A Subtractive Hybridization Study

PubMed Central

Hayward, David C.; Hetherington, Suzannah; Behm, Carolyn A.; Grasso, Lauretta C.; Forêt, Sylvain; Miller, David J.; Ball, Eldon E.

2011-01-01

Background A successful metamorphosis from a planktonic larva to a settled polyp, which under favorable conditions will establish a future colony, is critical for the survival of corals. However, in contrast to the situation in other animals, e.g., frogs and insects, little is known about the molecular basis of coral metamorphosis. We have begun to redress this situation with previous microarray studies, but there is still a great deal to learn. In the present paper we have utilized a different technology, subtractive hybridization, to characterize genes differentially expressed across this developmental transition and to compare the success of this method to microarray. Methodology/Principal Findings Suppressive subtractive hybridization (SSH) was used to identify two pools of transcripts from the coral, Acropora millepora. One is enriched for transcripts expressed at higher levels at the pre-settlement stage, and the other for transcripts expressed at higher levels at the post-settlement stage. Virtual northern blots were used to demonstrate the efficacy of the subtractive hybridization technique. Both pools contain transcripts coding for proteins in various functional classes but transcriptional regulatory proteins were represented more frequently in the post-settlement pool. Approximately 18% of the transcripts showed no significant similarity to any other sequence on the public databases. Transcripts of particular interest were further characterized by in situ hybridization, which showed that many are regulated spatially as well as temporally. Notably, many transcripts exhibit axially restricted expression patterns that correlate with the pool from which they were isolated. Several transcripts are expressed in patterns consistent with a role in calcification. Conclusions We have characterized over 200 transcripts that are differentially expressed between the planula larva and post-settlement polyp of the coral, Acropora millepora. Sequence, putative function, and in some cases temporal and spatial expression are reported. PMID:22065994

Pooled clustering of high-grade serous ovarian cancer gene expression leads to novel consensus subtypes associated with survival and surgical outcomes

PubMed Central

Wang, Chen; Armasu, Sebastian M.; Kalli, Kimberly R.; Maurer, Matthew J.; Heinzen, Ethan P.; Keeney, Gary L.; Cliby, William A.; Oberg, Ann L.; Kaufmann, Scott H.; Goode, Ellen L.

2017-01-01

Purpose Here we assess whether molecular subtyping identifies biological features of tumors that correlate with survival and surgical outcomes of high-grade serous ovarian cancer (HGSOC). Experimental Design Consensus clustering of pooled mRNA expression data from over 2,000 HGSOC cases was used to define molecular subtypes of HGSOCs. This de novo classification scheme was then applied to 381 Mayo Clinic HGSOC patients with detailed survival and surgical outcome information. Results Five molecular subtypes of HGSOC were identified. In the pooled dataset, three subtypes were largely concordant with prior studies describing proliferative, mesenchymal, and immunoreactive tumors (concordance > 70%), and the group of tumors previously described as differentiated type was segregated into two new types, one of which (anti-mesenchymal) had down-regulation of genes that were typically upregulated in the mesenchymal subtype. Molecular subtypes were significantly associated with overall survival (p<0.001) and with rate of optimal surgical debulking (≤1 cm, p=1.9E-4) in the pooled dataset. Among stage III-C or IV Mayo Clinic patients, molecular subtypes were also significantly associated with overall survival (p=0.001), as well as rate of complete surgical debulking (no residual disease; 16% in mesenchymal tumors comparing to >28% in other subtypes; p=0.02). Conclusions HGSOC tumors may be categorized into five molecular subtypes that associate with overall survival and the extent of residual disease following debulking surgery. Because mesenchymal tumors may have features that were associated with less favorable surgical outcome, molecular subtyping may have future utility in guiding neoadjuvant treatment decisions for women with HGSOC. PMID:28280090

A two-generation analysis of pollen pool genetic structure in flowering dogwood, Cornus florida (Cornaceae), in the Missouri Ozarks.

PubMed

Sork, Victoria L; Smouse, Peter E; Apsit, Victoria J; Dyer, Rodney J; Westfall, Robert D

2005-02-01

Anthropogenic landscape change can disrupt gene flow. As part of the Missouri Ozark Forest Ecosystem Project, this study examined whether silvicultural practices influence pollen-mediated gene movement in the insect-pollinated species, Cornus florida L., by comparing pollen pool structure (Φ(st)) among clear-cutting, selective cutting, and uncut regimes with the expectation that pollen movement should be least in the uncut regime. Using a sample of 1500 seedlings-10 each from 150 seed parents (43 in clear-cut, 74 in selective, and 33 in control sites) from six sites (each ranging from 266 to 527 ha), eight allozyme loci were analyzed with a pollen pool structure approach known as TwoGener (Smouse et al., 2001; Evolution 55: 260-271). This analysis revealed that pollen pool structure was less in clear-cut (Φ(C) = 0.090, P < 0.001) than in uncut areas (Φ(U) = 0.174, P < 0.001), with selective-cut intermediate (Φ(S) = 0.125, P < 0.001). These estimates translate into more effective pollen donors (N(ep)) in clear-cut (N(ep) = 5.56) and selective-cut (N(ep) = 4.00) areas than in uncut areas (N(ep) = 2.87). We demonstrate that Φ(C) ≤ Φ(S) ≤ Φ(U), with Φ(C) significantly smaller than Φ(U) (P < 0.034). The findings imply that, as long as a sufficiently large number of seed parents remain to provide adequate reproduction and to avoid a genetic bottleneck in the effective number of mothers, silvicultural management may not negatively affect the effective number of pollen parents, and hence subsequent genetic diversity in Cornus florida.

Regional gene pools for restoration, conservation, and genetic improvement of prairie grasses

USDA-ARS?s Scientific Manuscript database

Switchgrass (Panicum virgatum), big bluestem (Andropogon gerardii), and Indiangrass (Sorghastrum nutans) are native warm-season grasses that have been identified as potential cellulosic bioenergy feedstock crops due to their potential for high yields, perennial life habit, and nutrient use efficienc...

Chromonoodles: Jump into the Gene Pool

ERIC Educational Resources Information Center

Farrar, Jennifer; Barnhart, Kelsi

2011-01-01

Chromosomes, alleles, chromatids, genotype, phenotype, mitosis, meiosis, fertilization--this vocabulary can be overwhelming, confusing, and difficult for students to tie together. However, since these terms are commonplace in the high school biology classroom, and are the basis for understanding both DNA and heredity, students must understand…

Can We Communicate with Other Generations?

ERIC Educational Resources Information Center

Mellert, Robert B.

Communicating with future generations is not merely a question of "how" to communicate, but also of "what." Our major moral responsibilities to future generations concern the size of future populations, conservation of nonrenewable resources, diversity of the gene pool, and quality of the environment. To determine our…

Molecular characterization of Hepatozoon felis in Rhipicephalus sanguineus ticks infested on captive lions (Panthera leo).

PubMed

Bhusri, Benjaporn; Sariya, Ladawan; Mongkolphan, Chalisa; Suksai, Parut; Kaewchot, Supakarn; Changbunjong, Tanasak

2017-09-01

Hepatozoon spp. are protozoan parasites that infect a wide range of domestic and wild animals. The infection occurs by ingestion of an infected tick. This study was carried out to detect and characterize Hepatozoon spp. in ticks collected from captive lions ( Panthera leo ) in Thailand based on the partial 18S rRNA gene sequence. A total of 30 ticks were collected and identified as Rhipicephalus sanguineus . The collected ticks were separated into 10 tick pools by sex and life stages. Of the 10 tick pools examined, only one (10%) was found to be infected with the Hepatozoon species. Sequencing and phylogenetic analysis showed a clustering of the partial 18S rRNA gene sequence like that of H. felis from the GenBank database. This is the first report of H. felis in R. sanguineus ticks collected from captive lions in Thailand. Our results indicated that R. sanguineus may be a possible vector of feline Hepatozoon in Thailand.

Structural and functional diversity of microbial communities from a lake sediment contaminated with trenbolone, an endocrine-disrupting chemical.

PubMed

Radl, Viviane; Pritsch, Karin; Munch, Jean Charles; Schloter, Michael

2005-09-01

Effects of trenbolone (TBOH), a hormone used in cattle production, on the structure and function of microbial communities in a fresh water sediment from a lake in Southern Germany were studied in a microcosm experiment. The microbial community structure and the total gene pool of the sediment, assessed by 16S rRNA/rDNA and RAPD fingerprint analysis, respectively, were not significantly affected by TBOH. In contrast, the N-acetyl-glucosaminidase activity was almost 50% lower in TBOH treated samples (P<0.05). Also, the substrate utilization potential, measured using the BIOLOG system, was reduced after TBOH treatment. Interestingly, this potential did not recover at the end of the experiment, i.e. 19 days after the addition of the chemical. Repeated application of TBOH did not lead to an additional reduction in the substrate utilization potential. Overall results indicate that microbial community function was more sensitive to TBOH treatment than the community structure and the total gene pool.

Traditional Amerindian cultivators combine directional and ideotypic selection for sustainable management of cassava genetic diversity.

PubMed

Duputié, A; Massol, F; David, P; Haxaire, C; McKey, D

2009-06-01

Plant domestication provides striking examples of rapid evolution. Yet, it involves more complex processes than plain directional selection. Understanding the dynamics of diversity in traditional agroecosystems is both a fundamental goal in evolutionary biology and a practical goal in conservation. We studied how Amerindian cultivators maintain dynamically evolving gene pools in cassava. Farmers purposely maintain diversity in the form of phenotypically distinct, clonally propagated landraces. Landrace gene pools are continuously renewed by incorporating seedlings issued from spontaneous sexual reproduction. This poses two problems: agronomic quality may decrease because some seedlings are inbred, and landrace identity may be progressively lost through the incorporation of unrelated seedlings. Using a large microsatellite dataset, we show that farmers solve these problems by applying two kinds of selection: directional selection against inbred genotypes, and counter-selection of off-type phenotypes, which maintains high intra-landrace relatedness. Thus, cultural elements such as ideotypes (a representation of the ideal phenotype of a landrace) can shape genetic diversity.

Candida albicans and C. tropicalis Isolates from the Expired Breathes of Captive Dolphins and Their Environments in an Aquarium

PubMed Central

Takahashi, Hideo; Ueda, Keiichi; Itano, Eiko Nakagawa; Yanagisawa, Makio; Murata, Yoshiteru; Murata, Michiko; Yaguchi, Takashi; Murakami, Masaru; Kamei, Katsuhiko; Inomata, Tomo; Miyahara, Hirokazu; Sano, Ayako; Uchida, Senzo

2010-01-01

Genotypes of Candida spp. isolated from exhalation of 20 dolphins, 11 water samples from captive pools, and 24 oral cavities of staff members in an aquarium using a combination of multiple drug resistance 1 gene (MDR1) and the internal transcribed spacer (ITS) 1 5.8s-ITS 2 regions of ribosomal RNA gene (ITS rDNA) sequences were studied. The holding ratios of the dolphins, captive pools, and staff members were 70, 90, and 29%, respectively. Isolated pathogenic yeast species common to the dolphins and environments were Candida albicans and C. tropicalis. Identical genotypes in both Candida spp. based on the combination of MDR1 and ITSrDNA were found in some dolphins, between a dolphin and a staff, among dolphins and environments, and among environments. The results indicated the diffusion and exchange of pathogenic yeasts at the aquarium among dolphins and environments. The isolates at the aquarium showed higher rates of resistance to azole antifungals compared to reference isolates. PMID:21234394

Evaluating two-dimensional electrophoresis profiles of the protein phaseolin as markers of genetic differentiation and seed protein quality in common bean (Phaseolus vulgaris L.).

PubMed

López-Pedrouso, María; Bernal, Javier; Franco, Daniel; Zapata, Carlos

2014-07-23

High-resolution two-dimensional electrophoresis (2-DE) profiles of the protein phaseolin, the major seed storage protein of common bean, display great number of spots with differentially glycosylated and phosphorylated α- and β-type polypeptides. This work aims to test whether these complex profiles can be useful markers of genetic differentiation and seed protein quality in bean populations. The 2-DE phaseolin profile and the amino acid composition were examined in bean seeds from 18 domesticated and wild accessions belonging to the Mesoamerican and Andean gene pools. We found that proteomic distances based on 2-DE profiles were successful in identifying the accessions belonging to each gene pool and outliers distantly related. In addition, accessions identified as outliers from proteomic distances showed the highest levels of methionine content, an essential amino acid deficient in bean seeds. These findings suggest that 2-DE phaseolin profiles provide valuable information with potential of being used in common bean genetic improvement.

More to NAD+ than meets the eye: A regulator of metabolic pools and gene expression in Arabidopsis.

PubMed

Gakière, Bertrand; Fernie, Alisdair R; Pétriacq, Pierre

2018-01-05

Since its discovery more than a century ago, nicotinamide adenine dinucleotide (NAD + ) is recognised as a fascinating cornerstone of cellular metabolism. This ubiquitous energy cofactor plays vital roles in metabolic pathways and regulatory processes, a fact emphasised by the essentiality of a balanced NAD + metabolism for normal plant growth and development. Research on the role of NAD in plants has been predominantly carried out in the model plant Arabidopsis thaliana (Arabidopsis) with emphasis on the redox properties and cellular signalling functions of the metabolite. This review examines the current state of knowledge concerning how NAD can regulate both metabolic pools and gene expression in Arabidopsis. Particular focus is placed on recent studies highlighting the complexity of metabolic regulations involving NAD, more particularly in the mitochondrial compartment, and of signalling roles with respect to interactions with environmental fluctuations most specifically those involving plant immunity. Copyright © 2018 Elsevier Inc. All rights reserved.

Activity screening of carrier domains within nonribosomal peptide synthetases using complex substrate mixtures and large molecule mass spectrometry.

PubMed

Dorrestein, Pieter C; Blackhall, Jonathan; Straight, Paul D; Fischbach, Michael A; Garneau-Tsodikova, Sylvie; Edwards, Daniel J; McLaughlin, Shaun; Lin, Myat; Gerwick, William H; Kolter, Roberto; Walsh, Christopher T; Kelleher, Neil L

2006-02-14

For screening a pool of potential substrates that load carrier domains found in nonribosomal peptide synthetases, large molecule mass spectrometry is shown to be a new, unbiased assay. Combining the high resolving power of Fourier transform mass spectrometry with the ability of adenylation domains to select their own substrates, the mass change that takes place upon formation of a covalent intermediate thus identifies the substrate. This assay has an advantage over traditional radiochemical assays in that many substrates, the substrate pool, can be screened simultaneously. Using proteins on the nikkomycin, clorobiocin, coumermycin A1, yersiniabactin, pyochelin, and enterobactin biosynthetic pathways as proof of principle, preferred substrates are readily identified from substrate pools. Furthermore, this assay can be used to provide insight into the timing of tailoring events of biosynthetic pathways as demonstrated using the bromination reaction found on the jamaicamide biosynthetic pathway. Finally, this assay can provide insight into the role and function of orphan gene clusters for which the encoded natural product is unknown. This is demonstrated by identifying the substrates for two NRPS modules from the pksN and pksJ genes that are found on an orphan NRPS/PKS hybrid cluster from Bacillus subtilis. This new assay format is especially timely for activity screening in an era when new types of thiotemplate assembly lines that defy classification are being discovered at an accelerating rate.

Meta- and Pooled Analysis of GSTP1 Polymorphism and Lung Cancer: A HuGE-GSEC Review

PubMed Central

Chen, Wei; Smith, Daryn W.; Benhamou, Simone; Bouchardy, Christine; Butkiewicz, Dorota; Fong, Kwun M.; Gené, Manuel; Hirvonen, Ari; Kiyohara, Chikako; Larsen, Jill E.; Lin, Pinpin; Raaschou-Nielsen, Ole; Povey, Andrew C.; Reszka, Edyta; Risch, Angela; Schneider, Joachim; Schwartz, Ann G.; Sorensen, Mette; To-Figueras, Jordi; Tokudome, Shinkan; Pu, Yuepu; Yang, Ping; Wenzlaff, Angela S.; Wikman, Harriet; Taioli, Emanuela

2009-01-01

Lung cancer is the most common cancer worldwide. Polymorphisms in genes associated with carcinogen metabolism may modulate risk of disease. Glutathione S-transferase pi (GSTP1) detoxifies polycyclic aromatic hydrocarbons found in cigarette smoke and is the most highly expressed glutathione S-transferase in lung tissue. A polymorphism in the GSTP1 gene, an A-to-G transition in exon 5 (Ile105Val, 313A → 313G), results in lower activity among individuals who carry the valine allele. The authors present a meta- and a pooled analysis of case-control studies that examined the association between this polymorphism in GSTP1 and lung cancer risk (27 studies, 8,322 cases and 8,844 controls and 15 studies, 4,282 cases and 5,032 controls, respectively). Overall, the meta-analysis found no significant association between lung cancer risk and the GSTP1 exon 5 polymorphism. In the pooled analysis, there was an overall association (odds ratio = 1.11, 95% confidence interval: 1.03, 1.21) between lung cancer and carriage of the GSTP1 Val/Val or Ile/Val genotype compared with those carrying the Ile/Ile genotype. Increased risk varied by histologic type in Asians. There appears to be evidence for interaction between amount of smoking, the GSTP1 exon 5 polymorphism, and risk of lung cancer in whites. PMID:19240225

Molecular screening of blue mussels indicated high mid-summer prevalence of human genogroup II Noroviruses, including the pandemic "GII.4 2012" variants in UK coastal waters during 2013.

PubMed

Biswas, Subhajit; Jackson, Philippa; Shannon, Rebecca; Dulwich, Katherine; Sukla, Soumi; Dixon, Ronald A

This molecular study is the first report, to the best of our knowledge, on identification of norovirus, NoV GII.4 Sydney 2012 variants, from blue mussels collected from UK coastal waters. Blue mussels (three pooled samples from twelve mussels) collected during the 2013 summer months from UK coastal sites were screened by RT-PCR assays. PCR products of RdRP gene for noroviruses were purified, sequenced and subjected to phylogenetic analysis. All the samples tested positive for NoVs. Sequencing revealed that the NoV partial RdRP gene sequences from two pooled samples clustered with the pandemic "GII.4 Sydney variants" whilst the other pooled sample clustered with the NoV GII.2 variants. This molecular study indicated mussel contamination with pathogenic NoVs even during mid-summer in UK coastal waters which posed potential risk of NoV outbreaks irrespective of season. As the detection of Sydney 2012 NoV from our preliminary study of natural coastal mussels interestingly corroborated with NoV outbreaks in nearby areas during the same period, it emphasizes the importance of environmental surveillance work for forecast of high risk zones of NoV outbreaks. Copyright © 2017 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

Phleboviruses associated with sand flies in arid bio-geographical areas of Central Tunisia.

PubMed

Dachraoui, K; Fares, W; Bichaud, L; Barhoumi, W; Beier, J C; Derbali, M; Cherni, S; Lamballerie, X de; Chelbi, I; Charrel, R N; Zhioua, E

2016-06-01

An entomological investigation was carried out in 2014 at two sites located in Central Tunisia, one irrigated and another non-irrigated situated in arid bio-geographical areas. Sand flies of the subgenus Larroussius namely Phlebotomus perfiliewi, Phlebotomus perniciosus, and Phlebotomus longicuspis are the most abundant sand fly species in the irrigated site. However, in the non-irrigated site, Phlebotomus papatasi of the Phlebotomus genus is the most abundant species. A total of 3191 sand flies were collected and pooled with up to 30 specimens per pool based on sex, trapping location and collection date, were tested for the presence of phleboviruses by nested reverse transcriptase polymerase chain reaction in the polymerase gene and sequenced. Of a total of 117 pools, 4 were positive, yielding a minimum infection rate of sand flies with phleboviruses of 0.12%. Phylogenetic analysis performed using partial nucleotide and amino acid sequence in the polymerase gene showed that these phleboviruses belonged to four different clusters corresponding to Toscana virus (TOSV), Saddaguia virus (SADV), Sandfly Fever Sicilian Virus (SFSV) and Utique virus (UTIV). This study provides more evidence that the abundance of P. perfiliewi is associated with the development of irrigation in arid bio-geographical areas of Central Tunisia which may have led to the emergence of phleboviruses. We report the first detection of TOSV from sand flies collected from Central Tunisia. Copyright © 2016 Elsevier B.V. All rights reserved.

Use of a draft genome of coffee (Coffea arabica) to identify SNPs associated with caffeine content.

PubMed

Tran, Hue T M; Ramaraj, Thiruvarangan; Furtado, Agnelo; Lee, Leonard Slade; Henry, Robert J

2018-03-07

Arabica coffee (Coffea arabica) has a small gene pool limiting genetic improvement. Selection for caffeine content within this gene pool would be assisted by identification of the genes controlling this important trait. Sequencing of DNA bulks from 18 genotypes with extreme high- or low-caffeine content from a population of 232 genotypes was used to identify linked polymorphisms. To obtain a reference genome, a whole genome assembly of arabica coffee (variety K7) was achieved by sequencing using short read (Illumina) and long-read (PacBio) technology. Assembly was performed using a range of assembly tools resulting in 76 409 scaffolds with a scaffold N50 of 54 544 bp and a total scaffold length of 1448 Mb. Validation of the genome assembly using different tools showed high completeness of the genome. More than 99% of transcriptome sequences mapped to the C. arabica draft genome, and 89% of BUSCOs were present. The assembled genome annotated using AUGUSTUS yielded 99 829 gene models. Using the draft arabica genome as reference in mapping and variant calling allowed the detection of 1444 nonsynonymous single nucleotide polymorphisms (SNPs) associated with caffeine content. Based on Kyoto Encyclopaedia of Genes and Genomes pathway-based analysis, 65 caffeine-associated SNPs were discovered, among which 11 SNPs were associated with genes encoding enzymes involved in the conversion of substrates, which participate in the caffeine biosynthesis pathways. This analysis demonstrated the complex genetic control of this key trait in coffee. © 2018 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

AtLAZY1 is a signaling component required for gravitropism of the Arabidopsis thaliana inflorescence.

PubMed

Yoshihara, Takeshi; Spalding, Edgar P; Iino, Moritoshi

2013-04-01

The present study identified a family of six A. thaliana genes that share five limited regions of sequence similarity with LAZY1, a gene in Oryza sativa (rice) shown to participate in the early gravity signaling for shoot gravitropism. A T-DNA insertion into the Arabidopsis gene (At5g14090) most similar to LAZY1 increased the inflorescence branch angle to 81° from the wild type value of 42°. RNA interference lines and molecular rescue experiments confirmed the linkage between the branch-angle phenotype and the gene consequently named AtLAZY1. Time-resolved gravitropism measurements of atlazy1 hypocotyls and primary inflorescence stems showed a significantly reduced bending rate during the first hour of response. The subcellular localization of AtLAZY1 protein was investigated to determine if the nuclear localization predicted from the gene sequence was observable and important to its function in shoot gravity responses. AtLAZY1 fused to green fluorescent protein largely rescued the branch-angle phenotype of atlazy1, and was observed by confocal microscopy at the cell periphery and within the nucleus. Mutation of the nuclear localization signal prevented detectable levels of AtLAZY1 in the nucleus without affecting the ability of the gene to rescue the atlazy1 branch-angle phenotype. These results indicate that AtLAZY1 functions in gravity signaling during shoot gravitropism, being a functional ortholog of rice LAZY1. The nuclear pool of the protein appears to be unnecessary for this function, which instead relies on a pool that appears to reside at the cell periphery. © 2013 The Authors The Plant Journal © 2013 Blackwell Publishing Ltd.

Genome-wide analysis of trans-splicing in the nematode Pristionchus pacificus unravels conserved gene functions for germline and dauer development in divergent operons.

PubMed

Sinha, Amit; Langnick, Claudia; Sommer, Ralf J; Dieterich, Christoph

2014-09-01

Discovery of trans-splicing in multiple metazoan lineages led to the identification of operon-like gene organization in diverse organisms, including trypanosomes, tunicates, and nematodes, but the functional significance of such operons is not completely understood. To see whether the content or organization of operons serves similar roles across species, we experimentally defined operons in the nematode model Pristionchus pacificus. We performed affinity capture experiments on mRNA pools to specifically enrich for transcripts that are trans-spliced to either the SL1- or SL2-spliced leader, using spliced leader-specific probes. We obtained distinct trans-splicing patterns from the analysis of three mRNA pools (total mRNA, SL1 and SL2 fraction) by RNA-seq. This information was combined with a genome-wide analysis of gene orientation and spacing. We could confirm 2219 operons by RNA-seq data out of 6709 candidate operons, which were predicted by sequence information alone. Our gene order comparison of the Caenorhabditis elegans and P. pacificus genomes shows major changes in operon organization in the two species. Notably, only 128 out of 1288 operons in C. elegans are conserved in P. pacificus. However, analysis of gene-expression profiles identified conserved functions such as an enrichment of germline-expressed genes and higher expression levels of operonic genes during recovery from dauer arrest in both species. These results provide support for the model that a necessity for increased transcriptional efficiency in the context of certain developmental processes could be a selective constraint for operon evolution in metazoans. Our method is generally applicable to other metazoans to see if similar functional constraints regulate gene organization into operons. © 2014 Sinha et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Prognostic utility of gene therapy with herpes simplex virus thymidine kinase for patients with high-grade malignant gliomas: a systematic review and meta analysis.

PubMed

Zhao, Fei; Tian, Jinhui; An, Lifeng; Yang, Kehu

2014-06-01

The aim of this study was to assess the effectiveness of adding viral vector-mediated gene therapy with herpes simplex virus thymidine kinase (HSV-tk) to standard treatment, in comparison with standard treatment alone to treat patients with high-grade gliomas (HGGs). A literature search of the databases PubMed, Embase, the Cochrane Library, Web of Science, and Chinese biomedicine was performed to identify eligible studies. Three randomized controlled trials (involving a total of 532 patients) were included in this systematic review. A meta-analysis of included studies demonstrated a significant increase in median survival time (MST) in patients who were treated with HSV-tk gene therapy (mean deviation 0.59, 95% CI: 0.41-0.76, p < 0.0001). The results of pooled analysis for different patient groups show that overall survival (OS) for all HGG patients was improved by adding gene therapy [hazard ratio (HR) = 0.91, 95% CI: 0.74-1.13, p = 0.42], while a different result was seen for glioblastoma multiforme (GBM) patients (HR = 1.06, 95% CI: 0.80-1.41, p = 0.70). Furthermore, the combined results for tumor progression implied that standard therapy was superior to gene therapy [odds ratio (OR) = 1.31, p = 0.09]; yet differences in HR and OR between experimental groups and control groups had no statistical significance (p > 0.05). Based on the best available evidence, it appears that adding gene therapy with HSV-tk has some effect in treating HGG patients, especially with respect to MST. However, neither the pooled analysis of OS, nor the combined analysis of tumor progress indicates any significant advantage to adding gene therapy compared with standard treatment alone. More prospective studies are needed to draw solid conclusions about whether gene therapy has significant prognostic advantage.

Tissue-Specific Effects of Reduced β-catenin Expression on Adenomatous Polyposis Coli Mutation-Instigated Tumorigenesis in Mouse Colon and Ovarian Epithelium

PubMed Central

Feng, Ying; Sakamoto, Naoya; Wu, Rong; Liu, Jie-yu; Wiese, Alexandra; Green, Maranne E.; Green, Megan; Akyol, Aytekin; Roy, Badal C.; Zhai, Yali; Cho, Kathleen R.; Fearon, Eric R.

2015-01-01

Adenomatous polyposis coli (APC) inactivating mutations are present in most human colorectal cancers and some other cancers. The APC protein regulates the β-catenin protein pool that functions as a co-activator of T cell factor (TCF)-regulated transcription in Wnt pathway signaling. We studied effects of reduced dosage of the Ctnnb1 gene encoding β-catenin in Apc-mutation-induced colon and ovarian mouse tumorigenesis and cell culture models. Concurrent somatic inactivation of one Ctnnb1 allele, dramatically inhibited Apc mutation-induced colon polyposis and greatly extended Apc-mutant mouse survival. Ctnnb1 hemizygous dose markedly inhibited increases in β-catenin levels in the cytoplasm and nucleus following Apc inactivation in colon epithelium, with attenuated expression of key β-catenin/TCF-regulated target genes, including those encoding the EphB2/B3 receptors, the stem cell marker Lgr5, and Myc, leading to maintenance of crypt compartmentalization and restriction of stem and proliferating cells to the crypt base. A critical threshold for β-catenin levels in TCF-regulated transcription was uncovered for Apc mutation-induced effects in colon epithelium, along with evidence of a feed-forward role for β-catenin in Ctnnb1 gene expression and CTNNB1 transcription. The active β-catenin protein pool was highly sensitive to CTNNB1 transcript levels in colon cancer cells. In mouse ovarian endometrioid adenocarcinomas (OEAs) arising from Apc- and Pten-inactivation, while Ctnnb1 hemizygous dose affected β-catenin levels and some β-catenin/TCF target genes, Myc induction was retained and OEAs arose in a fashion akin to that seen with intact Ctnnb1 gene dose. Our findings indicate Ctnnb1 gene dose exerts tissue-specific differences in Apc mutation-instigated tumorigenesis. Differential expression of selected β-catenin/TCF-regulated genes, such as Myc, likely underlies context-dependent effects of Ctnnb1 gene dosage in tumorigenesis. PMID:26528816

Cloning of the Pseudomonas aeruginosa outer membrane porin protein P gene: evidence for a linked region of DNA homology.

PubMed Central

Siehnel, R J; Worobec, E A; Hancock, R E

1988-01-01

The gene encoding the outer membrane phosphate-selective porin protein P from Pseudomonas aeruginosa was cloned into Escherichia coli. The protein product was expressed and transported to the outer membrane of an E. coli phoE mutant and assembled into functional trimers. Expression of a product of the correct molecular weight was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot (immunoblot) analysis, using polyclonal antibodies to protein P monomer and trimer forms. Protein P trimers were partially purified from the E. coli clone and shown to form channels with the same conductance as those formed by protein P from P. aeruginosa. The location and orientation of the protein P-encoding (oprP) gene on the cloned DNA was identified by three methods: (i) mapping the insertion point of transposon Tn501 in a previously isolated P. aeruginosa protein P-deficient mutant; (ii) hybridization of restriction fragments from the cloned DNA to an oligonucleotide pool synthesized on the basis of the amino-terminal protein sequence of protein P; and (iii) fusion of a PstI fragment of the cloned DNA to the amino terminus of the beta-galactosidase gene of pUC8, producing a fusion protein that contained protein P-antigenic epitopes. Structural analysis of the cloned DNA and P. aeruginosa chromosomal DNA revealed the presence of two adjacent PstI fragments which cross-hybridized, suggesting a possible gene duplication. The P-related (PR) region hybridized to the oligonucleotide pool described above. When the PstI fragment which contained the PR region was fused to the beta-galactosidase gene of pUC8, a fusion protein was produced which reacted with a protein P-specific antiserum. However, the restriction endonuclease patterns of the PR region and the oprP gene differed significantly beyond the amino-terminal one-third of the two genes. Images PMID:2834340

Prophinder: a computational tool for prophage prediction in prokaryotic genomes.

PubMed

Lima-Mendez, Gipsi; Van Helden, Jacques; Toussaint, Ariane; Leplae, Raphaël

2008-03-15

Prophinder is a prophage prediction tool coupled with a prediction database, a web server and web service. Predicted prophages will help to fill the gaps in the current sparse phage sequence space, which should cover an estimated 100 million species. Systematic and reliable predictions will enable further studies of prophages contribution to the bacteriophage gene pool and to better understand gene shuffling between prophages and phages infecting the same host. Softare is available at http://aclame.ulb.ac.be/prophinder

The association between LEPR Q223R polymorphisms and breast cancer risk.

PubMed

Wang, Yadong; Yang, Haiyan; Gao, Huiyan; Wang, Haiyu

2015-05-01

Recently, we have read with great interest the article entitled "The association between polymorphisms in the leptin receptor (LEPR) gene and risk of breast cancer: a systematic review and pooled analysis" published online by Wang et al. (Breast Cancer Res Treat 136:231-239, 2012). This article suggests that the A allele of LEPR gene rs1137101 variant was low-penetrant risk factor for developing breast cancer. The result is encouraging. Nevertheless, several key issues are worth noticing.

Infection of rhesus macaques with a pool of simian immunodeficiency virus with the envelope genes from acute HIV-1 infections.

PubMed

Krebs, Kendall C; Tian, Meijuan; Asmal, Mohammed; Ling, Binhua; Nelson, Kenneth; Henry, Kenneth; Gibson, Richard; Li, Yuejin; Han, Weining; Shattock, Robin J; Veazey, Ronald S; Letvin, Norman; Arts, Eric J; Gao, Yong

2016-11-25

New simian-human immunodeficiency chimeric viruses with an HIV-1 env (SHIVenv) are critical for studies on HIV pathogenesis, vaccine development, and microbicide testing. Macaques are typically exposed to single CCR5-using SHIVenv which in most instances does not reflect the conditions during acute/early HIV infection (AHI) in humans. Instead of individual and serial testing new SHIV constructs, a pool of SHIVenv_B derived from 16 acute HIV-1 infections were constructed using a novel yeast-based SHIV cloning approach and then used to infect macaques. Even though none of the 16 SHIVenvs contained the recently reported mutations in env genes that could significantly enhance their binding affinity to RhCD4, one SHIVenv (i.e. SHIVenv_B3-PRB926) established infection in macaques exposed to this pool. AHI SHIVenv_B viruses as well as their HIVenv_B counterparts were analyzed for viral protein content, function, and fitness to identify possible difference between SHIVenv_B3-PRB926 and the other 15 SHIVenvs in the pool. All of the constructs produced SHIV or HIV chimeric with wild type levels of capsid (p27 and p24) content, reverse transcriptase (RT) activity, and expressed envelope glycoproteins that could bind to cell receptors CD4/CCR5 and mediate virus entry. HIV-1env_B chimeric viruses were propagated in susceptible cell lines but the 16 SHIVenv_B variants showed only limited replication in macaque peripheral blood mononuclear cells (PBMCs) and 174×CEM.CCR5 cell line. AHI chimeric viruses including HIVenv_B3 showed only minor variations in cell entry efficiency and kinetics as well as replicative fitness in human PBMCs. Reduced number of N-link glycosylation sites and slightly greater CCR5 affinity/avidity was the only distinguishing feature of env_B3 versus other AHI env's in the pool, a feature also observed in the HIV establishing new infections in humans. Despite the inability to propagate in primary cells and cell lines, a pool of 16 SHIVenv viruses could establish infection but only one virus, SHIVenv_B3 was isolated in the macaque and then shown to repeatedly infected macaques. This SHIVenv_B3 virus did not show any distinct phenotypic property from the other 15 SHIVenv viruses but did have the fewest N-linked glycosylation sites.

Selection of Suitable Internal Control Genes for Accurate Normalization of Real-Time Quantitative PCR Data of Buffalo (Bubalus bubalis) Blastocysts Produced by SCNT and IVF.

PubMed

Sood, Tanushri Jerath; Lagah, Swati Viviyan; Sharma, Ankita; Singla, Suresh Kumar; Mukesh, Manishi; Chauhan, Manmohan Singh; Manik, Radheysham; Palta, Prabhat

2017-10-01

We evaluated the suitability of 10 candidate internal control genes (ICGs), belonging to different functional classes, namely ACTB, EEF1A1, GAPDH, HPRT1, HMBS, RPS15, RPS18, RPS23, SDHA, and UBC for normalizing the real-time quantitative polymerase chain reaction (qPCR) data of blastocyst-stage buffalo embryos produced by hand-made cloning and in vitro fertilization (IVF). Total RNA was isolated from three pools, each of cloned and IVF blastocysts (n = 50/pool) for cDNA synthesis. Two different statistical algorithms geNorm and NormFinder were used for evaluating the stability of these genes. Based on gene stability measure (M value) and pairwise variation (V value), calculated by geNorm analysis, the most stable ICGs were RPS15, HPRT1, and ACTB for cloned blastocysts, HMBS, UBC, and HPRT1 for IVF blastocysts and RPS15, GAPDH, and HPRT1 for both the embryo types analyzed together. RPS18 was the least stable gene for both cloned and IVF blastocysts. Following NormFinder analysis, the order of stability was RPS15 = HPRT1>GAPDH for cloned blastocysts, HMBS = UBC>RPS23 for IVF blastocysts, and HPRT1>GAPDH>RPS15 for cloned and IVF blastocysts together. These results suggest that despite overlapping of the three most stable ICGs between cloned and IVF blastocysts, the panel of ICGs selected for normalization of qPCR data of cloned and IVF blastocyst-stage embryos should be different.

Genesis and genetic constellations of swine influenza viruses in Thailand.

PubMed

Poonsuk, Sukontip; Sangthong, Pradit; Petcharat, Nantawan; Lekcharoensuk, Porntippa

2013-12-27

Swine influenza virus (SIV) is one of the most important zoonotic agents and the origin of the most recent pandemic virus. Asia is considered to be the epicenter for genetic exchanging of influenza A viruses and Southeast Asia including Thailand serves as a reservoir to maintain the persistence of the viruses for seeding other regions. Therefore, searching for new reassortants in this area has been routinely required. Although SIVs in Thailand have been characterized, collective information regarding their genetic evolution and gene constellations is limited. In this study, whole genomes of 30 SIVs isolated during clinical target surveillance plus all available sequences of past and currently circulating Thai SIVs were genetically characterized based on their evolutionary relationships. All genetic pools of Thai SIVs are comprised of four lineages including classical swine (CS), Eurasian swine (EAs), Triple reassortants (TRIG) and Seasonal human (Shs). Out of 84 isolates, nine H1N1, six H3N2 and one H1N2 strains were identified. Gene constellations of SIVs in Thailand are highly complex resulting from multiple reassortments among concurrently circulating SIVs and temporally introduced foreign genes. Most strains contain gene segments from both EAs and CS lineages and appeared transiently. TRIG lineage has been recently introduced into Thai SIV gene pools. The existence of EAs and TRIG lineages in this region may increase rates of genetic exchange and diversity while Southeast Asia is a persistent reservoir for influenza A viruses. Continual monitoring of SIV evolution in this region is crucial in searching for the next potential pandemic viruses. Copyright © 2013 Elsevier B.V. All rights reserved.

Pregnane X Receptor Polymorphisms and Risk of Inflammatory Bowel Disease: A Meta-Analysis.

PubMed

Guo, Xiaolan; Yan, Ming

2017-08-01

Pregnane X receptor (PXR) gene polymorphisms have been widely studied in terms of the association with inflammatory bowel disease (IBD), with inconsistent results. The present meta-analysis was performed to assess the association between PXR gene polymorphisms and the susceptibility of IBD, Crohn's disease (CD), and ulcerative colitis (UC). PubMed, Wanfang, and CNKI databases were searched for eligible studies before November 1, 2016. Pooled odds ratios (ORs) and 95% confidence intervals (95% CIs) were used to calculate the various genetic models using either a fixed-effect or a random-effect model. The heterogeneity of the included studies was examined with Cochran Q and I 2 statistics. Begg's rank correlation test and Egger's linear regression test were used to assess the publication bias. A total of six studies with 4248 cases and 3853 controls were included in this meta-analysis. Three PXR gene polymorphisms were evaluated: rs1523127, rs2276707, and rs6785049. Our analyses of rs1523127, rs2276707, and rs6785049 suggested that PXR gene polymorphism had no obvious influence on the risk of IBD in Caucasians. Subgroup analyses based on disease type showed similar results. Our meta-analysis revealed that PXR gene polymorphism may not be significantly associated with IBD susceptibility. However, the number of original studies was limited and further studies with large samples are needed to verify the results. PXR = pregnane X receptor, IBD = inflammatory bowel disease, CD = Crohn's disease, UC = ulcerative colitis, ORs = pooled odds ratios, 95% CIs = 95% confidence intervals, NOS = Newcastle-Ottawa scale, HWE = Hardy-Weinberg equilibrium.

Population Structure of Barley Landrace Populations and Gene-Flow with Modern Varieties

PubMed Central

Bellucci, Elisa; Bitocchi, Elena; Rau, Domenico; Nanni, Laura; Ferradini, Nicoletta; Giardini, Alessandro; Rodriguez, Monica; Attene, Giovanna; Papa, Roberto

2013-01-01

Landraces are heterogeneous plant varieties that are reproduced by farmers as populations that are subject to both artificial and natural selection. Landraces are distinguished by farmers due to their specific traits, and different farmers often grow different populations of the same landrace. We used simple sequence repeats (SSRs) to analyse 12 barley landrace populations from Sardinia from two collections spanning 10 years. We analysed the population structure, and compared the population diversity of the landraces that were collected at field level (population). We used a representative pool of barley varieties for diversity comparisons and to analyse the effects of gene flow from modern varieties. We found that the Sardinian landraces are a distinct gene pool from those of both two-row and six-row barley varieties. There is also a low, but significant, mean level and population-dependent level of introgression from the modern varieties into the Sardinian landraces. Moreover, we show that the Sardinian landraces have the same level of gene diversity as the representative sample of modern commercial varieties grown in Italy in the last decades, even within population level. Thus, these populations represent crucial sources of germplasm that will be useful for crop improvement and for population genomics studies and association mapping, to identify genes, loci and genome regions responsible for adaptive variations. Our data also suggest that landraces are a source of valuable germplasm for sustainable agriculture in the context of future climate change, and that in-situ conservation strategies based on farmer use can preserve the genetic identity of landraces while allowing adaptation to local environments. PMID:24386303

Pool-based genome-wide association study identified novel candidate regions on BTA9 and 14 for oleic acid percentage in Japanese Black cattle.

PubMed

Kawaguchi, Fuki; Kigoshi, Hiroto; Nakajima, Ayaka; Matsumoto, Yuta; Uemoto, Yoshinobu; Fukushima, Moriyuki; Yoshida, Emi; Iwamoto, Eiji; Akiyama, Takayuki; Kohama, Namiko; Kobayashi, Eiji; Honda, Takeshi; Oyama, Kenji; Mannen, Hideyuki; Sasazaki, Shinji

2018-05-17

Fatty acid composition is an important indicator of beef quality. The objective of this study was to search the potential candidate region for fatty acid composition. We performed pool-based genome-wide association studies (GWAS) for oleic acid percentage (C18:1) in a Japanese Black cattle population from the Hyogo prefecture. GWAS analysis revealed two novel candidate regions on BTA9 and BTA14. The most significant single nucleotide polymorphisms (SNPs) in each region were genotyped in a population (n = 899) to verify their effect on C18:1. Statistical analysis revealed that both SNPs were significantly associated with C18:1 (p = .0080 and .0003), validating the quantitative trait loci (QTLs) detected in GWAS. We subsequently selected VNN1 and LYPLA1 genes as candidate genes from each region on BTA9 and BTA14, respectively. We sequenced full-length coding sequence (CDS) of these genes in eight individuals and identified a nonsynonymous SNP T66M on VNN1 gene as a putative candidate polymorphism. The polymorphism was also significantly associated with C18:1, but the p value (p = .0162) was higher than the most significant SNP on BTA9, suggesting that it would not be responsible for the QTL. Although further investigation will be needed to determine the responsible gene and polymorphism, our findings would contribute to development of selective markers for fatty acid composition in the Japanese Black cattle of Hyogo. © 2018 Japanese Society of Animal Science.

Phenotypic assessments of peanut nested association mapping (NAM) populations

USDA-ARS?s Scientific Manuscript database

Nested association mapping (NAM) is a valuable innovation and multi-parental mapping population strategy in peanut genetics which increases the power to map quantitative trait loci and assists in extending the gene pool of elite peanut lines. In the peanut research community, two structured mapping ...

Breeding value of primary synthetic wheat genotypes for grain yield

USDA-ARS?s Scientific Manuscript database

To introduce new genetic diversity into the bread wheat gene pool from its progenitor, Aegilops tauschii (Coss.) Schmalh, 33 primary synthetic hexaploid wheat genotypes (SYN) were crossed to 20 spring bread wheat (BW) cultivars at the International Wheat and Maize Improvement Center. Modified single...

Discovering Protein-Coding Genes from the Environment: Time for the Eukaryotes?

PubMed

Marmeisse, Roland; Kellner, Harald; Fraissinet-Tachet, Laurence; Luis, Patricia

2017-09-01

Eukaryotic microorganisms from diverse environments encompass a large number of taxa, many of them still unknown to science. One strategy to mine these organisms for genes of biotechnological relevance is to use a pool of eukaryotic mRNA directly extracted from environmental samples. Recent reports demonstrate that the resulting metatranscriptomic cDNA libraries can be screened by expression in yeast for a wide range of genes and functions from many of the different eukaryotic taxa. In combination with novel emerging high-throughput technologies, we anticipate that this approach should contribute to exploring the functional diversity of the eukaryotic microbiota. Copyright © 2017 Elsevier Ltd. All rights reserved.

Molecular identification of Plasmodium spp. and blood meal sources of anophelines in environmental reserves on São Luís Island, state of Maranhão, Brazil.

PubMed

Figueiredo, Mayra Araguaia Pereira; Di Santi, Silvia Maria; Manrique, Wilson Gómez; Gonçalves, Luiz Ricardo; André, Marcos Rogério; Machado, Rosangela Zacarias

2017-04-26

Considering the diversity of feeding habits that females of some species of anophelines present, it is important to understand which vertebrates are part of blood food sources and how important is the role of each in the ecoepidemiology of malaria. There are many vector species for Plasmodium spp. in the State of Maranhão, Brazil. In São Luís Island, Anopheles aquasalis is the main vector for human malaria; this species is abundant in areas with primates that are positive for Plasmodium. Anopheles aquasalis has natural exophilic and zoophilic feeding behavior, but in cases of high density and absence of animals, presents quite varied behavior, and feeds on human blood. In this context, the objective of the present study was to identify Plasmodium spp. and the blood meal sources of anophelines in two environmental reserves on São Luís Island, state of Maranhão, using molecular methods. Between June and July 2013, female anophelines were collected in the Sítio Aguahy Private Reserve, in the municipality of São José de Ribamar, and in the Sítio Mangalho Reserve, located within the Maracanã Environmental Protection Area, in the municipality of São Luís. CDC-type light traps, Shannon traps and protected human bait were used during three consecutive hours in peridomestic and wooded areas. Pools of anophelines were formed using mosquitoes of the same species that had been caught at the same site on the same date. A genus-specific amplification protocol based on the 18S rRNA gene was used for qPCR and cPCR. A total of 416 anophelines were collected, of the following species: An. aquasalis (399), An. mediopunctatus (3), An. shannoni (1), An. nuneztovari (sensu lato) (1), An. goeldii (1), An. evansae (2) and An. (Nyssorhynchus) sp. (9), comprising 54 pools. Two pools were positive for Plasmodium (2/54) based on the 18S rRNA gene. In the phylogenetic analysis using the maximum likelihood method, based on a 240 bp fragment of the 18S rRNA gene, it was found that the sequences of Plasmodium sp. amplified from pools of An. aquasalis (pool 2) and An. nuneztovari (s.l.) (pool 10) were phylogenetically related to a clade of P. falciparum isolates from India, and to a clade of Plasmodium sp. isolates from psittacines in Brazil, respectively. Cat, dog and human DNA were identified in the blood meals of the anophelines sampled. The species An. aquasalis was the most abundant anopheline species in São Luís Island. Plasmodium spp. DNA was detected, thus confirming the importance of this species as the main vector on São Luís Island, Brazil. In addition, the presence of An. nuneztovari (s.l.) with DNA positive for Plasmodium spp. confirms its importance as a secondary vector.

Lunar Gene Bank For Endangered Species

NASA Astrophysics Data System (ADS)

Swain, R.; Behera, D.; Sahoo, P. K.; Swain, S. K.; Sasmal, A.

2012-09-01

Before the dawn of the 22nd century, we face the huge risk of losing our genetic heritage accumulated during aeons of evolution. The losses include hundreds of vertebrates, hundreds of thousands of plants and over a million insect species. The gene pools of many human ethnic groups are also threatened. As we have observed, adequate conservation of habitat is unfeasible and active breeding programs cover only a handful of the many thousand species threatened. Against such indispensable losses scientists are starting cryopreservation of germplasms by creation of gene banks. I propose to construct a cDNA library based gene bank for endangered species in the permanently shadowed polar lunar craters that would provide immunity from both natural disadvantages and humanitarian intrusions [4].

Vaccinium Species of Section Hemimyrtillus: Their value to cultivated blueberry and approaches to utilization

USDA-ARS?s Scientific Manuscript database

Section Hemimyrtillus represents species that are part of the tertiary gene pool of Vaccinium. Two species of Section Hemimyrtillus, native to the Portuguese islands of Madeira (V. padifolium Smith), and the Azores (V. cylindraceum Smith) have features of notable value to conventional blueberry deve...

The Gene Pool Concept Applied to Crop Wild Relatives: An Evolutionary Perspective

USDA-ARS?s Scientific Manuscript database

Crop wild relatives (CWR) can provide important resources for the genetic improvement of cultivated species. Because crops are often closely related to many wild species, and because exploration of CWR for useful traits can take many years and substantial resources, the categorization of CWR based o...

Genetic diversity for wheat improvement as a conduit to food security

USDA-ARS?s Scientific Manuscript database

Genetic diversity is paramount for any crops genetic improvement and this resides in three gene pools of the Triticeae for wheat. Access to the diversity and its exploitation is based upon genetic distance of the species relatives from the wheat genomes. Apart from the conventional genetic base fo...

A Phaseolus vulgaris diversity panel for Andean bean improvement

USDA-ARS?s Scientific Manuscript database

Dry beans (Phaseolus vulgaris L.) of the Andean gene pool, including red mottled, kidney, cranberry, and yellow seed types are important in Africa and in the Americas. Andean dry bean breeding gains have lagged behind those of Mesoamerican beans. These differences may be due to a narrower genetic b...

Molecular confirmation of Gossypium hirsutum chromosome substitution lines

USDA-ARS?s Scientific Manuscript database

The primary gene pool for tetraploid cotton species includes G. hirsutum L., as well as the other four 2n=52 species of Gossypium (G. barbadense, G. mustellinum, G. tomentosum and G. darwinii). To help overcome barriers to effective introgression, we have developed a number of alien chromosome subst...

Molecular Characterization of Cultivated Pawpaw (Asimina triloba) Using RAPD Markers

Treesearch

Hongwen Huang; Desmond R. Layne; Thomas L. Kubisiak

2003-01-01

Thirty-four extant pawpaw [Asimina triloba (L.) Dunal] cultivars and advanced selections representing a large portion of the gene pool of cultivated pawpaws were investigated using 71 randomly amplified polymorphic DNA (RAPD) markers to establish genetic identities and evaluate genetic relatedness. All 34 cultivated pawpaws were uniquely...

Dana-Farber Cancer Institute: Genome-wide shRNA Screens with DEMETER Inferred Gene Effects | Office of Cancer Genomics

Cancer.gov

In this study RNA interference (RNAi) screens were performed on 285 cell lines and combined with 216 lines previously screened, which were then analyzed together with DEMETER to discover genetic dependencies across the entire pool of cell lines. Read the abstract

Synthetic hexaploids: Harnessing species of the primary gene pool for wheat improvement

USDA-ARS?s Scientific Manuscript database

Incorporation of genetic diversity into elite wheat cultivars has long been recognized as a means of improving wheat productivity and securing the global wheat supply. Synthetic hexaploid wheat (SHW) recreated from its two progenitor species, the tetraploid, Triticum turgidum and its diploid wild r...

A Systems Biology Study in Tomato Fruit Reveals Correlations between the Ascorbate Pool and Genes Involved in Ribosome Biogenesis, Translation, and the Heat-Shock Response

PubMed Central

Stevens, Rebecca G.; Baldet, Pierre; Bouchet, Jean-Paul; Causse, Mathilde; Deborde, Catherine; Deschodt, Claire; Faurobert, Mireille; Garchery, Cécile; Garcia, Virginie; Gautier, Hélène; Gouble, Barbara; Maucourt, Mickaël; Moing, Annick; Page, David; Petit, Johann; Poëssel, Jean-Luc; Truffault, Vincent; Rothan, Christophe

2018-01-01

Changing the balance between ascorbate, monodehydroascorbate, and dehydroascorbate in plant cells by manipulating the activity of enzymes involved in ascorbate synthesis or recycling of oxidized and reduced forms leads to multiple phenotypes. A systems biology approach including network analysis of the transcriptome, proteome and metabolites of RNAi lines for ascorbate oxidase, monodehydroascorbate reductase and galactonolactone dehydrogenase has been carried out in orange fruit pericarp of tomato (Solanum lycopersicum). The transcriptome of the RNAi ascorbate oxidase lines is inversed compared to the monodehydroascorbate reductase and galactonolactone dehydrogenase lines. Differentially expressed genes are involved in ribosome biogenesis and translation. This transcriptome inversion is also seen in response to different stresses in Arabidopsis. The transcriptome response is not well correlated with the proteome which, with the metabolites, are correlated to the activity of the ascorbate redox enzymes—ascorbate oxidase and monodehydroascorbate reductase. Differentially accumulated proteins include metacaspase, protein disulphide isomerase, chaperone DnaK and carbonic anhydrase and the metabolites chlorogenic acid, dehydroascorbate and alanine. The hub genes identified from the network analysis are involved in signaling, the heat-shock response and ribosome biogenesis. The results from this study therefore reveal one or several putative signals from the ascorbate pool which modify the transcriptional response and elements downstream. PMID:29491875

Migration strategy affects avian influenza dynamics in mallards (Anas platyrhynchos).

USGS Publications Warehouse

Takekawa, John Y.; Hill, Nichola J.; Ackerman, Joshua T.; Herring, Garth; Hobson, Keith; Cardona, Carol J.; Runstadler, Jonathan; Boyce, Walter M.

2012-01-01

Studies of pathogen transmission typically overlook that wildlife hosts can include both migrant and resident populations when attempting to model circulation. Through the application of stable isotopes in flight feathers, we estimated the migration strategy of mallards (Anas platyrhynchos) occurring on California wintering grounds. Our study demonstrates that mallards- a principal host of avian influenza virus (AIV) in nature, contribute differently to virus gene flow depending on migration strategy. No difference in AIV prevalence was detected between resident (9.6%), intermediate-distance (9.6%) and long-distance migrants (7.4%). Viral diversity among the three groups was also comparable, possibly owing to viral pool mixing when birds converge at wetlands during winter. However, migrants and residents contributed differently to the virus gene pool at wintering wetlands. Migrants introduced virus from northern breeding grounds (Alaska and the NW Pacific Rim) into the wintering population, facilitating gene flow at continental scales, but circulation of imported virus appeared to be limited. In contrast, resident mallards acted as AIV reservoirs facilitating year-round circulation of limited subtypes (i.e. H5N2) at lower latitudes. This study supports a model of virus exchange in temperate regions driven by the convergence of wild birds with separate geographic origins and exposure histories.

Two showy traits, scent emission and pigmentation, are finely coregulated by the MYB transcription factor PH4 in petunia flowers.

PubMed

Cna'ani, Alon; Spitzer-Rimon, Ben; Ravid, Jasmin; Farhi, Moran; Masci, Tania; Aravena-Calvo, Javiera; Ovadis, Marianna; Vainstein, Alexander

2015-11-01

The mechanism underlying the emission of phenylpropanoid volatiles is poorly understood. Here, we reveal the involvement of PH4, a petunia MYB-R2R3 transcription factor previously studied for its role in vacuolar acidification, in floral volatile emission. We used the virus-induced gene silencing (VIGS) approach to knock down PH4 expression in petunia, measured volatile emission and internal pool sizes by GC-MS, and analyzed transcript abundances of scent-related phenylpropanoid genes in flowers. Silencing of PH4 resulted in a marked decrease in floral phenylpropanoid volatile emission, with a concurrent increase in internal pool levels. Expression of scent-related phenylpropanoid genes was not affected. To identify putative scent-related targets of PH4, we silenced PH5, a tonoplast-localized H(+) -ATPase that maintains vacuolar pH homeostasis. Suppression of PH5 did not yield the reduced-emission phenotype, suggesting that PH4 does not operate in the context of floral scent through regulation of vacuolar pH. We conclude that PH4 is a key floral regulator that integrates volatile production and emission processes and interconnects two essential floral traits - color and scent. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

Population genomic analysis uncovers environmental stress-driven selection and adaptation of Lentinula edodes population in China.

PubMed

Xiao, Yang; Cheng, Xuanjin; Liu, Jun; Li, Chuang; Nong, Wenyan; Bian, Yinbing; Cheung, Man Kit; Kwan, Hoi Shan

2016-11-10

The elucidation of genome-wide variations could help reveal aspects of divergence, domestication, and adaptation of edible mushrooms. Here, we resequenced the whole genomes of 39 wild and 21 cultivated strains of Chinese Lentinula edodes, the shiitake mushroom. We identified three distinct genetic groups in the Chinese L. edodes population with robust differentiation. Results of phylogenetic and population structure analyses suggest that the cultivated strains and most of the wild trains of L. edodes in China possess different gene pools and two outlier strains show signatures of hybridization between groups. Eighty-four candidate genes contributing to population divergence were detected in outlier analysis, 18 of which are involved in response to environmental stresses. Gene enrichment analysis of group-specific single nucleotide polymorphisms showed that the cultivated strains were genetically diversified in biological processes related to stress response. As the formation of fruiting bodies is a stress-response process, we postulate that environment factors, such as temperature, drove the population divergence of L. edodes in China by natural or artificial selection. We also found phenotypic variations between groups and identified some wild strains that have potential to diversify the genetic pool for improving agricultural traits of L. edodes cultivars in China.

Population genomic analysis uncovers environmental stress-driven selection and adaptation of Lentinula edodes population in China

PubMed Central

Xiao, Yang; Cheng, Xuanjin; Liu, Jun; Li, Chuang; Nong, Wenyan; Bian, Yinbing; Cheung, Man Kit; Kwan, Hoi Shan

2016-01-01

The elucidation of genome-wide variations could help reveal aspects of divergence, domestication, and adaptation of edible mushrooms. Here, we resequenced the whole genomes of 39 wild and 21 cultivated strains of Chinese Lentinula edodes, the shiitake mushroom. We identified three distinct genetic groups in the Chinese L. edodes population with robust differentiation. Results of phylogenetic and population structure analyses suggest that the cultivated strains and most of the wild trains of L. edodes in China possess different gene pools and two outlier strains show signatures of hybridization between groups. Eighty-four candidate genes contributing to population divergence were detected in outlier analysis, 18 of which are involved in response to environmental stresses. Gene enrichment analysis of group-specific single nucleotide polymorphisms showed that the cultivated strains were genetically diversified in biological processes related to stress response. As the formation of fruiting bodies is a stress-response process, we postulate that environment factors, such as temperature, drove the population divergence of L. edodes in China by natural or artificial selection. We also found phenotypic variations between groups and identified some wild strains that have potential to diversify the genetic pool for improving agricultural traits of L. edodes cultivars in China. PMID:27830835

Spatial and temporal dynamics of organohalide-respiring bacteria in a heterogeneous PCE-DNAPL source zone

NASA Astrophysics Data System (ADS)

Cápiro, Natalie L.; Löffler, Frank E.; Pennell, Kurt D.

2015-11-01

Effective treatment of sites contaminated with dense non-aqueous phase liquids (DNAPLs) requires detailed understanding of the microbial community responses to changes in source zone strength and architecture. Changes in the spatial and temporal distributions of the organohalide-respiring Dehalococcoides mccartyi (Dhc) strains and Geobacter lovleyi strain SZ (GeoSZ) were examined in a heterogeneous tetrachloroethene- (PCE-) DNAPL source zone within a two-dimensional laboratory-scale aquifer flow cell. As part of a combined remedy approach, flushing with 2.3 pore volumes (PVs) of 4% (w/w) solution of the nonionic, biodegradable surfactant Tween® 80 removed 55% of the initial contaminant mass, and resulted in a PCE-DNAPL distribution that contained 51% discrete ganglia and 49% pools (ganglia-to-pool ratio of 1.06). Subsequent bioaugmentation with the PCE-to-ethene-dechlorinating consortium BDI-SZ resulted in cis-1,2-dichloroethene (cis-DCE) formation after 1 PV (ca. 7 days), while vinyl chloride (VC) and ethene were detected 10 PVs after bioaugmentation. Maximum ethene yields (ca. 90 μM) within DNAPL pool and ganglia regions coincided with the detection of the vcrA reductive dehalogenase (RDase) gene that exceeded the Dhc 16S rRNA genes by 2.0 ± 1.3 and 4.0 ± 1.7 fold in the pool and ganglia regions, respectively. Dhc and GeoSZ cell abundance increased by up to 4 orders-of-magnitude after 28 PVs of steady-state operation, with 1 to 2 orders-of-magnitude increases observed in close proximity to residual PCE-DNAPL. These observations suggest the involvement of these dechlorinators the in observed PCE dissolution enhancements of up to 2.3 and 6.0-fold within pool and ganglia regions, respectively. Analysis of the solid and aqueous samples at the conclusion of the experiment revealed that the highest VC (≥ 155 μM) and ethene (≥ 65 μM) concentrations were measured in zones where Dhc and GeoSZ were predominately attached to the solids. These findings demonstrate dynamic responses of organohalide-respiring bacteria in a heterogeneous DNAPL source zone, and emphasize the influence of source zone architecture on bioremediation performance.

DNA methylation changes measured in pre-diagnostic peripheral blood samples are associated with smoking and lung cancer risk.

PubMed

Baglietto, Laura; Ponzi, Erica; Haycock, Philip; Hodge, Allison; Bianca Assumma, Manuela; Jung, Chol-Hee; Chung, Jessica; Fasanelli, Francesca; Guida, Florence; Campanella, Gianluca; Chadeau-Hyam, Marc; Grankvist, Kjell; Johansson, Mikael; Ala, Ugo; Provero, Paolo; Wong, Ee Ming; Joo, Jihoon; English, Dallas R; Kazmi, Nabila; Lund, Eiliv; Faltus, Christian; Kaaks, Rudolf; Risch, Angela; Barrdahl, Myrto; Sandanger, Torkjel M; Southey, Melissa C; Giles, Graham G; Johansson, Mattias; Vineis, Paolo; Polidoro, Silvia; Relton, Caroline L; Severi, Gianluca

2017-01-01

DNA methylation changes are associated with cigarette smoking. We used the Illumina Infinium HumanMethylation450 array to determine whether methylation in DNA from pre-diagnostic, peripheral blood samples is associated with lung cancer risk. We used a case-control study nested within the EPIC-Italy cohort and a study within the MCCS cohort as discovery sets (a total of 552 case-control pairs). We validated the top signals in 429 case-control pairs from another 3 studies. We identified six CpGs for which hypomethylation was associated with lung cancer risk: cg05575921 in the AHRR gene (p-value pooled  = 4 × 10 -17 ), cg03636183 in the F2RL3 gene (p-value pooled  = 2 × 10 - 13 ), cg21566642 and cg05951221 in 2q37.1 (p-value pooled  = 7 × 10 -16 and 1 × 10 -11 respectively), cg06126421 in 6p21.33 (p-value pooled  = 2 × 10 -15 ) and cg23387569 in 12q14.1 (p-value pooled  = 5 × 10 -7 ). For cg05951221 and cg23387569 the strength of association was virtually identical in never and current smokers. For all these CpGs except for cg23387569, the methylation levels were different across smoking categories in controls (p-values heterogeneity  ≤ 1.8 x10 - 7 ), were lowest for current smokers and increased with time since quitting for former smokers. We observed a gain in discrimination between cases and controls measured by the area under the ROC curve of at least 8% (p-values ≥ 0.003) in former smokers by adding methylation at the 6 CpGs into risk prediction models including smoking status and number of pack-years. Our findings provide convincing evidence that smoking and possibly other factors lead to DNA methylation changes measurable in peripheral blood that may improve prediction of lung cancer risk. © 2016 UICC.

Caught in the middle with multiple displacement amplification: the myth of pooling for avoiding multiple displacement amplification bias in a metagenome.

PubMed

Marine, Rachel; McCarren, Coleen; Vorrasane, Vansay; Nasko, Dan; Crowgey, Erin; Polson, Shawn W; Wommack, K Eric

2014-01-30

Shotgun metagenomics has become an important tool for investigating the ecology of microorganisms. Underlying these investigations is the assumption that metagenome sequence data accurately estimates the census of microbial populations. Multiple displacement amplification (MDA) of microbial community DNA is often used in cases where it is difficult to obtain enough DNA for sequencing; however, MDA can result in amplification biases that may impact subsequent estimates of population census from metagenome data. Some have posited that pooling replicate MDA reactions negates these biases and restores the accuracy of population analyses. This assumption has not been empirically tested. Using mock viral communities, we examined the influence of pooling on population-scale analyses. In pooled and single reaction MDA treatments, sequence coverage of viral populations was highly variable and coverage patterns across viral genomes were nearly identical, indicating that initial priming biases were reproducible and that pooling did not alleviate biases. In contrast, control unamplified sequence libraries showed relatively even coverage across phage genomes. MDA should be avoided for metagenomic investigations that require quantitative estimates of microbial taxa and gene functional groups. While MDA is an indispensable technique in applications such as single-cell genomics, amplification biases cannot be overcome by combining replicate MDA reactions. Alternative library preparation techniques should be utilized for quantitative microbial ecology studies utilizing metagenomic sequencing approaches.

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

Villiers, Etienne P. de, E-mail: e.villiers@cgiar.or; Gallardo, Carmina; Arias, Marisa

Viral molecular epidemiology has traditionally analyzed variation in single genes. Whole genome phylogenetic analysis of 123 concatenated genes from 11 ASFV genomes, including E75, a newly sequenced virulent isolate from Spain, identified two clusters. One contained South African isolates from ticks and warthog, suggesting derivation from a sylvatic transmission cycle. The second contained isolates from West Africa and the Iberian Peninsula. Two isolates, from Kenya and Malawi, were outliers. Of the nine genomes within the clusters, seven were within p72 genotype 1. The 11 genomes sequenced comprised only 5 of the 22 p72 genotypes. Comparison of synonymous and non-synonymous mutationsmore » at the genome level identified 20 genes subject to selection pressure for diversification. A novel gene of the E75 virus evolved by the fusion of two genes within the 360 multicopy family. Comparative genomics reveals high diversity within a limited sample of the ASFV viral gene pool.« less

Impact of selective logging on inbreeding and gene dispersal in an Amazonian tree population of Carapa guianensis Aubl.

PubMed

Cloutier, D; Kanashiro, M; Ciampi, A Y; Schoen, D J

2007-02-01

Selective logging may impact patterns of genetic diversity within populations of harvested forest tree species by increasing distances separating conspecific trees, and modifying physical and biotic features of the forest habitat. We measured levels of gene diversity, inbreeding, pollen dispersal and spatial genetic structure (SGS) of an Amazonian insect-pollinated Carapa guianensis population before and after commercial selective logging. Similar levels of gene diversity and allelic richness were found before and after logging in both the adult and the seed generations. Pre- and post-harvest outcrossing rates were high, and not significantly different from one another. We found no significant levels of biparental inbreeding either before or after logging. Low levels of pollen pool differentiation were found, and the pre- vs. post-harvest difference was not significant. Pollen dispersal distance estimates averaged between 75 m and 265 m before logging, and between 76 m and 268 m after logging, depending on the value of tree density and the dispersal model used. There were weak and similar levels of differentiation of allele frequencies in the adults and in the pollen pool, before and after logging occurred, as well as weak and similar pre- and post-harvest levels of SGS among adult trees. The large neighbourhood sizes estimated suggest high historical levels of gene flow. Overall our results indicate that there is no clear short-term genetic impact of selective logging on this population of C. guianensis.

Nonmetric cranial trait variation and population history of medieval East Slavic tribes.

PubMed

Movsesian, Alla A

2013-12-01

The population history of the East Slavs is complicated. There are still many unanswered questions relating to the origins and formation of the East Slavic gene pool. The aims of the current study were as follows: (1) to assess the degree of biological affinity in medieval East Slavic tribes and to test the hypothesis that East Slavic peoples have a common origin; (2) to show their genetic connections to the autochthonous populations of the northern part of Eastern Europe (Baltic and Finno-Ugric tribes); and (3) to identify a genetic continuity between the bearers of Chernyakhov culture and medieval Eastern Slavs. In this study, nonmetric cranial trait data for medieval East Slavic tribes and comparative samples from unrelated groups were examined. Analyzes of phenotypic differentiation were based on Nei's standard genetic distance and hierarchical GST statistics. The results obtained suggest that the genetic affinity of the East Slavic tribes is due not only to inter-tribal gene flow, but is, more importantly, a result of their common population history. Evidence of gene flow from the Baltic and Finno-Ugric groups was showed in the gene pool of Eastern Slavs, as was genetic continuity between medieval East Slavic tribes and the populations of the preceding Chernyakhov culture. These findings support a "generalizing" hypothesis of East Slavic origin, in which a Slavic community was formed in some particular ancestral area, and subsequently spread throughout Eastern Europe. Copyright © 2013 Wiley Periodicals, Inc.

The transcriptome of Spodoptera exigua larvae exposed to different types of microbes.

PubMed

Pascual, Laura; Jakubowska, Agata K; Blanca, Jose M; Cañizares, Joaquin; Ferré, Juan; Gloeckner, Gernot; Vogel, Heiko; Herrero, Salvador

2012-08-01

We have obtained and characterized the transcriptome of Spodoptera exigua larvae with special emphasis on pathogen-induced genes. In order to obtain a highly representative transcriptome, we have pooled RNA from diverse insect colonies, conditions and tissues. Sequenced cDNA included samples from 3 geographically different colonies. Enrichment of RNA from pathogen-related genes was accomplished by exposing larvae to different pathogenic and non-pathogenic microbial agents such as the bacteria Bacillus thuringiensis, Micrococcus luteus, and Escherichia coli, the yeast Saccharomyces cerevisiae, and the S. exigua nucleopolyhedrovirus (SeMNPV). In addition, to avoid the loss of tissue-specific genes we included cDNA from the midgut, fat body, hemocytes and integument derived from pathogen exposed insects. RNA obtained from the different types of samples was pooled, normalized and sequenced. Analysis of the sequences obtained using the Roche 454 FLX and Sanger methods has allowed the generation of the largest public set of ESTs from S. exigua, including a large group of immune genes, and the identification of an important number of SSR (simple sequence repeats) and SNVs (single nucleotide variants: SNPs and INDELs) with potential use as genetic markers. Moreover, data mining has allowed the discovery of novel RNA viruses with potential influence in the insect population dynamics and the larval interactions with the microbial pesticides that are currently in use for the biological control of this pest. Copyright © 2012 Elsevier Ltd. All rights reserved.

A genome-wide association study for fat-related traits computed by image analysis in Japanese Black cattle.

PubMed

Nakajima, Ayaka; Kawaguchi, Fuki; Uemoto, Yoshinobu; Fukushima, Moriyuki; Yoshida, Emi; Iwamoto, Eiji; Akiyama, Takayuki; Kohama, Namiko; Kobayashi, Eiji; Honda, Takeshi; Oyama, Kenji; Mannen, Hideyuki; Sasazaki, Shinji

2018-05-01

The objective of this study was to identify genomic regions associated with fat-related traits using a Japanese Black cattle population in Hyogo. From 1836 animals, those with high or low values were selected on the basis of corrected phenotype and then pooled into high and low groups (n = 100 each), respectively. DNA pool-based genome-wide association study (GWAS) was performed using Illumina BovineSNP50 BeadChip v2 with three replicate assays for each pooled sample. GWAS detected that two single nucleotide polymorphisms (SNPs) on BTA7 (ARS-BFGL-NGS-35463 and Hapmap23838-BTA-163815) and one SNP on BTA12 (ARS-BFGL-NGS-2915) significantly affected fat percentage (FAR). The significance of ARS-BFGL-NGS-35463 on BTA7 was confirmed by individual genotyping in all pooled samples. Moreover, association analysis between SNP and FAR in 803 Japanese Black cattle revealed a significant effect of SNP on FAR. Thus, further investigation of these regions is required to identify FAR-associated genes and mutations, which can lead to the development of DNA markers for marker-assisted selection for the genetic improvement of beef quality. © 2018 Japanese Society of Animal Science.

Screening for residual disease in pediatric burkitt lymphoma using consensus primer pools.

PubMed

Agsalda, Melissa; Kusao, Ian; Troelstrup, David; Shiramizu, Bruce

2009-01-01

Assessing molecular persistent or minimal residual disease (PD/MRD) in childhood Burkitt lymphoma (BL) is challenging because access to original tumor is usually needed to design patient-specific primers (PSPs). Because BL is characterized by rearranged immunoglobulin heavy chain (IgV(H)) genes, IgV(H) primer pools from IgV(H1)-IgV(H7) regions were tested to detect PD/MRD, thus eliminating the need for original tumor. The focus of the current study was to assess the feasibility of using IgV(H) primer pools to detect disease in clinical specimens. Fourteen children diagnosed with B-NHL had follow-up repository specimens available to assess PD/MRD. Of the 14 patients, 12 were PD/MRD negative after 2 months of therapy and remained in remission at the end of therapy; 2/14 patients were PD/MRD positive at 2-3 months and later relapsed. PSP-based assays from these 14 patients showed 100% concordance with the current assay. This feasibility study warrants further investigation to assess PD/MRD using IgV(H) primer pools, which could have clinical significance as a real-time assessment tool to monitor pediatric BL and possibly other B-cell non-Hodgkin lymphoma therapy.

IP{sub 3}-dependent intracellular Ca{sup 2+} release is required for cAMP-induced c-fos expression in hippocampal neurons

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

Zhang, Wenting; Tingare, Asmita; Ng, David Chi-Heng

2012-08-24

Highlights: Black-Right-Pointing-Pointer cAMP-induced c-fos expression in hippocampal neurons requires a submembraneous Ca{sup 2+} pool. Black-Right-Pointing-Pointer The submembraneous Ca{sup 2+} pool derives from intracellular ER stores. Black-Right-Pointing-Pointer Expression of IP{sub 3}-metabolizing enzymes inhibits cAMP-induced c-fos expression. Black-Right-Pointing-Pointer SRE-mediated and CRE-mediated gene expression is sensitive to IP{sub 3}-metabolizing enzymes. Black-Right-Pointing-Pointer Intracellular Ca{sup 2+} release is required for cAMP-induced nuclear translocation of TORC1. -- Abstract: Ca{sup 2+} and cAMP are widely used in concert by neurons to relay signals from the synapse to the nucleus, where synaptic activity modulates gene expression required for synaptic plasticity. Neurons utilize different transcriptional regulators to integrate informationmore » encoded in the spatiotemporal dynamics and magnitude of Ca{sup 2+} and cAMP signals, including some that are Ca{sup 2+}-responsive, some that are cAMP-responsive and some that detect coincident Ca{sup 2+} and cAMP signals. Because Ca{sup 2+} and cAMP can influence each other's amplitude and spatiotemporal characteristics, we investigated how cAMP acts to regulate gene expression when increases in intracellular Ca{sup 2+} are buffered. We show here that cAMP-mobilizing stimuli are unable to induce expression of the immediate early gene c-fos in hippocampal neurons in the presence of the intracellular Ca{sup 2+} buffer BAPTA-AM. Expression of enzymes that attenuate intracellular IP{sub 3} levels also inhibited cAMP-dependent c-fos induction. Synaptic activity induces c-fos transcription through two cis regulatory DNA elements - the CRE and the SRE. We show here that in response to cAMP both CRE-mediated and SRE-mediated induction of a luciferase reporter gene is attenuated by IP{sub 3} metabolizing enzymes. Furthermore, cAMP-induced nuclear translocation of the CREB coactivator TORC1 was inhibited by depletion of intracellular Ca{sup 2+} stores. Our data indicate that Ca{sup 2+} release from IP{sub 3}-sensitive pools is required for cAMP-induced transcription in hippocampal neurons.« less

Escherichia coli O26 in feedlot cattle: fecal prevalence, isolation, characterization, and effects of an E. coli O157 vaccine and a direct-fed microbial.

PubMed

Paddock, Zac D; Renter, David G; Cull, Charley A; Shi, Xiarong; Bai, Jianfa; Nagaraja, Tiruvoor G

2014-03-01

Escherichia coli O26 is second only to O157 in causing foodborne, Shiga toxin-producing E. coli (STEC) infections. Our objectives were to determine fecal prevalence and characteristics of E. coli O26 in commercial feedlot cattle (17,148) that were enrolled in a study to evaluate an E. coli O157:H7 siderophore receptor and porin (SRP(®)) vaccine (VAC) and a direct-fed microbial (DFM; 10(6) colony-forming units [CFU]/animal/day of Lactobacillus acidophilus and 10(9) CFU/animal/day of Propionibacterium freudenreichii). Cattle were randomly allocated to 40 pens within 10 complete blocks; pens were randomly assigned to control, VAC, DFM, or VAC+DFM treatments. Vaccine was administered on days 0 and 21, and DFM was fed throughout the study. Pen-floor fecal samples (30/pen) were collected weekly for the last 4 study weeks. Samples were enriched in E. coli broth and subjected to a multiplex polymerase chain reaction (PCR) designed to detect O26-specific wzx gene and four major virulence genes (stx1, stx2, eae, and ehxA) and to a culture-based procedure that involved immunomagnetic separation and plating on MacConkey agar. Ten presumptive E. coli colonies were randomly picked, pooled, and tested by the multiplex PCR. Pooled colonies positive for O26 serogroup were streaked on sorbose MacConkey agar, and 10 randomly picked colonies per sample were tested individually by the multiplex PCR. The overall prevalence of E. coli O26 was higher (p<0.001) by the culture-based method compared to the PCR assay (22.7 versus 10.5%). The interventions (VAC and or DFM) had no impact on fecal shedding of O26. Serogroup O26 was recovered in pure culture from 23.9% (260 of 1089) of O26 PCR-positive pooled colonies. Only 7 of the 260 isolates were positive for the stx gene and 90.1% of the isolates possessed an eaeβ gene that codes for intimin subtype β, but not the bfpA gene, which codes for bundle-forming pilus. Therefore, the majority of the O26 recovered from feedlot cattle feces was atypical enteropathogenic E. coli, and not STEC.

Screening for mutations in two exons of FANCG gene in Pakistani population.

PubMed

Aymun, Ujala; Iram, Saima; Aftab, Iram; Khaliq, Saba; Nadir, Ali; Nisar, Ahmed; Mohsin, Shahida

2017-06-01

Fanconi anemia is a rare autosomal recessive disorder of genetic instability. It is both molecularly and clinically, a heterogeneous disorder. Its incidence is 1 in 129,000 births and relatively high in some ethnic groups. Sixteen genes have been identified among them mutations in FANCG gene are most common after FANCA and FANCC gene mutations. To study mutations in exon 3 and 4 of FANCG gene in Pakistani population. Thirty five patients with positive Diepoxybutane test were included in the study. DNA was extracted and amplified for exons 3 and 4. Thereafter Sequencing was done and analyzed for the presence of mutations. No mutation was detected in exon 3 whereas a carrier of known mutation c.307+1 G>T was found in exon 4 of the FANCG gene. Absence of any mutation in exon 3 and only one heterozygous mutation in exon 4 of FANCG gene points to a different spectrum of FA gene pool in Pakistan that needs extensive research in this area.

Driver gene classification reveals a substantial overrepresentation of tumor suppressors among very large chromatin-regulating proteins.

PubMed

Waks, Zeev; Weissbrod, Omer; Carmeli, Boaz; Norel, Raquel; Utro, Filippo; Goldschmidt, Yaara

2016-12-23

Compiling a comprehensive list of cancer driver genes is imperative for oncology diagnostics and drug development. While driver genes are typically discovered by analysis of tumor genomes, infrequently mutated driver genes often evade detection due to limited sample sizes. Here, we address sample size limitations by integrating tumor genomics data with a wide spectrum of gene-specific properties to search for rare drivers, functionally classify them, and detect features characteristic of driver genes. We show that our approach, CAnceR geNe similarity-based Annotator and Finder (CARNAF), enables detection of potentially novel drivers that eluded over a dozen pan-cancer/multi-tumor type studies. In particular, feature analysis reveals a highly concentrated pool of known and putative tumor suppressors among the <1% of genes that encode very large, chromatin-regulating proteins. Thus, our study highlights the need for deeper characterization of very large, epigenetic regulators in the context of cancer causality.

Systematic Integration of Brain eQTL and GWAS Identifies ZNF323 as a Novel Schizophrenia Risk Gene and Suggests Recent Positive Selection Based on Compensatory Advantage on Pulmonary Function.

PubMed

Luo, Xiong-Jian; Mattheisen, Manuel; Li, Ming; Huang, Liang; Rietschel, Marcella; Børglum, Anders D; Als, Thomas D; van den Oord, Edwin J; Aberg, Karolina A; Mors, Ole; Mortensen, Preben Bo; Luo, Zhenwu; Degenhardt, Franziska; Cichon, Sven; Schulze, Thomas G; Nöthen, Markus M; Su, Bing; Zhao, Zhongming; Gan, Lin; Yao, Yong-Gang

2015-11-01

Genome-wide association studies have identified multiple risk variants and loci that show robust association with schizophrenia. Nevertheless, it remains unclear how these variants confer risk to schizophrenia. In addition, the driving force that maintains the schizophrenia risk variants in human gene pool is poorly understood. To investigate whether expression-associated genetic variants contribute to schizophrenia susceptibility, we systematically integrated brain expression quantitative trait loci and genome-wide association data of schizophrenia using Sherlock, a Bayesian statistical framework. Our analyses identified ZNF323 as a schizophrenia risk gene (P = 2.22×10(-6)). Subsequent analyses confirmed the association of the ZNF323 and its expression-associated single nucleotide polymorphism rs1150711 in independent samples (gene-expression: P = 1.40×10(-6); single-marker meta-analysis in the combined discovery and replication sample comprising 44123 individuals: P = 6.85×10(-10)). We found that the ZNF323 was significantly downregulated in hippocampus and frontal cortex of schizophrenia patients (P = .0038 and P = .0233, respectively). Evidence for pleiotropic effects was detected (association of rs1150711 with lung function and gene expression of ZNF323 in lung: P = 6.62×10(-5) and P = 9.00×10(-5), respectively) with the risk allele (T allele) for schizophrenia acting as protective allele for lung function. Subsequent population genetics analyses suggest that the risk allele (T) of rs1150711 might have undergone recent positive selection in human population. Our findings suggest that the ZNF323 is a schizophrenia susceptibility gene whose expression may influence schizophrenia risk. Our study also illustrates a possible mechanism for maintaining schizophrenia risk variants in the human gene pool. © The Author 2015. Published by Oxford University Press on behalf of the Maryland Psychiatric Research Center. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Patient-Level DNA Damage and Repair Pathway Profiles and Prognosis After Prostatectomy for High-Risk Prostate Cancer.

PubMed

Evans, Joseph R; Zhao, Shuang G; Chang, S Laura; Tomlins, Scott A; Erho, Nicholas; Sboner, Andrea; Schiewer, Matthew J; Spratt, Daniel E; Kothari, Vishal; Klein, Eric A; Den, Robert B; Dicker, Adam P; Karnes, R Jeffrey; Yu, Xiaochun; Nguyen, Paul L; Rubin, Mark A; de Bono, Johann; Knudsen, Karen E; Davicioni, Elai; Feng, Felix Y

2016-04-01

A substantial number of patients diagnosed with high-risk prostate cancer are at risk for metastatic progression after primary treatment. Better biomarkers are needed to identify patients at the highest risk to guide therapy intensification. To create a DNA damage and repair (DDR) pathway profiling method for use as a prognostic signature biomarker in high-risk prostate cancer. A cohort of 1090 patients with high-risk prostate cancer who underwent prostatectomy and were treated at 3 different academic institutions were divided into a training cohort (n = 545) and 3 pooled validation cohorts (n = 232, 130, and 183) assembled for case-control or case-cohort studies. Profiling of 9 DDR pathways using 17 gene sets for GSEA (Gene Set Enrichment Analysis) of high-density microarray gene expression data from formalin-fixed paraffin-embedded prostatectomy samples with median 10.3 years follow-up was performed. Prognostic signature development from DDR pathway profiles was studied, and DDR pathway gene mutation in published cohorts was analyzed. Biochemical recurrence-free, metastasis-free, and overall survival. Across the training cohort and pooled validation cohorts, 1090 men were studied; mean (SD) age at diagnosis was 65.3 (6.4) years. We found that there are distinct clusters of DDR pathways within the cohort, and DDR pathway enrichment is only weakly correlated with clinical variables such as age (Spearman ρ [ρ], range, -0.07 to 0.24), Gleason score (ρ, range, 0.03 to 0.20), prostate-specific antigen level (ρ, range, -0.07 to 0.10), while 13 of 17 DDR gene sets are strongly correlated with androgen receptor pathway enrichment (ρ, range, 0.33 to 0.82). In published cohorts, DDR pathway genes are rarely mutated. A DDR pathway profile prognostic signature built in the training cohort was significantly associated with biochemical recurrence-free, metastasis-free, and overall survival in the pooled validation cohorts independent of standard clinicopathological variables. The prognostic performance of the signature for metastasis-free survival appears to be stronger in the younger patients (HR, 1.67; 95% CI, 1.12-2.50) than in the older patients (HR, 0.77; 95% CI, 0.29-2.07) on multivariate Cox analysis. DNA damage and repair pathway profiling revealed patient-level variations and the DDR pathways are rarely affected by mutation. A DDR pathway signature showed strong prognostic performance with the long-term outcomes of metastasis-free and overall survival that may be useful for risk stratification of high-risk prostate cancer patients.

Pigsties near dwellings as a potential risk factor for the prevalence of Japanese encephalitis virus in adult in Shanxi, China.

PubMed

Ren, Xiaojie; Fu, Shihong; Dai, Peifang; Wang, Huanyu; Li, Yuanyuan; Li, Xiaolong; Lei, Wenwen; Gao, Xiaoyan; He, Ying; Lv, Zhi; Cheng, Jingxia; Wang, Guiqin; Liang, Guodong

2017-06-08

The increasing trend of adult cases of Japanese encephalitis (JE) in China, particularly in northern China, has become an important public health issue. We conducted an epidemiological investigation in the south of Shanxi Province to examine the relationships between mosquitoes, Japanese encephalitis virus (JEV), and adult JE cases. Mosquito specimens were collected from the courtyards of farmers' households and pig farms in Shanxi Province. Mosquitoes were pooled, homogenized, and centrifuged. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect mosquito-borne arbovirus genes in homogenates. Specimens positive for these genes were inoculated into the baby hamster kidney cell line (BHK-21) to isolate virus. Minimum infection rate was calculated and phylogenetic analyses were performed. A total of 7 943 mosquitoes belonging to six species in four genera were collected; Culex tritaeniorhynchus accounted for 73.08% (5 805/7 943), C. pipiens pallens for 24.75% (1 966/7 943), and the remaining 3% (104/ 7943) consisted of Anopheles sinensis, Aedes vexans, Ae. dorsalis, and Armigeres subalbatus. Sixteen pools were positive for JEV based on RT-PCR using JEV pre-membrane gene nested primers. Phylogenetic analyses showed that all JEVs belonged to genotype I; two pools were positive using Getah Virus (GETV) gene primers. In addition, one JEV strain (SXYC1523) was isolated from C. pipiens pallens specimens. These results indicate that the minimum infection rate of JEV in mosquito specimens collected from the courtyards of farmers' households with pigsties was 7.39/1 000; the rate for pig farms was 2.68/1 000; and the rate for farmers' courtyards without pigsties was zero. The high-prevalence regions of adult JE investigated in this study are still the natural epidemic focus of JEV. Having pigsties near dwellings is a potential risk factor contributing to the prevalence of adult JE. To prevent the occurrence of local adult JE cases, a recommendation was raised that, besides continuing to implement the Expanded Program on Immunization for children, the government should urge local farmers to cease raising pigs in their own courtyards to reduce the probability of infection with JEV.

Associations Between Genetic Variants in 19p13 and 19q13 Regions and Susceptibility to Alzheimer Disease: A Meta-Analysis.

PubMed

Bao, Jie; Wang, Xiao-jie; Mao, Zong-fu

2016-01-22

Alzheimer disease (AD) has become an epidemic within the growing elderly population and effective therapies of AD have not been discovered. Genetic factors accounted for over 70% of the incidence of AD and the disease-related polymorphisms are located on chromosome 19, which is one of several prominent chromosomes related to the development of AD. Many inconsistent associations between polymorphisms in ABCA7, CD33, and TOMM40 genes and the susceptibility to AD have been suggested by several independent studies. A comprehensive literature search for studies involving the association between gene polymorphisms and AD was performed, and we finally selected 3 genes (4 polymorphisms) for the meta-analysis: ABCA7 (rs3764650), CD33 (rs3865444), and TOMM40 (rs157580, rs2075650). A total of 25 articles investigating 3 genes (4 polymorphisms) were included in the meta-analysis. The pooled results of 4 polymorphisms were all significantly associated with the susceptibility to AD. The pooled effect of ABCA7 rs3764605 allele G was significantly associated with an increased the risk of AD (OR=1.20, 95% CI: 1.14-1.26, P value <0.001). Similarly, our evidence suggested that allele A of TOMM40 rs2075650 polymorphism was a risk factor for AD (OR=2.87, 95% CI: 2.46-3.34, P value <0.001). Alleles A of CD33 rs3865444 and A of TOMM40 rs157580 were both protective factors for AD onset (OR=0.94, 95% CI: 0.90-0.98, P value=0.003; OR=0.62, 95% CI: 0.57-0.66, P value <0.001). CONCLUSIONS" Results from the meta-analysis revealed that the pooled ABCA7 rs376465, CD33 rs3865444, TOMM40 rs157580, and rs2075650 variants were significantly associated with the susceptibility to AD. However, the association differed significantly between Asian and Caucasian groups for SNPs of CD33 rs3865444, TOMM40 rs157580, and rs2075650.

Lung cancer signature biomarkers: tissue specific semantic similarity based clustering of digital differential display (DDD) data.

PubMed

Srivastava, Mousami; Khurana, Pankaj; Sugadev, Ragumani

2012-11-02

The tissue-specific Unigene Sets derived from more than one million expressed sequence tags (ESTs) in the NCBI, GenBank database offers a platform for identifying significantly and differentially expressed tissue-specific genes by in-silico methods. Digital differential display (DDD) rapidly creates transcription profiles based on EST comparisons and numerically calculates, as a fraction of the pool of ESTs, the relative sequence abundance of known and novel genes. However, the process of identifying the most likely tissue for a specific disease in which to search for candidate genes from the pool of differentially expressed genes remains difficult. Therefore, we have used 'Gene Ontology semantic similarity score' to measure the GO similarity between gene products of lung tissue-specific candidate genes from control (normal) and disease (cancer) sets. This semantic similarity score matrix based on hierarchical clustering represents in the form of a dendrogram. The dendrogram cluster stability was assessed by multiple bootstrapping. Multiple bootstrapping also computes a p-value for each cluster and corrects the bias of the bootstrap probability. Subsequent hierarchical clustering by the multiple bootstrapping method (α = 0.95) identified seven clusters. The comparative, as well as subtractive, approach revealed a set of 38 biomarkers comprising four distinct lung cancer signature biomarker clusters (panel 1-4). Further gene enrichment analysis of the four panels revealed that each panel represents a set of lung cancer linked metastasis diagnostic biomarkers (panel 1), chemotherapy/drug resistance biomarkers (panel 2), hypoxia regulated biomarkers (panel 3) and lung extra cellular matrix biomarkers (panel 4). Expression analysis reveals that hypoxia induced lung cancer related biomarkers (panel 3), HIF and its modulating proteins (TGM2, CSNK1A1, CTNNA1, NAMPT/Visfatin, TNFRSF1A, ETS1, SRC-1, FN1, APLP2, DMBT1/SAG, AIB1 and AZIN1) are significantly down regulated. All down regulated genes in this panel were highly up regulated in most other types of cancers. These panels of proteins may represent signature biomarkers for lung cancer and will aid in lung cancer diagnosis and disease monitoring as well as in the prediction of responses to therapeutics.

Pivotal Role of Inosine Triphosphate Pyrophosphatase in Maintaining Genome Stability and the Prevention of Apoptosis in Human Cells

PubMed Central

Lopez-Bertoni, Hernando; Luo, Xu; Pavlov, Youri I.

2012-01-01

Pure nucleotide precursor pools are a prerequisite for high-fidelity DNA replication and the suppression of mutagenesis and carcinogenesis. ITPases are nucleoside triphosphate pyrophosphatases that clean the precursor pools of the non-canonical triphosphates of inosine and xanthine. The precise role of the human ITPase, encoded by the ITPA gene, is not clearly defined. ITPA is clinically important because a widespread polymorphism, 94C>A, leads to null ITPase activity in erythrocytes and is associated with an adverse reaction to thiopurine drugs. We studied the cellular function of ITPA in HeLa cells using the purine analog 6-N hydroxylaminopurine (HAP), whose triphosphate is also a substrate for ITPA. In this study, we demonstrate that ITPA knockdown sensitizes HeLa cells to HAP-induced DNA breaks and apoptosis. The HAP-induced DNA damage and cytotoxicity observed in ITPA knockdown cells are rescued by an overexpression of the yeast ITPase encoded by the HAM1 gene. We further show that ITPA knockdown results in elevated mutagenesis in response to HAP treatment. Our studies reveal the significance of ITPA in preventing base analog-induced apoptosis, DNA damage and mutagenesis in human cells. This implies that individuals with defective ITPase are predisposed to genome damage by impurities in nucleotide pools, which is drastically augmented by therapy with purine analogs. They are also at an elevated risk for degenerative diseases and cancer. PMID:22384212

A pooling-based approach to mapping genetic variants associated with DNA methylation

PubMed Central

Kaplow, Irene M.; MacIsaac, Julia L.; Mah, Sarah M.; McEwen, Lisa M.; Kobor, Michael S.; Fraser, Hunter B.

2015-01-01

DNA methylation is an epigenetic modification that plays a key role in gene regulation. Previous studies have investigated its genetic basis by mapping genetic variants that are associated with DNA methylation at specific sites, but these have been limited to microarrays that cover <2% of the genome and cannot account for allele-specific methylation (ASM). Other studies have performed whole-genome bisulfite sequencing on a few individuals, but these lack statistical power to identify variants associated with DNA methylation. We present a novel approach in which bisulfite-treated DNA from many individuals is sequenced together in a single pool, resulting in a truly genome-wide map of DNA methylation. Compared to methods that do not account for ASM, our approach increases statistical power to detect associations while sharply reducing cost, effort, and experimental variability. As a proof of concept, we generated deep sequencing data from a pool of 60 human cell lines; we evaluated almost twice as many CpGs as the largest microarray studies and identified more than 2000 genetic variants associated with DNA methylation. We found that these variants are highly enriched for associations with chromatin accessibility and CTCF binding but are less likely to be associated with traits indirectly linked to DNA, such as gene expression and disease phenotypes. In summary, our approach allows genome-wide mapping of genetic variants associated with DNA methylation in any tissue of any species, without the need for individual-level genotype or methylation data. PMID:25910490

A pooling-based approach to mapping genetic variants associated with DNA methylation

DOE PAGES

Kaplow, Irene M.; MacIsaac, Julia L.; Mah, Sarah M.; ...

2015-04-24

DNA methylation is an epigenetic modification that plays a key role in gene regulation. Previous studies have investigated its genetic basis by mapping genetic variants that are associated with DNA methylation at specific sites, but these have been limited to microarrays that cover <2% of the genome and cannot account for allele-specific methylation (ASM). Other studies have performed whole-genome bisulfite sequencing on a few individuals, but these lack statistical power to identify variants associated with DNA methylation. We present a novel approach in which bisulfite-treated DNA from many individuals is sequenced together in a single pool, resulting in a trulymore » genome-wide map of DNA methylation. Compared to methods that do not account for ASM, our approach increases statistical power to detect associations while sharply reducing cost, effort, and experimental variability. As a proof of concept, we generated deep sequencing data from a pool of 60 human cell lines; we evaluated almost twice as many CpGs as the largest microarray studies and identified more than 2000 genetic variants associated with DNA methylation. Here we found that these variants are highly enriched for associations with chromatin accessibility and CTCF binding but are less likely to be associated with traits indirectly linked to DNA, such as gene expression and disease phenotypes. In summary, our approach allows genome-wide mapping of genetic variants associated with DNA methylation in any tissue of any species, without the need for individual-level genotype or methylation data.« less

Isolation of Ganjam virus from ticks collected off domestic animals around Pune, Maharashtra, India.

PubMed

Joshi, M V; Geevarghese, G; Joshi, G D; Ghodke, Y S; Mourya, D T; Mishra, A C

2005-03-01

Studies on viruses of zoonotic importance in certain villages around Pune were undertaken between December 2000 and January 2002. A total of 1,138 adult ticks belonging to six different species were collected off domestic animals and processed for virus isolation. Six virus isolates were obtained. All six isolates were identified as Ganjam virus by Quick Complement Fixation test and reverse transcriptase-polymerase chain reaction using RNA nucleocapsid gene amplification. Five isolates were from the pools of adult Hemaphysalis intermedia ticks, and one isolate was from a pool of adult Rhipecephalus hemaphysaloides. This is the first report of isolation of Ganjam virus from Maharashtra state of India.

Targeted 'next-generation' sequencing in anophthalmia and microphthalmia patients confirms SOX2, OTX2 and FOXE3 mutations.

PubMed

Jimenez, Nelson Lopez; Flannick, Jason; Yahyavi, Mani; Li, Jiang; Bardakjian, Tanya; Tonkin, Leath; Schneider, Adele; Sherr, Elliott H; Slavotinek, Anne M

2011-12-28

Anophthalmia/microphthalmia (A/M) is caused by mutations in several different transcription factors, but mutations in each causative gene are relatively rare, emphasizing the need for a testing approach that screens multiple genes simultaneously. We used next-generation sequencing to screen 15 A/M patients for mutations in 9 pathogenic genes to evaluate this technology for screening in A/M. We used a pooled sequencing design, together with custom single nucleotide polymorphism (SNP) calling software. We verified predicted sequence alterations using Sanger sequencing. We verified three mutations - c.542delC in SOX2, resulting in p.Pro181Argfs*22, p.Glu105X in OTX2 and p.Cys240X in FOXE3. We found several novel sequence alterations and SNPs that were likely to be non-pathogenic - p.Glu42Lys in CRYBA4, p.Val201Met in FOXE3 and p.Asp291Asn in VSX2. Our analysis methodology gave one false positive result comprising a mutation in PAX6 (c.1268A > T, predicting p.X423LeuextX*15) that was not verified by Sanger sequencing. We also failed to detect one 20 base pair (bp) deletion and one 3 bp duplication in SOX2. Our results demonstrated the power of next-generation sequencing with pooled sample groups for the rapid screening of candidate genes for A/M as we were correctly able to identify disease-causing mutations. However, next-generation sequencing was less useful for small, intragenic deletions and duplications. We did not find mutations in 10/15 patients and conclude that there is a need for further gene discovery in A/M.

Targeted 'Next-Generation' sequencing in anophthalmia and microphthalmia patients confirms SOX2, OTX2 and FOXE3 mutations

PubMed Central

2011-01-01

Background Anophthalmia/microphthalmia (A/M) is caused by mutations in several different transcription factors, but mutations in each causative gene are relatively rare, emphasizing the need for a testing approach that screens multiple genes simultaneously. We used next-generation sequencing to screen 15 A/M patients for mutations in 9 pathogenic genes to evaluate this technology for screening in A/M. Methods We used a pooled sequencing design, together with custom single nucleotide polymorphism (SNP) calling software. We verified predicted sequence alterations using Sanger sequencing. Results We verified three mutations - c.542delC in SOX2, resulting in p.Pro181Argfs*22, p.Glu105X in OTX2 and p.Cys240X in FOXE3. We found several novel sequence alterations and SNPs that were likely to be non-pathogenic - p.Glu42Lys in CRYBA4, p.Val201Met in FOXE3 and p.Asp291Asn in VSX2. Our analysis methodology gave one false positive result comprising a mutation in PAX6 (c.1268A > T, predicting p.X423LeuextX*15) that was not verified by Sanger sequencing. We also failed to detect one 20 base pair (bp) deletion and one 3 bp duplication in SOX2. Conclusions Our results demonstrated the power of next-generation sequencing with pooled sample groups for the rapid screening of candidate genes for A/M as we were correctly able to identify disease-causing mutations. However, next-generation sequencing was less useful for small, intragenic deletions and duplications. We did not find mutations in 10/15 patients and conclude that there is a need for further gene discovery in A/M. PMID:22204637

Variants of the FADS1 FADS2 Gene Cluster, Blood Levels of Polyunsaturated Fatty Acids and Eczema in Children within the First 2 Years of Life

PubMed Central

Rzehak, Peter; Thijs, Carel; Standl, Marie; Mommers, Monique; Glaser, Claudia; Jansen, Eugène; Klopp, Norman; Koppelman, Gerard H.; Singmann, Paula; Postma, Dirkje S.; Sausenthaler, Stefanie; Dagnelie, Pieter C.; van den Brandt, Piet A.; Koletzko, Berthold; Heinrich, Joachim

2010-01-01

Background Association of genetic-variants in the FADS1-FADS2-gene-cluster with fatty-acid-composition in blood of adult-populations is well established. We analyze this genetic-association in two children-cohort-studies. In addition, the association between variants in the FADS-gene-cluster and blood-fatty-acid-composition with eczema was studied. Methods and Principal Findings Data of two population-based-birth-cohorts in the Netherlands and Germany (KOALA, LISA) were pooled (n = 879) and analyzed by (logistic) regression regarding the mutual influence of single-nucleotide-polymorphisms (SNPs) in the FADS-gene-cluster (rs174545, rs174546, rs174556, rs174561, rs3834458), on polyunsaturated fatty acids (PUFA) in blood and parent-reported eczema until the age of 2 years. All SNPs were highly significantly associated with all PUFAs except for alpha-linolenic-acid and eicosapentaenoic-acid, also after correction for multiple-testing. All tested SNPs showed associations with eczema in the LISA-study, but not in the KOALA-study. None of the PUFAs was significantly associated with eczema neither in the pooled nor in the analyses stratified by study-cohort. Conclusions and Significance PUFA-composition in young children's blood is under strong control of the FADS-gene-cluster. Inconsistent results were found for a link between these genetic-variants with eczema. PUFA in blood was not associated with eczema. Thus the hypothesis of an inflammatory-link between PUFA and eczema by the metabolic-pathway of LC-PUFAs as precursors for inflammatory prostaglandins and leukotrienes could not be confirmed by these data. PMID:20948998

Diagnostic accuracy of PCR for detecting ALK gene rearrangement in NSCLC patients: A systematic review and meta-analysis

PubMed Central

Zhang, Xia; Zhou, Jian-Guo; Wu, Hua-Lian; Ma, Hu; Jiang, Zhi-Xia

2017-01-01

Background Anaplastic lymphoma kinase (ALK) gene fusion has been reported in 3∼5% non-small cell lung carcinoma (NSCLC) patients, and polymerase chain reaction (PCR) is commonly used to detecting the gene status, but the diagnostic capacity of it is still controversial. A systematic review and meta-analysis was conducted to clarify the diagnostic accuracy of PCR for detecting ALK gene rearrangement in NSCLC patients. Results 18 articles were enrolled, which included 21 studies, involving 2800 samples from NSCLC patients. The overall pooled parameters were calculated: sensitivity was 92.4% [95% confidence interval (CI): 82.2%–97.0%], specificity was 97.8% [95% CI: 95.1%–99.0%], PLR was 41.51 [95% CI: 18.10–95.22], NLR was 0.08 [95% CI: 0.03–0.19], DOR was 535.72 [95% CI: 128.48–2233.79], AUROC was 0.99 [95% CI: 0.98–1.00]. Materials and Methods Relevant articles were searched from PubMed, EMBASE, Web of Science, Cochrane library, American Society of Clinical Oncology (ASCO), European Society for Medical Oncology (ESMO), China National Knowledge Infrastructure (CNKI), China Wan Fang databases and Chinese biomedical literature database (CBM). Diagnostic capacity of PCR test was assessed by the pooled sensitivity and specificity, positive likelihood ratio (PLR), negative likelihood ratio (NLR), diagnostic odds ratio (DOR), area under the summary receiver operating characteristic (AUROC). Conclusions Based on the results from this review, PCR has good diagnostic performance for detecting the ALK gene fusion in NSCLC patients. Moreover, due to the poor methodology quality of the enrolled trials, more well-designed multi-center trials should be performed. PMID:29088875

Fatty Acid Biosynthesis Pathways in Methylomicrobium buryatense 5G(B1).

PubMed

Demidenko, Aleksandr; Akberdin, Ilya R; Allemann, Marco; Allen, Eric E; Kalyuzhnaya, Marina G

2016-01-01

Methane utilization by methanotrophic bacteria is an attractive application for biotechnological conversion of natural or biogas into high-added-value products. Haloalcaliphilic methanotrophic bacteria belonging to the genus Methylomicrobium are among the most promising strains for methane-based biotechnology, providing easy and inexpensive cultivation, rapid growth, and the availability of established genetic tools. A number of methane bioconversions using these microbial cultures have been discussed, including the derivation of biodiesel, alkanes, and OMEGA-3 supplements. These compounds are derived from bacterial fatty acid pools. Here, we investigate fatty acid biosynthesis in Methylomicrobium buryatense 5G(B1) . Most of the genes homologous to typical Type II fatty acid biosynthesis pathways could be annotated by bioinformatics analyses, with the exception of fatty acid transport and regulatory elements. Different approaches for improving fatty acid accumulation were investigated. These studies indicated that both fatty acid degradation and acetyl- and malonyl-CoA levels are bottlenecks for higher level fatty acid production. The best strain generated in this study synthesizes 111 ± 2 mg/gDCW of extractable fatty acids, which is ~20% more than the original strain. A candidate gene for fatty acid biosynthesis regulation, farE , was identified and studied. Its deletion resulted in drastic changes to the fatty acid profile, leading to an increased pool of C18-fatty acid methyl ester. The FarE-regulon was further investigated by RNA-seq analysis of gene expression in farE -knockout mutants and farE -overexpressing strains. These gene profiles highlighted a novel set of enzymes and regulators involved in fatty acid biosynthesis. The gene expression and fatty acid profiles of the different farE -strains support the hypothesis that metabolic fluxes upstream of fatty acid biosynthesis restrict fatty acid production in the methanotroph.

Fatty acid biosynthesis pathways in Methylomicrobium buryatense 5G(B1)

DOE PAGES

Demidenko, Aleksandr; Akberdin, Ilya R.; Allemann, Marco; ...

2017-01-10

Methane utilization by methanotrophic bacteria is an attractive application for biotechnological conversion of natural or biogas into high-added-value products. Haloalcaliphilic methanotrophic bacteria belonging to the genus Methylomicrobium are among the most promising strains for methane-based biotechnology, providing easy and inexpensive cultivation, rapid growth, and the availability of established genetic tools. A number of methane bioconversions using these microbial cultures have been discussed, including the derivation of biodiesel, alkanes, and OMEGA-3 supplements. These compounds are derived from bacterial fatty acid pools. Here, we investigate fatty acid biosynthesis in Methylomicrobium buryatense 5G(B1). Most of the genes homologous to typical Type II fattymore » acid biosynthesis pathways could be annotated by bioinformatics analyses, with the exception of FA transport and regulatory elements. Different approaches for improving fatty acid accumulation were investigated. These studies indicated that both fatty acid degradation and acetyl- and malonyl-CoA levels are bottlenecks for higher level fatty acid production. The best strain generated in this study synthesizes 111 ± 2 mg/gDCW of extractable fatty acids, which is ~20% more than the original strain. A candidate gene for FA-biosynthesis regulation, farE, was identified and studied. Its deletion resulted in drastic changes to the FA profile, leading to an increased pool of C18-fatty acid methyl ester. The FarE-regulon was further investigated by RNA-seq analysis of gene expression in farE-knockout mutants and farE-overexpressing strains. These gene profiles highlighted a novel set of enzymes and regulators involved in fatty acid biosynthesis. As a result, the gene expression and fatty acid profiles of the different farE-strains support the hypothesis that metabolic fluxes upstream of fatty acid biosynthesis restrict fatty acid production in the methanotroph.« less

Fatty acid biosynthesis pathways in Methylomicrobium buryatense 5G(B1)

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

Demidenko, Aleksandr; Akberdin, Ilya R.; Allemann, Marco

Methane utilization by methanotrophic bacteria is an attractive application for biotechnological conversion of natural or biogas into high-added-value products. Haloalcaliphilic methanotrophic bacteria belonging to the genus Methylomicrobium are among the most promising strains for methane-based biotechnology, providing easy and inexpensive cultivation, rapid growth, and the availability of established genetic tools. A number of methane bioconversions using these microbial cultures have been discussed, including the derivation of biodiesel, alkanes, and OMEGA-3 supplements. These compounds are derived from bacterial fatty acid pools. Here, we investigate fatty acid biosynthesis in Methylomicrobium buryatense 5G(B1). Most of the genes homologous to typical Type II fattymore » acid biosynthesis pathways could be annotated by bioinformatics analyses, with the exception of FA transport and regulatory elements. Different approaches for improving fatty acid accumulation were investigated. These studies indicated that both fatty acid degradation and acetyl- and malonyl-CoA levels are bottlenecks for higher level fatty acid production. The best strain generated in this study synthesizes 111 ± 2 mg/gDCW of extractable fatty acids, which is ~20% more than the original strain. A candidate gene for FA-biosynthesis regulation, farE, was identified and studied. Its deletion resulted in drastic changes to the FA profile, leading to an increased pool of C18-fatty acid methyl ester. The FarE-regulon was further investigated by RNA-seq analysis of gene expression in farE-knockout mutants and farE-overexpressing strains. These gene profiles highlighted a novel set of enzymes and regulators involved in fatty acid biosynthesis. As a result, the gene expression and fatty acid profiles of the different farE-strains support the hypothesis that metabolic fluxes upstream of fatty acid biosynthesis restrict fatty acid production in the methanotroph.« less

A meta-analysis of association between glutathione S-transferase M1 gene polymorphism and Parkinson's disease susceptibility.

PubMed

Weikang, Chen; Jie, Li; Likang, Lan; Weiwen, Qiu; Liping, Lu

2016-01-01

The aim of this meta-analysis was to evaluate whether there was an association between glutathione S-transferase M1(GSTM1)gene polymorphism and Parkinson's disease (PD) susceptibility by pooling published data. We performed comprehensive electronic database search for articles published between February12,2015 and April30 2016. The published case-control or cohort studies related to GSTM1 gene polymorphism and Parkinson's disease susceptibility were screened, reviewed, and included in this meta-analysis. The correlation between GSTM1 gene polymorphism and PD susceptibility was expressed by odds ratio (OR) and its corresponding 95% confidence interval (95%CI). Publication bias was evaluated by Begg's funnel plot and Egger's line regression test. All analysis was done by stata11.0 software. After searching the PubMed, EMBASE, and CNKI databases, seventeen case-control studies with 3,538 PD and 5,180 controls were included in the final meta-analysis. The data was pooled by a fixed-effect model for lack of statistical heterogeneity across the studies; the results showed GSTM1 null expression can significant increase the susceptibility of PD (OR=1.11, 95% CI:1.01-1.21, P<0.05). Subgroup analysis indicated GSTM1 gene polymorphism was associated with PD susceptibility in the Caucasian ethnic group (OR=1.15, 95% CI:1.05-1.27, P<0.05) but not in the Asian ethnic group (OR=0.89, 95% CI:0.70-1.12, P>0.05). Begg's funnel plot and Egger's line regression test showed no significant publication bias. Based on the present evidence, GSTM1 null expression can significant increase the susceptibility of PD in persons of Caucasian ethnicity.

Allele-specific expression of the MAOA gene and X chromosome inactivation in in vitro produced bovine embryos.

PubMed

Ferreira, A R; Machado, G M; Diesel, T O; Carvalho, J O; Rumpf, R; Melo, E O; Dode, M A N; Franco, M M

2010-07-01

During embryogenesis, one of the two X chromosomes is inactivated in embryos. The production of embryos in vitro may affect epigenetic mechanisms that could alter the expression of genes related to embryo development and X chromosome inactivation (XCI). The aim of this study was to understand XCI during in vitro, pre-implantation bovine embryo development by characterizing the allele-specific expression pattern of the X chromosome-linked gene, monoamine oxidase A (MAOA). Two pools of ten embryos, comprised of the 4-, 8- to 16-cell, morula, blastocyst, and expanded blastocyst stages, were collected. Total RNA from embryos was isolated, and the RT-PCR-RFLP technique was used to observe expression of the MAOA gene. The DNA amplicons were also sequenced using the dideoxy sequencing method. MAOA mRNA was detected, and allele-specific expression was identified in each pool of embryos. We showed the presence of both the maternal and paternal alleles in the 4-, 8- to 16-cell, blastocyst and expanded blastocyst embryos, but only the maternal allele was present in the morula stage. Therefore, we can affirm that the paternal X chromosome is totally inactivated at the morula stage and reactivated at the blastocyst stage. To our knowledge, this is the first report of allele-specific expression of an X-linked gene that is subject to XCI in in vitro bovine embryos from the 4-cell to expanded blastocyst stages. We have established a pattern of XCI in our in vitro embryo production system that can be useful as a marker to assist the development of new protocols for in vitro embryo production. (c) 2010 Wiley-Liss, Inc.

Fatty Acid Biosynthesis Pathways in Methylomicrobium buryatense 5G(B1)

PubMed Central

Demidenko, Aleksandr; Akberdin, Ilya R.; Allemann, Marco; Allen, Eric E.; Kalyuzhnaya, Marina G.

2017-01-01

Methane utilization by methanotrophic bacteria is an attractive application for biotechnological conversion of natural or biogas into high-added-value products. Haloalcaliphilic methanotrophic bacteria belonging to the genus Methylomicrobium are among the most promising strains for methane-based biotechnology, providing easy and inexpensive cultivation, rapid growth, and the availability of established genetic tools. A number of methane bioconversions using these microbial cultures have been discussed, including the derivation of biodiesel, alkanes, and OMEGA-3 supplements. These compounds are derived from bacterial fatty acid pools. Here, we investigate fatty acid biosynthesis in Methylomicrobium buryatense 5G(B1). Most of the genes homologous to typical Type II fatty acid biosynthesis pathways could be annotated by bioinformatics analyses, with the exception of fatty acid transport and regulatory elements. Different approaches for improving fatty acid accumulation were investigated. These studies indicated that both fatty acid degradation and acetyl- and malonyl-CoA levels are bottlenecks for higher level fatty acid production. The best strain generated in this study synthesizes 111 ± 2 mg/gDCW of extractable fatty acids, which is ~20% more than the original strain. A candidate gene for fatty acid biosynthesis regulation, farE, was identified and studied. Its deletion resulted in drastic changes to the fatty acid profile, leading to an increased pool of C18-fatty acid methyl ester. The FarE-regulon was further investigated by RNA-seq analysis of gene expression in farE-knockout mutants and farE-overexpressing strains. These gene profiles highlighted a novel set of enzymes and regulators involved in fatty acid biosynthesis. The gene expression and fatty acid profiles of the different farE-strains support the hypothesis that metabolic fluxes upstream of fatty acid biosynthesis restrict fatty acid production in the methanotroph. PMID:28119683

Polymorphism discovery and allele frequency estimation using high-throughput DNA sequencing of target-enriched pooled DNA samples

PubMed Central

2012-01-01

Background The central role of the somatotrophic axis in animal post-natal growth, development and fertility is well established. Therefore, the identification of genetic variants affecting quantitative traits within this axis is an attractive goal. However, large sample numbers are a pre-requisite for the identification of genetic variants underlying complex traits and although technologies are improving rapidly, high-throughput sequencing of large numbers of complete individual genomes remains prohibitively expensive. Therefore using a pooled DNA approach coupled with target enrichment and high-throughput sequencing, the aim of this study was to identify polymorphisms and estimate allele frequency differences across 83 candidate genes of the somatotrophic axis, in 150 Holstein-Friesian dairy bulls divided into two groups divergent for genetic merit for fertility. Results In total, 4,135 SNPs and 893 indels were identified during the resequencing of the 83 candidate genes. Nineteen percent (n = 952) of variants were located within 5' and 3' UTRs. Seventy-two percent (n = 3,612) were intronic and 9% (n = 464) were exonic, including 65 indels and 236 SNPs resulting in non-synonymous substitutions (NSS). Significant (P < 0.01) mean allele frequency differentials between the low and high fertility groups were observed for 720 SNPs (58 NSS). Allele frequencies for 43 of the SNPs were also determined by genotyping the 150 individual animals (Sequenom® MassARRAY). No significant differences (P > 0.1) were observed between the two methods for any of the 43 SNPs across both pools (i.e., 86 tests in total). Conclusions The results of the current study support previous findings of the use of DNA sample pooling and high-throughput sequencing as a viable strategy for polymorphism discovery and allele frequency estimation. Using this approach we have characterised the genetic variation within genes of the somatotrophic axis and related pathways, central to mammalian post-natal growth and development and subsequent lactogenesis and fertility. We have identified a large number of variants segregating at significantly different frequencies between cattle groups divergent for calving interval plausibly harbouring causative variants contributing to heritable variation. To our knowledge, this is the first report describing sequencing of targeted genomic regions in any livestock species using groups with divergent phenotypes for an economically important trait. PMID:22235840

Habitat heterogeneity and connectivity shape microbial communities in South American peatlands.

PubMed

Oloo, Felix; Valverde, Angel; Quiroga, María Victoria; Vikram, Surendra; Cowan, Don; Mataloni, Gabriela

2016-05-10

Bacteria play critical roles in peatland ecosystems. However, very little is known of how habitat heterogeneity affects the structure of the bacterial communities in these ecosystems. Here, we used amplicon sequencing of the 16S rRNA and nifH genes to investigate phylogenetic diversity and bacterial community composition in three different sub-Antarctic peat bog aquatic habitats: Sphagnum magellanicum interstitial water, and water from vegetated and non-vegetated pools. Total and putative nitrogen-fixing bacterial communities from Sphagnum interstitial water differed significantly from vegetated and non-vegetated pool communities (which were colonized by the same bacterial populations), probably as a result of differences in water chemistry and biotic interactions. Total bacterial communities from pools contained typically aquatic taxa, and were more dissimilar in composition and less species rich than those from Sphagnum interstitial waters (which were enriched in taxa typically from soils), probably reflecting the reduced connectivity between the former habitats. These results show that bacterial communities in peatland water habitats are highly diverse and structured by multiple concurrent factors.

The active translation of MHCII mRNA during dendritic cells maturation supplies new molecules to the cell surface pool.

PubMed

Malanga, Donatella; Barba, Pasquale; Harris, Paul E; Maffei, Antonella; Del Pozzo, Giovanna

2007-04-01

The transition of human dendritic cells (DCs) from the immature to the mature phenotype is characterized by an increased density of MHC class II (MHCII) molecules on the plasma membrane, a key requirement of their competence as professional antigen presenting cells (APCs). MHCII molecules on the cell surface derive from newly synthesized as well as from preexisting proteins. So far, all the studies done on DCs during maturation, to establish the relative contribution of newly synthesized MHCII molecules to the cell surface pool did not produced a clear, unified scenario. We report that, in human DCs stimulated ex vivo with LPS, the changes in the RNA accumulation specific for at least two MHCII genes (HLA-DRA and HLA-DQA1) due to transcriptional upregulation, is associated with the active translation at high rate of these transcripts. Our finding reveals that, across the 24h of the maturation process in human DCs, newly synthesized MHCII proteins are supplied to the APCs cell surface pool.

Continental-scale assessment of genetic diversity and population structure in quaking aspen (Populus tremuloides)

Treesearch

Colin M. Callahan; Carol A. Rowe; Ronald J. Ryel; John D. Shaw; Michael D. Madritch; Karen E. Mock

2013-01-01

Aspen populations in the south-western portion of the range are consistent with expectations for a historically stable edge, with low within-population diversity, significant geographical population structuring, and little evidence of northward expansion. Structuring within the southwestern cluster may result from distinct gene pools separated during the Pleistocene...

ECTOMYCORRHIZAL FUNGI IDENTIFICATION IN SINGLE AND POOLED ROOT SAMPLES: TERMINAL RESTRICTION FRAGMENT LENGTH POLYMORPHISM (TRFLP) AND MORPHOTYPING COMPARED

EPA Science Inventory

PCR-TRFLP methodology targeting rRNA genes has effectively been used to discriminate between microbial communities but to date has not been used specifically for the analysis of ectomycorrhizal communities colonizing plant roots. We describe here results of a study conducted to a...

Breeding implications of boll distribution responses to water stress deficits in Upland cotton (Gossypium hirsutum L.)

USDA-ARS?s Scientific Manuscript database

To identify the impact of different water stress deficits on the boll distribution of elite Upland cotton (Gossypium hirsutum L.) cultivars in the Texas High Plains, cultivars which represent the diverse gene-pools of the private sector were subjected to three different water deficit regimes. This s...

Diversity for cooking time in Andean dry bean

USDA-ARS?s Scientific Manuscript database

A diversity panel of 250 dry bean lines from the Andean gene pool was evaluated for cooking time. Cooking time ranged from 17 to 90 min with an average of 36 min. A faster cooking time was also correlated with a number of other seed characteristics, most notably, higher levels of boron and potassium...

Efect of tri-species chromosome shuffling on agronomic and fiber traits in Upland cotton

USDA-ARS?s Scientific Manuscript database

Gossypium barbadense (L.), G. tomentosum (Seem.), G. mustelinum (Watt.) and G. darwinii (Watt.) are in the primary gene pool of Upland cotton (G. hirsutum). They share a common chromosome number (2n=52), similar AD-genome architecture, and form reasonably fertile F1 hybrids. However, reduced transm...

Alleles conferring improved fiber quality from EMS mutagenesis of elite cotton genotypes

USDA-ARS?s Scientific Manuscript database

The elite gene pool of cotton (Gossypium spp.) has less diversity than those of most other major crops, making identification of novel alleles important to ongoing crop improvement. A total of 3,164 M5 lines resulting from ethyl methanesulfonate mutagenesis of two G. hirsutum breeding lines, TAM 94L...

Epidemiology, ecology and gene pool of influenza A virus in Egypt: will Egypt be the epicentre of the next influenza pandemic?

PubMed

Abdelwhab, E M; Abdel-Moneim, Ahmed S

2015-01-01

Outside Asia, Egypt is considered to be an influenza H5N1 epicentre and presents a far greater pandemic risk than other countries. The long-term endemicity of H5N1 and the recent emergence of H9N2 in poultry call attention to the need for unravelling the epidemiology, ecology and highly diverse gene pool of influenza A virus (IAV) in Egypt which is the aim of this review. Isolation of a considerable number of IAV subtypes from several avian and mammalian hosts was described. Co-infections of poultry with H5N1 and H9N2 and subclinical infections of pigs and humans with H1N1 and H5N1 may raise the potential for the reassortment of these viruses. Moreover, the adjustment of IAV genomes, particularly H5N1, to optimize their evolution toward efficient transmission in human is progressing in Egypt. Understanding the present situation of influenza viruses in Egypt will help in the control of the disease and can potentially prevent a possible pandemic.

The impact of natural transformation on adaptation in spatially structured bacterial populations.

PubMed

Moradigaravand, Danesh; Engelstädter, Jan

2014-06-20

Recent studies have demonstrated that natural transformation and the formation of highly structured populations in bacteria are interconnected. In spite of growing evidence about this connection, little is known about the dynamics of natural transformation in spatially structured bacterial populations. In this work, we model the interdependency between the dynamics of the bacterial gene pool and those of environmental DNA in space to dissect the effect of transformation on adaptation. Our model reveals that even with only a single locus under consideration, transformation with a free DNA fragment pool results in complex adaptation dynamics that do not emerge in previous models focusing only on the gene shuffling effect of transformation at multiple loci. We demonstrate how spatial restriction on population growth and DNA diffusion in the environment affect the impact of transformation on adaptation. We found that in structured bacterial populations intermediate DNA diffusion rates predominantly cause transformation to impede adaptation by spreading deleterious alleles in the population. Overall, our model highlights distinctive evolutionary consequences of bacterial transformation in spatially restricted compared to planktonic bacterial populations.

[Genetic ecological monitoring in human populations: heterozygosity, mtDNA haplotype variation, and genetic load].

PubMed

Balanovskiĭ, O P; Koshel', S M; Zaporozhchenko, V V; Pshenichnov, A S; Frolova, S A; Kuznetsova, M A; Baranova, E E; Teuchezh, I E; Kuznetsova, A A; Romashkina, M V; Utevskaia, O M; Churnosov, M I; Villems, R; Balanovskaia, E V

2011-11-01

Yu. P. Altukhov suggested that heterozygosity is an indicator of the state of the gene pool. The idea and a linked concept of genetic ecological monitoring were applied to a new dataset on mtDNA variation in East European ethnic groups. Haplotype diversity (an analog of the average heterozygosity) was shown to gradually decrease northwards. Since a similar trend is known for population density, interlinked changes were assumed for a set of parameters, which were ordered to form a causative chain: latitude increases, land productivity decreases, population density decreases, effective population size decreases, isolation of subpopulations increases, genetic drift increases, and mtDNA haplotype diversity decreases. An increase in genetic drift increases the random inbreeding rate and, consequently, the genetic load. This was confirmed by a significant correlation observed between the incidence of autosomal recessive hereditary diseases and mtDNA haplotype diversity. Based on the findings, mtDNA was assumed to provide an informative genetic system for genetic ecological monitoring; e.g., analyzing the ecology-driven changes in the gene pool.

Apoptosis of Limb Innervating Motor Neurons and Erosion of Motor Pool Identity Upon Lineage Specific Dicer Inactivation

PubMed Central

Chen, Jun-An; Wichterle, Hynek

2012-01-01

Diversification of mammalian spinal motor neurons into hundreds of subtypes is critical for the maintenance of body posture and coordination of complex movements. Motor neuron differentiation is controlled by extrinsic signals that regulate intrinsic genetic programs specifying and consolidating motor neuron subtype identity. While transcription factors have been recognized as principal regulators of the intrinsic program, the role of posttranscriptional regulations has not been systematically tested. MicroRNAs produced by Dicer mediated cleavage of RNA hairpins contribute to gene regulation by posttranscriptional silencing. Here we used Olig2-cre conditional deletion of Dicer gene in motor neuron progenitors to examine effects of miRNA biogenesis disruption on postmitotic spinal motor neurons. We report that despite the initial increase in the number of motor neuron progenitors, disruption of Dicer function results in a loss of many limb- and sympathetic ganglia-innervating spinal motor neurons. Furthermore, it leads to defects in motor pool identity specification. Thus, our results indicate that miRNAs are an integral part of the genetic program controlling motor neuron survival and acquisition of subtype specific properties. PMID:22629237

Systems Biology-Based Identification of Mycobacterium tuberculosis Persistence Genes in Mouse Lungs

PubMed Central

Dutta, Noton K.; Bandyopadhyay, Nirmalya; Veeramani, Balaji; Lamichhane, Gyanu; Karakousis, Petros C.; Bader, Joel S.

2014-01-01

ABSTRACT Identifying Mycobacterium tuberculosis persistence genes is important for developing novel drugs to shorten the duration of tuberculosis (TB) treatment. We developed computational algorithms that predict M. tuberculosis genes required for long-term survival in mouse lungs. As the input, we used high-throughput M. tuberculosis mutant library screen data, mycobacterial global transcriptional profiles in mice and macrophages, and functional interaction networks. We selected 57 unique, genetically defined mutants (18 previously tested and 39 untested) to assess the predictive power of this approach in the murine model of TB infection. We observed a 6-fold enrichment in the predicted set of M. tuberculosis genes required for persistence in mouse lungs relative to randomly selected mutant pools. Our results also allowed us to reclassify several genes as required for M. tuberculosis persistence in vivo. Finally, the new results implicated additional high-priority candidate genes for testing. Experimental validation of computational predictions demonstrates the power of this systems biology approach for elucidating M. tuberculosis persistence genes. PMID:24549847

Differentially-Expressed Pseudogenes in HIV-1 Infection.

PubMed

Gupta, Aditi; Brown, C Titus; Zheng, Yong-Hui; Adami, Christoph

2015-09-29

Not all pseudogenes are transcriptionally silent as previously thought. Pseudogene transcripts, although not translated, contribute to the non-coding RNA pool of the cell that regulates the expression of other genes. Pseudogene transcripts can also directly compete with the parent gene transcripts for mRNA stability and other cell factors, modulating their expression levels. Tissue-specific and cancer-specific differential expression of these "functional" pseudogenes has been reported. To ascertain potential pseudogene:gene interactions in HIV-1 infection, we analyzed transcriptomes from infected and uninfected T-cells and found that 21 pseudogenes are differentially expressed in HIV-1 infection. This is interesting because parent genes of one-third of these differentially-expressed pseudogenes are implicated in HIV-1 life cycle, and parent genes of half of these pseudogenes are involved in different viral infections. Our bioinformatics analysis identifies candidate pseudogene:gene interactions that may be of significance in HIV-1 infection. Experimental validation of these interactions would establish that retroviruses exploit this newly-discovered layer of host gene expression regulation for their own benefit.

Extensive female-mediated gene flow and low phylogeography among seventeen goat breeds in southwest China.

PubMed

Zhao, Wei; Zhong, Tao; Wang, Lin Jie; Li, Li; Zhang, Hong Ping

2014-08-01

Indigenous Chinese goat mtDNA is highly diverse but lacks geographic specificity; however, whether gene flow or gene exchange contributed to this remains unknown. We reanalyzed a consensus fragment of 481 bp in the D-loop region from 339 individuals. The network and neighbor-joining tree revealed three divergent maternal haplogroups (A, B1, and B2) in 17 local breeds. Although high polymorphism resulting in 198 different haplotypes was observed (h = 0.984 ± 0.002; π = 0.0336 ± 0.0008), neither the distribution of haplotypes nor PCA analysis revealed any obvious geographic structure in the local breeds. Extensive gene flow was widely detected among breeds from southwest China. High levels of gene exchange were detected between Qianbei Brown goats and the other breeds, indicating either more contribution or introgression to their gene pools. This study will be helpful in understanding the phylogeography and gene flow among the goat breeds of southwest China.

Probability of identity by descent in metapopulations.

PubMed Central

Kaj, I; Lascoux, M

1999-01-01

Equilibrium probabilities of identity by descent (IBD), for pairs of genes within individuals, for genes between individuals within subpopulations, and for genes between subpopulations are calculated in metapopulation models with fixed or varying colony sizes. A continuous-time analog to the Moran model was used in either case. For fixed-colony size both propagule and migrant pool models were considered. The varying population size model is based on a birth-death-immigration (BDI) process, to which migration between colonies is added. Wright's F statistics are calculated and compared to previous results. Adding between-island migration to the BDI model can have an important effect on the equilibrium probabilities of IBD and on Wright's index. PMID:10388835

Sinu Virus, a Novel and divergent Orthomyxovirus Related to Members of the Genus Thogotovirus, Isolated from Mosquitoes in Colombia

PubMed Central

Contreras-Gutiérrez, María Angélica; Nunes, Marcio R.T.; Guzman, Hilda; Uribe, Sandra; Gómez, Juan Carlos Gallego; Vasco, Juan David Suaza; Cardoso, Jedson F.; Popov, Vsevolod L.; Widen, Steven G.; Wood, Thomas G.; Vasilakis, Nikos; Tesh, Robert B.

2016-01-01

The genome and structural organization of a novel insect-specific orthomyxovirus, designated Sinu virus, is described. Sinu virus (SINUV) was isolated in cultures of C6/36 cells from a pool of mosquitoes collected in northwestern Colombia. The virus has six negative-sense ssRNA segments. Genetic analysis of each segment demonstrated the presence of six distinct ORFs encoding the following genes: PB2 (Segment 1), PB1, (Segment 2), PA protein (Segment 3), envelope GP gene (Segment 4), the NP (Segment 5), and M-like gene (Segment 6). Phylogenetically, SINUV appears to be most closed related to viruses in the genus Thogotovirus. PMID:27936462

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

PubMed Central

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

2018-01-01

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

Prevalence and Evolution of Core Photosystem II Genes in Marine Cyanobacterial Viruses and Their Hosts

PubMed Central

Lee, Jessica A; Thompson, Luke R; Bielawski, Joseph P

2006-01-01

Cyanophages (cyanobacterial viruses) are important agents of horizontal gene transfer among marine cyanobacteria, the numerically dominant photosynthetic organisms in the oceans. Some cyanophage genomes carry and express host-like photosynthesis genes, presumably to augment the host photosynthetic machinery during infection. To study the prevalence and evolutionary dynamics of this phenomenon, 33 cultured cyanophages of known family and host range and viral DNA from field samples were screened for the presence of two core photosystem reaction center genes, psbA and psbD. Combining this expanded dataset with published data for nine other cyanophages, we found that 88% of the phage genomes contain psbA, and 50% contain both psbA and psbD. The psbA gene was found in all myoviruses and Prochlorococcus podoviruses, but could not be amplified from Prochlorococcus siphoviruses or Synechococcus podoviruses. Nearly all of the phages that encoded both psbA and psbD had broad host ranges. We speculate that the presence or absence of psbA in a phage genome may be determined by the length of the latent period of infection. Whether it also carries psbD may reflect constraints on coupling of viral- and host-encoded PsbA–PsbD in the photosynthetic reaction center across divergent hosts. Phylogenetic clustering patterns of these genes from cultured phages suggest that whole genes have been transferred from host to phage in a discrete number of events over the course of evolution (four for psbA, and two for psbD), followed by horizontal and vertical transfer between cyanophages. Clustering patterns of psbA and psbD from Synechococcus cells were inconsistent with other molecular phylogenetic markers, suggesting genetic exchanges involving Synechococcus lineages. Signatures of intragenic recombination, detected within the cyanophage gene pool as well as between hosts and phages in both directions, support this hypothesis. The analysis of cyanophage psbA and psbD genes from field populations revealed significant sequence diversity, much of which is represented in our cultured isolates. Collectively, these findings show that photosynthesis genes are common in cyanophages and that significant genetic exchanges occur from host to phage, phage to host, and within the phage gene pool. This generates genetic diversity among the phage, which serves as a reservoir for their hosts, and in turn influences photosystem evolution. PMID:16802857

RNA interference-mediated survivin gene knockdown induces growth arrest and reduced migration of vascular smooth muscle cells.

PubMed

Nabzdyk, Christoph S; Lancero, Hope; Nguyen, Khanh P; Salek, Sherveen; Conte, Michael S

2011-11-01

Survivin (SVV) is a multifunctional protein that has been implicated in the development of neointimal hyperplasia. Nuclear SVV is essential for mitosis, whereas in mitochondria SVV has a cytoprotective function. Here, we investigated the effects of RNA interference (RNAi)-mediated SVV knockdown on cell cycle kinetics, apoptosis, migration, and gene expression in primary cultured vascular smooth muscle cells (VSMCs) from the human saphenous vein. Primary Human VSMCs were obtained from saphenous veins and cultured under standard conditions. SVV knockdown was achieved by either small interfering RNA or lentiviral transduction of short hairpin RNA, reducing SVV gene expression by quantitative PCR (>75%, P < 0.01) without a loss of cell viability. Subcellular fractionation revealed that RNAi treatment effectively targeted the nuclear SVV pool, whereas the larger mitochondrial pool was much less sensitive to transient knockdown. Both p53 and p27 protein levels were notably increased. SVV RNAi treatment significantly blocked VSMC proliferation in response to serum and PDGF-AB, arresting VSMC growth. Cell cycle analysis revealed an increased G(2)/M fraction consistent with a mitotic defect; 4',6-diamidino-2-phenylindole staining confirmed an increased frequency of polyploid and abnormal nuclei. In a transwell assay, SVV knockdown reduced migration to PDGF-AB, and actin-phalloidin staining revealed disorganized actin filaments and polygonal cell shape. However, apoptosis (DNA content and annexin V flow cytometry) was not directly induced by SVV RNAi, and sensitivity to apoptotic agonists (e.g., staurosporine and cytokines) was unchanged. In conclusion, RNAi-mediated SVV knockdown in VSMCs leads to profound cell cycle arrest at G(2)/M and impaired chemotaxis without cytotoxicity. The regulation of mitosis and apoptosis in VSMC involves differentially regulated subcellular pools of SVV. Thus, treatment of VSMC with RNAi targeting SVV might limit the response to vascular injury without destabilizing the vessel wall.

RNA interference-mediated survivin gene knockdown induces growth arrest and reduced migration of vascular smooth muscle cells

PubMed Central

Nabzdyk, Christoph S.; Lancero, Hope; Nguyen, Khanh P.; Salek, Sherveen

2011-01-01

Survivin (SVV) is a multifunctional protein that has been implicated in the development of neointimal hyperplasia. Nuclear SVV is essential for mitosis, whereas in mitochondria SVV has a cytoprotective function. Here, we investigated the effects of RNA interference (RNAi)-mediated SVV knockdown on cell cycle kinetics, apoptosis, migration, and gene expression in primary cultured vascular smooth muscle cells (VSMCs) from the human saphenous vein. Primary Human VSMCs were obtained from saphenous veins and cultured under standard conditions. SVV knockdown was achieved by either small interfering RNA or lentiviral transduction of short hairpin RNA, reducing SVV gene expression by quantitative PCR (>75%, P < 0.01) without a loss of cell viability. Subcellular fractionation revealed that RNAi treatment effectively targeted the nuclear SVV pool, whereas the larger mitochondrial pool was much less sensitive to transient knockdown. Both p53 and p27 protein levels were notably increased. SVV RNAi treatment significantly blocked VSMC proliferation in response to serum and PDGF-AB, arresting VSMC growth. Cell cycle analysis revealed an increased G2/M fraction consistent with a mitotic defect; 4′,6-diamidino-2-phenylindole staining confirmed an increased frequency of polyploid and abnormal nuclei. In a transwell assay, SVV knockdown reduced migration to PDGF-AB, and actin-phalloidin staining revealed disorganized actin filaments and polygonal cell shape. However, apoptosis (DNA content and annexin V flow cytometry) was not directly induced by SVV RNAi, and sensitivity to apoptotic agonists (e.g., staurosporine and cytokines) was unchanged. In conclusion, RNAi-mediated SVV knockdown in VSMCs leads to profound cell cycle arrest at G2/M and impaired chemotaxis without cytotoxicity. The regulation of mitosis and apoptosis in VSMC involves differentially regulated subcellular pools of SVV. Thus, treatment of VSMC with RNAi targeting SVV might limit the response to vascular injury without destabilizing the vessel wall. PMID:21856925

Involvement of a cyclic adenosine monophosphate-dependent signal in the diet-induced canalicular trafficking of adenosine triphosphate-binding cassette transporter g5/g8.

PubMed

Yamazaki, Yasuhiro; Yasui, Kenta; Hashizume, Takahiro; Suto, Arisa; Mori, Ayaka; Murata, Yuzuki; Yamaguchi, Masahiko; Ikari, Akira; Sugatani, Junko

2015-10-01

The adenosine triphosphate-binding cassette (ABC) half-transporters Abcg5 and Abcg8 promote the secretion of neutral sterol into bile. Studies have demonstrated the diet-induced gene expression of these transporters, but the regulation of their trafficking when the nutritional status changes in the liver remains to be elucidated. Here, we generated a novel in vivo kinetic analysis that can monitor the intracellular trafficking of Abcg5/Abcg8 in living mouse liver by in vivo transfection of the genes of fluorescent protein-tagged transporters and investigated how hypernutrition affects the canalicular trafficking of these transporters. The kinetic analysis showed that lithogenic diet consumption accelerated the translocation of newly synthesized fluorescent-tagged transporters to intracellular pools in an endosomal compartment and enhanced the recruitment of these pooled gene products into the bile canalicular membrane in mouse liver. Because some ABC transporters are reported to be recruited from intracellular pools to the bile canaliculi by cyclic adenosine monophosphate (cAMP) signaling, we next evaluated the involvement of this machinery in a diet-induced event. Administration of a protein kinase A inhibitor, N-(2-{[3-(4-bromophenyl)-2-propenyl]amino}ethyl)-5-isoquinolinesulfonamide, decreased the canalicular expression of native Abcg5/Abcg8 in lithogenic diet-fed mice, and injection of a cAMP analog, dibutyryl cAMP, transiently increased their levels in standard diet-fed mice, indicating the involvement of cAMP signaling. Indeed, canalicular trafficking of the fluorescent-tagged Abcg5/Abcg8 was enhanced by dibutyryl cAMP administration. These observations suggest that diet-induced lipid loading into liver accelerates the trafficking of Abcg5/Abcg8 to the bile canalicular membrane through cAMP signaling machinery. © 2015 by the American Association for the Study of Liver Diseases.

Geographical and environmental determinants of the genetic structure of wild barley in southeastern Anatolia.

PubMed

Pournosrat, Reza; Kaya, Selma; Shaaf, Salar; Kilian, Benjamin; Ozkan, Hakan

2018-01-01

Despite the global value of barley, compared to its wild progenitor, genetic variation in this crop has been drastically reduced due to the process of domestication, selection and improvement. In the medium term, this will negatively impact both the vulnerability and yield stability of barley against biotic and abiotic stresses under climate change. Returning to the crop wild relatives (CWR) as sources of new and beneficial alleles is a clear option for enhancing the resilience of diversity and adaptation to climate change. Southeastern Anatolia constitutes an important part of the natural distribution of wild barley in the Fertile Crescent where important crops were initially domesticated. In this study, we investigated genetic diversity in a comprehensive collection of 281 geo-referenced wild barley individuals from 92 collection sites with sample sizes ranging from 1 to 9 individuals per site, collected from southeastern Anatolia and 131 domesticated genotypes from 49 different countries using 40 EST-SSR markers. A total of 375 alleles were detected across entire collection, of which 283 were carried by domesticated genotypes and 316 alleles were present in the wild gene pool. The number of unique alleles in the wild and in the domesticated gene pool was 92 and 59, respectively. The population structure at K = 3 suggested two groups of wild barley namely G1-W consisting wild barley genotypes from the western part and G1-E comprising those mostly from the eastern part of the study area, with a sharp separation from the domesticated gene pool. The geographic and climatic factors jointly showed significant effects on the distribution of wild barley. Using a Latent Factor Mixed Model, we identified four candidate loci potentially involved in adaptation of wild barley to three environmental factors: temperature seasonality, mean temperature of driest quarter, and precipitation of coldest quarter. These loci are probably the targets of genomic regions, with potential roles against abiotic stresses.

Genetic Patterns of Common-Bean Seed Acquisition and Early-Stage Adoption Among Farmer Groups in Western Uganda

PubMed Central

Wilkus, Erin L.; Berny Mier y Teran, Jorge C.; Mukankusi, Clare M.; Gepts, Paul

2018-01-01

Widespread adoption of new varieties can be valuable, especially where improved agricultural production technologies are hard to access. However, as farmers adopt new varieties, in situ population structure and genetic diversity of their seed holdings can change drastically. Consequences of adoption are still poorly understood due to a lack of crop genetic diversity assessments and detailed surveys of farmers' seed management practices. Common bean (Phaseolus vulgaris) is an excellent model for these types of studies, as it has a long history of cultivation among smallholder farmers, exhibits eco-geographic patterns of diversity (e.g., Andean vs. Mesoamerican gene-pools), and has been subjected to post-Columbian dispersal and recent introduction of improved cultivars. The Hoima district of western Uganda additionally provides an excellent social setting for evaluating consequences of adoption because access to improved varieties has varied across farmer groups in this production region. This study establishes a baseline understanding of the common bean diversity found among household producers in Uganda and compares the crop population structure, diversity and consequences of adoption of household producers with different adoption practices. Molecular diversity analysis, based on 4,955 single nucleotide polymorphism (SNP) markers, evaluated a total of 1,156 seed samples that included 196 household samples collected from household producers in the Hoima district, 19 breeder-selected varieties used in participatory breeding activities that had taken place prior to the study in the region, and a global bean germplasm collection. Households that had participated in regional participatory breeding efforts were more likely to adopt new varieties and, consequently, diversify their seed stocks than those that had not participated. Of the three farmer groups that participated in breeding efforts, households from the farmer group with the longest history of bean production were more likely to conserve “Seed Engufu”, a local “Calima”-type variety of the Andean bean gene pool, and, at the same time, introduce rare Mesoamerican gene pool varieties into household seed stocks. PMID:29868053

Genetic Patterns of Common-Bean Seed Acquisition and Early-Stage Adoption Among Farmer Groups in Western Uganda.

PubMed

Wilkus, Erin L; Berny Mier Y Teran, Jorge C; Mukankusi, Clare M; Gepts, Paul

2018-01-01

Widespread adoption of new varieties can be valuable, especially where improved agricultural production technologies are hard to access. However, as farmers adopt new varieties, in situ population structure and genetic diversity of their seed holdings can change drastically. Consequences of adoption are still poorly understood due to a lack of crop genetic diversity assessments and detailed surveys of farmers' seed management practices. Common bean ( Phaseolus vulgaris ) is an excellent model for these types of studies, as it has a long history of cultivation among smallholder farmers, exhibits eco-geographic patterns of diversity (e.g., Andean vs. Mesoamerican gene-pools), and has been subjected to post-Columbian dispersal and recent introduction of improved cultivars. The Hoima district of western Uganda additionally provides an excellent social setting for evaluating consequences of adoption because access to improved varieties has varied across farmer groups in this production region. This study establishes a baseline understanding of the common bean diversity found among household producers in Uganda and compares the crop population structure, diversity and consequences of adoption of household producers with different adoption practices. Molecular diversity analysis, based on 4,955 single nucleotide polymorphism (SNP) markers, evaluated a total of 1,156 seed samples that included 196 household samples collected from household producers in the Hoima district, 19 breeder-selected varieties used in participatory breeding activities that had taken place prior to the study in the region, and a global bean germplasm collection. Households that had participated in regional participatory breeding efforts were more likely to adopt new varieties and, consequently, diversify their seed stocks than those that had not participated. Of the three farmer groups that participated in breeding efforts, households from the farmer group with the longest history of bean production were more likely to conserve "Seed Engufu", a local "Calima"-type variety of the Andean bean gene pool, and, at the same time, introduce rare Mesoamerican gene pool varieties into household seed stocks.

Presence of an emerging subclone of Francisella tularensis holarctica in Ixodes ricinus ticks from south-western Germany.

PubMed

Gehringer, Heike; Schacht, Erik; Maylaender, Nicole; Zeman, Ella; Kaysser, Philipp; Oehme, Rainer; Pluta, Silvia; Splettstoesser, Wolf D

2013-02-01

The zoonotic disease tularaemia is caused by the bacterial pathogen Francisella tularensis. Although the causative agent is known for 100 years, knowledge of its enzootic cycles is still rudimentary. Apart from tabanids and mosquitoes, hard ticks have been described as important vectors and potential reservoirs for F. tularensis. Available data on the incidence of human tularaemia indicate an increase in cases in the federal state of Baden-Wuerttemberg. To determine whether ticks are involved in the reported increase in F. tularensis infections in humans and wildlife in this south-western part of Germany, 916 Ixodes ricinus and 211 adult Dermacentor marginatus and D. reticulatus ticks were collected in two different locations. Screening for the presence of F. tularensis was performed by real-time PCR of the 16S rRNA gene. Of the 95 pools of I. ricinus ticks (representing 916 individual ticks), 8 tick pools (8.4%) were positive in this PCR. 30-bp deletion PCR confirmed that the F. tularensis subspecies holarctica was present. FtM24 VNTR analysis revealed that they belong to the emerging Franco-Iberian subclone group of F. tularensis holarctica. Of the 211 ticks of the genus Dermacentor, 35 randomly chosen DNAs were subjected to 16S rRNA gene screening PCR; 20 of these (57%) gave positive signals. For cluster analysis, the lpnA gene region of all Francisella-positive I. ricinus pools and 6 Dermacentor ticks with a positive reaction in the screening PCR was amplified and sequenced. In the resulting neighbour-joining tree, all Francisella-positive I. ricinus samples clustered with sequences of F. tularensis, whilst all Dermacentor tick samples clustered with FLE (Francisella-like endosymbiont) sequences. This study shows that I. ricinus ticks may serve as vectors and/or reservoirs of F. tularensis in Germany and supports the hypothesis that the state of Baden-Wuerttemberg represents an emerging endemic focus of tularaemia. Copyright © 2012 Elsevier GmbH. All rights reserved.

Complete Mitochondrial DNA Analysis of Eastern Eurasian Haplogroups Rarely Found in Populations of Northern Asia and Eastern Europe

PubMed Central

Derenko, Miroslava; Malyarchuk, Boris; Denisova, Galina; Perkova, Maria; Rogalla, Urszula; Grzybowski, Tomasz; Khusnutdinova, Elza; Dambueva, Irina; Zakharov, Ilia

2012-01-01

With the aim of uncovering all of the most basal variation in the northern Asian mitochondrial DNA (mtDNA) haplogroups, we have analyzed mtDNA control region and coding region sequence variation in 98 Altaian Kazakhs from southern Siberia and 149 Barghuts from Inner Mongolia, China. Both populations exhibit the prevalence of eastern Eurasian lineages accounting for 91.9% in Barghuts and 60.2% in Altaian Kazakhs. The strong affinity of Altaian Kazakhs and populations of northern and central Asia has been revealed, reflecting both influences of central Asian inhabitants and essential genetic interaction with the Altai region indigenous populations. Statistical analyses data demonstrate a close positioning of all Mongolic-speaking populations (Mongolians, Buryats, Khamnigans, Kalmyks as well as Barghuts studied here) and Turkic-speaking Sojots, thus suggesting their origin from a common maternal ancestral gene pool. In order to achieve a thorough coverage of DNA lineages revealed in the northern Asian matrilineal gene pool, we have completely sequenced the mtDNA of 55 samples representing haplogroups R11b, B4, B5, F2, M9, M10, M11, M13, N9a and R9c1, which were pinpointed from a massive collection (over 5000 individuals) of northern and eastern Asian, as well as European control region mtDNA sequences. Applying the newly updated mtDNA tree to the previously reported northern Asian and eastern Asian mtDNA data sets has resolved the status of the poorly classified mtDNA types and allowed us to obtain the coalescence age estimates of the nodes of interest using different calibrated rates. Our findings confirm our previous conclusion that northern Asian maternal gene pool consists of predominantly post-LGM components of eastern Asian ancestry, though some genetic lineages may have a pre-LGM/LGM origin. PMID:22363811

Geographical and environmental determinants of the genetic structure of wild barley in southeastern Anatolia

PubMed Central

Shaaf, Salar; Kilian, Benjamin

2018-01-01

Despite the global value of barley, compared to its wild progenitor, genetic variation in this crop has been drastically reduced due to the process of domestication, selection and improvement. In the medium term, this will negatively impact both the vulnerability and yield stability of barley against biotic and abiotic stresses under climate change. Returning to the crop wild relatives (CWR) as sources of new and beneficial alleles is a clear option for enhancing the resilience of diversity and adaptation to climate change. Southeastern Anatolia constitutes an important part of the natural distribution of wild barley in the Fertile Crescent where important crops were initially domesticated. In this study, we investigated genetic diversity in a comprehensive collection of 281 geo-referenced wild barley individuals from 92 collection sites with sample sizes ranging from 1 to 9 individuals per site, collected from southeastern Anatolia and 131 domesticated genotypes from 49 different countries using 40 EST-SSR markers. A total of 375 alleles were detected across entire collection, of which 283 were carried by domesticated genotypes and 316 alleles were present in the wild gene pool. The number of unique alleles in the wild and in the domesticated gene pool was 92 and 59, respectively. The population structure at K = 3 suggested two groups of wild barley namely G1-W consisting wild barley genotypes from the western part and G1-E comprising those mostly from the eastern part of the study area, with a sharp separation from the domesticated gene pool. The geographic and climatic factors jointly showed significant effects on the distribution of wild barley. Using a Latent Factor Mixed Model, we identified four candidate loci potentially involved in adaptation of wild barley to three environmental factors: temperature seasonality, mean temperature of driest quarter, and precipitation of coldest quarter. These loci are probably the targets of genomic regions, with potential roles against abiotic stresses. PMID:29420597

Prevalence and antibiotic resistance of Pseudomonas aeruginosa in water samples in central Italy and molecular characterization of oprD in imipenem resistant isolates

PubMed Central

Carloni, Elisa; Andreoni, Francesca; Magi, Silvia; Chironna, Maria; Brandi, Giorgio; Amagliani, Giulia

2017-01-01

Scope This study aimed to analyse the prevalence, antibiotic resistance and genetic relatedness of P. aeruginosa isolates obtained from potable and recreational water samples (n. 8,351) collected from different settings (swimming pools, n. 207; healthcare facilities, n 1,684; accommodation facilities, n. 1,518; municipal waterworks, n. 4,500; residential buildings, n. 235). Possible mechanisms underlying resistance to imipenem, with particular focus on those involving oprD-based uptake, were also explored. Methods and results Isolation and identification of Pseudomonas aeruginosa was performed according to the standardized procedure UNI EN ISO 16266:2008 followed by PCR confirmation. Antibiotic Susceptibility testing was conducted according to EUCAST standardized disk diffusion method. Genetic relatedness of strains was carried out by RAPD. The sequence of the oprD gene was analyzed by standard method. Fifty-three samples (0.63%) were positive for P. aeruginosa, of which 10/207 (4.83%) were from swimming pools. Five isolates (9.43%) were resistant to imipenem, one to Ticarcillin + Clavulanate, one to both Piperacillin and Ticarcillin + Clavulanate. The highest isolation rate of imipenem resistant P. aeruginosa was observed in swimming pool water. Identical RAPD profiles were found in isolates from the same location in the same year or even in different years. Conclusions Imipenem resistant strains were identified as carbapenemase-negative and resistance has been associated with inactivating mutations within the oprD gene, with a concomitant loss of porin. RAPD results proved that a water system can remain colonized by one strain for long periods and the contamination may be difficult to eradicate. This study has revealed the presence of P. aeruginosa in different water samples, including resistant strains, especially in swimming pools, and confirmed the role of porins as a contributing factor in carbapenem resistance in Gram-negative bacteria. PMID:29211780

Prevalence and antibiotic resistance of Pseudomonas aeruginosa in water samples in central Italy and molecular characterization of oprD in imipenem resistant isolates.

PubMed

Schiavano, Giuditta Fiorella; Carloni, Elisa; Andreoni, Francesca; Magi, Silvia; Chironna, Maria; Brandi, Giorgio; Amagliani, Giulia

2017-01-01

This study aimed to analyse the prevalence, antibiotic resistance and genetic relatedness of P. aeruginosa isolates obtained from potable and recreational water samples (n. 8,351) collected from different settings (swimming pools, n. 207; healthcare facilities, n 1,684; accommodation facilities, n. 1,518; municipal waterworks, n. 4,500; residential buildings, n. 235). Possible mechanisms underlying resistance to imipenem, with particular focus on those involving oprD-based uptake, were also explored. Isolation and identification of Pseudomonas aeruginosa was performed according to the standardized procedure UNI EN ISO 16266:2008 followed by PCR confirmation. Antibiotic Susceptibility testing was conducted according to EUCAST standardized disk diffusion method. Genetic relatedness of strains was carried out by RAPD. The sequence of the oprD gene was analyzed by standard method. Fifty-three samples (0.63%) were positive for P. aeruginosa, of which 10/207 (4.83%) were from swimming pools. Five isolates (9.43%) were resistant to imipenem, one to Ticarcillin + Clavulanate, one to both Piperacillin and Ticarcillin + Clavulanate. The highest isolation rate of imipenem resistant P. aeruginosa was observed in swimming pool water. Identical RAPD profiles were found in isolates from the same location in the same year or even in different years. Imipenem resistant strains were identified as carbapenemase-negative and resistance has been associated with inactivating mutations within the oprD gene, with a concomitant loss of porin. RAPD results proved that a water system can remain colonized by one strain for long periods and the contamination may be difficult to eradicate. This study has revealed the presence of P. aeruginosa in different water samples, including resistant strains, especially in swimming pools, and confirmed the role of porins as a contributing factor in carbapenem resistance in Gram-negative bacteria.

Detection of genetic variation affecting milk coagulation properties in Danish Holstein dairy cattle by analyses of pooled whole-genome sequences from phenotypically extreme samples (pool-seq).

PubMed

Bertelsen, H P; Gregersen, V R; Poulsen, N; Nielsen, R O; Das, A; Madsen, L B; Buitenhuis, A J; Holm, L-E; Panitz, F; Larsen, L B; Bendixen, C

2016-04-01

Rennet-induced milk coagulation is an important trait for cheese production. Recent studies have reported an alarming frequency of cows producing poorly coagulating milk unsuitable for cheese production. Several genetic factors are known to affect milk coagulation, including variation in the major milk proteins; however, recent association studies indicate genetic effects from other genomic regions as well. The aim of this study was to detect genetic variation affecting milk coagulation properties, measured as curd-firming rate (CFR) and milk pH. This was achieved by examining allele frequency differences between pooled whole-genome sequences of phenotypically extreme samples (pool-seq).. Curd-firming rate and raw milk pH were measured for 415 Danish Holstein cows, and each animal was sequenced at low coverage. Pools were created containing whole genome sequence reads from samples with "extreme" values (high or low) for both phenotypic traits. A total of 6,992,186 and 5,295,501 SNP were assessed in relation to CFR and milk pH, respectively. Allele frequency differences were calculated between pools and 32 significantly different SNP were detected, 1 for milk pH and 31 for CFR, of which 19 are located on chromosome 6. A total of 9 significant SNP, which were selected based on the possible function of proximal candidate genes, were genotyped in the entire sample set ( = 415) to test for an association. The most significant SNP was located proximal to , explaining 33% of the phenotypic variance. , coding for κ-casein, is the most studied in relation to milk coagulation due to its position on the surface of the casein micelles and the direct involvement in milk coagulation. Three additional SNP located on chromosome 6 showed significant associations explaining 7, 3.6, and 1.3% of the phenotypic variance of CFR. The significant SNP on chromosome 6 were shown to be in linkage disequilibrium with the SNP peaking proximal to ; however, after accounting for the genotype of the peak SNP within this QTL, significant effects (-value < 0.1) could still be detected for 2 of the SNP accounting for 2 and 1% of the phenotypic variance. These 2 interesting SNP were located within introns or proximal to the candidate genes-solute carrier family 4 (sodium bicarbonate cotransporter), member 4 () and LIM and calponin homology domains 1 (), respectively-making them interesting targets for further analysis.

Artificial genetic selection for an efficient translation initiation site for expression of human RACK1 gene in Escherichia coli

PubMed Central

Zhelyabovskaya, Olga B.; Berlin, Yuri A.; Birikh, Klara R.

2004-01-01

In bacterial expression systems, translation initiation is usually the rate limiting and the least predictable stage of protein synthesis. Efficiency of a translation initiation site can vary dramatically depending on the sequence context. This is why many standard expression vectors provide very poor expression levels of some genes. This notion persuaded us to develop an artificial genetic selection protocol, which allows one to find for a given target gene an individual efficient ribosome binding site from a random pool. In order to create Darwinian pressure necessary for the genetic selection, we designed a system based on translational coupling, in which microorganism survival in the presence of antibiotic depends on expression of the target gene, while putting no special requirements on this gene. Using this system we obtained superproducing constructs for the human protein RACK1 (receptor for activated C kinase). PMID:15034151

Inherited Mitochondrial Diseases of DNA Replication

PubMed Central

Copeland, William C.

2007-01-01

Mitochondrial genetic diseases can result from defects in mitochondrial DNA (mtDNA) in the form of deletions, point mutations, or depletion, which ultimately cause loss of oxidative phosphorylation. These mutations may be spontaneous, maternally inherited, or a result of inherited nuclear defects in genes that maintain mtDNA. This review focuses on our current understanding of nuclear gene mutations that produce mtDNA alterations and cause mitochondrial depletion syndrome (MDS), progressive external ophthalmoplegia (PEO), ataxia-neuropathy, or mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). To date, all of these etiologic nuclear genes fall into one of two categories: genes whose products function directly at the mtDNA replication fork, such as POLG, POLG2, and TWINKLE, or genes whose products supply the mitochondria with deoxynucleotide triphosphate pools needed for DNA replication, such as TK2, DGUOK, TP, SUCLA2, ANT1, and possibly the newly identified MPV17. PMID:17892433

Tumor Hypoxia and Genetic Alterations in Sporadic Cancers

PubMed Central

Koi, Minoru; Boland, C.R.

2011-01-01

The cancer genome contains many gene alterations. How cancer cells acquire these alterations is a matter for discussion. One hypothesis is that cancer cells obtain mutations in genome stability genes at an early stage of tumor development, which results in genetic instability and generates a gene pool that enhances cellular proliferation and survival. Another hypothesis puts its emphasis on the natural selection of gene mutations for fitness. Recent data for systematic cancer genome sequencing shows that mutations in stability genes are rare in human sporadic cancers. Instead, many “passenger” mutations that do not drive the carcinogenesis process have been found in the cancer genome. Both the hypotheses mentioned above fall short in explaining recent data. Recently, many studies demonstrate the role of the tumor microenvironment, especially hypoxia and reoxygenation, in genetic instability. In this review, literature will be presented which supports a third hypothesis, i.e. that hypoxia/re-oxygenation induces genetic instability. PMID:21272156

On the Origin of Reverse Transcriptase-Using CRISPR-Cas Systems and Their Hyperdiverse, Enigmatic Spacer Repertoires.

PubMed

Silas, Sukrit; Makarova, Kira S; Shmakov, Sergey; Páez-Espino, David; Mohr, Georg; Liu, Yi; Davison, Michelle; Roux, Simon; Krishnamurthy, Siddharth R; Fu, Becky Xu Hua; Hansen, Loren L; Wang, David; Sullivan, Matthew B; Millard, Andrew; Clokie, Martha R; Bhaya, Devaki; Lambowitz, Alan M; Kyrpides, Nikos C; Koonin, Eugene V; Fire, Andrew Z

2017-07-11

Cas1 integrase is the key enzyme of the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas adaptation module that mediates acquisition of spacers derived from foreign DNA by CRISPR arrays. In diverse bacteria, the cas1 gene is fused (or adjacent) to a gene encoding a reverse transcriptase (RT) related to group II intron RTs. An RT-Cas1 fusion protein has been recently shown to enable acquisition of CRISPR spacers from RNA. Phylogenetic analysis of the CRISPR-associated RTs demonstrates monophyly of the RT-Cas1 fusion, and coevolution of the RT and Cas1 domains. Nearly all such RTs are present within type III CRISPR-Cas loci, but their phylogeny does not parallel the CRISPR-Cas type classification, indicating that RT-Cas1 is an autonomous functional module that is disseminated by horizontal gene transfer and can function with diverse type III systems. To compare the sequence pools sampled by RT-Cas1-associated and RT-lacking CRISPR-Cas systems, we obtained samples of a commercially grown cyanobacterium- Arthrospira platensis Sequencing of the CRISPR arrays uncovered a highly diverse population of spacers. Spacer diversity was particularly striking for the RT-Cas1-containing type III-B system, where no saturation was evident even with millions of sequences analyzed. In contrast, analysis of the RT-lacking type III-D system yielded a highly diverse pool but reached a point where fewer novel spacers were recovered as sequencing depth was increased. Matches could be identified for a small fraction of the non-RT-Cas1-associated spacers, and for only a single RT-Cas1-associated spacer. Thus, the principal source(s) of the spacers, particularly the hypervariable spacer repertoire of the RT-associated arrays, remains unknown. IMPORTANCE While the majority of CRISPR-Cas immune systems adapt to foreign genetic elements by capturing segments of invasive DNA, some systems carry reverse transcriptases (RTs) that enable adaptation to RNA molecules. From analysis of available bacterial sequence data, we find evidence that RT-based RNA adaptation machinery has been able to join with CRISPR-Cas immune systems in many, diverse bacterial species. To investigate whether the abilities to adapt to DNA and RNA molecules are utilized for defense against distinct classes of invaders in nature, we sequenced CRISPR arrays from samples of commercial-scale open-air cultures of Arthrospira platensis , a cyanobacterium that contains both RT-lacking and RT-containing CRISPR-Cas systems. We uncovered a diverse pool of naturally occurring immune memories, with the RT-lacking locus acquiring a number of segments matching known viral or bacterial genes, while the RT-containing locus has acquired spacers from a distinct sequence pool for which the source remains enigmatic. Copyright © 2017 Silas et al.

Evaluation of acetylcholinesterase in an animal model of maple syrup urine disease.

PubMed

Scaini, Giselli; de Rochi, Natália; Jeremias, Isabela C; Deroza, Pedro F; Zugno, Alexandra I; Pereira, Talita C B; Oliveira, Giovanna M T; Kist, Luiza W; Bogo, Maurício R; Schuck, Patrícia F; Ferreira, Gustavo C; Streck, Emilio L

2012-04-01

Maple syrup urine disease is an inherited metabolic disease predominantly characterized by neurological dysfunction. However, the mechanisms underlying the neuropathology of this disease are still not defined. Therefore, the aim of this study was to investigate the effect of acute and chronic administration of a branched-chain amino acids (BCAA) pool (leucine, isoleucine, and valine) on acetylcholinesterase (AChE) activity and gene expression in the brain and serum of rats and to assess if antioxidant treatment prevented the alterations induced by BCAA administration. Our results show that the acute administration of a BCAA pool in 10- and 30-day-old rats increases AChE activity in the cerebral cortex, striatum, hippocampus, and serum. Moreover, chronic administration of the BCAA pool also increases AChE activity in the structures studied, and antioxidant treatment prevents this increase. In addition, we show a significant decrease in the mRNA expression of AChE in the hippocampus following acute administration in 10- and 30-day-old rats. On the other hand, AChE expression increased significantly after chronic administration of the BCAA pool. Interestingly, the antioxidant treatment was able to prevent the increased AChE activity without altering AChE expression. In conclusion, the results from the present study demonstrate a marked increase in AChE activity in all brain structures following the administration of a BCAA pool. Moreover, the increased AChE activity is prevented by the coadministration of N-acetylcysteine and deferoxamine as antioxidants.

A reserve stem cell population in small intestine renders Lgr5-positive cells dispensable

PubMed Central

Tian, Hua; Biehs, Brian; Warming, Soren; Leong, Kevin G.; Rangell, Linda; Klein, Ophir D.; de Sauvage, Frederic J.

2014-01-01

The small intestine epithelium renews every 2 to 5 days, making it one of the most regenerative mammalian tissues. Genetic inducible fate mapping studies have identified two principal epithelial stem cell pools in this tissue. One pool consists of columnar Lgr5-expressing cells that cycle rapidly and are present predominantly at the crypt base1. The other pool consists of Bmi1-expressing cells that largely reside above the crypt base2. However, the relative functions of these two pools and their interrelationship are not understood. Here, we specifically ablated Lgr5-expressing cells using a diphtheria toxin receptor (DTR) gene knocked into the Lgr5 locus. We found that complete loss of the Lgr5-expressing cells did not perturb homeostasis of the epithelium, indicating that other cell types can compensate for elimination of this population. After ablation of Lgr5-expressing cells, progeny production by Bmi1-expressing cells increased, suggesting that Bmi1-expressing stem cells compensate for the loss of Lgr5-expressing cells. Indeed, lineage tracing showed that Bmi1-expressing cells gave rise to Lgr5-expressing cells, pointing to a hierarchy of stem cells in the intestinal epithelium. Our results demonstrate that Lgr5-expressing cells are dispensable for normal intestinal homeostasis. In the absence of these cells, the Bmi1-expressing cells can serve as an alternative stem cell pool, providing the first experimental evidence for the interrelationship between these populations. The Bmi1-expressing stem cells may represent both a reserve stem cell pool in case of injury to the small intestine epithelium and a source for replenishment of the Lgr5-expressing cells under non-pathological conditions. PMID:21927002

High-density SNP assay development for genetic analysis in maritime pine (Pinus pinaster).

PubMed

Plomion, C; Bartholomé, J; Lesur, I; Boury, C; Rodríguez-Quilón, I; Lagraulet, H; Ehrenmann, F; Bouffier, L; Gion, J M; Grivet, D; de Miguel, M; de María, N; Cervera, M T; Bagnoli, F; Isik, F; Vendramin, G G; González-Martínez, S C

2016-03-01

Maritime pine provides essential ecosystem services in the south-western Mediterranean basin, where it covers around 4 million ha. Its scattered distribution over a range of environmental conditions makes it an ideal forest tree species for studies of local adaptation and evolutionary responses to climatic change. Highly multiplexed single nucleotide polymorphism (SNP) genotyping arrays are increasingly used to study genetic variation in living organisms and for practical applications in plant and animal breeding and genetic resource conservation. We developed a 9k Illumina Infinium SNP array and genotyped maritime pine trees from (i) a three-generation inbred (F2) pedigree, (ii) the French breeding population and (iii) natural populations from Portugal and the French Atlantic coast. A large proportion of the exploitable SNPs (2052/8410, i.e. 24.4%) segregated in the mapping population and could be mapped, providing the densest ever gene-based linkage map for this species. Based on 5016 SNPs, natural and breeding populations from the French gene pool exhibited similar level of genetic diversity. Population genetics and structure analyses based on 3981 SNP markers common to the Portuguese and French gene pools revealed high levels of differentiation, leading to the identification of a set of highly differentiated SNPs that could be used for seed provenance certification. Finally, we discuss how the validated SNPs could facilitate the identification of ecologically and economically relevant genes in this species, improving our understanding of the demography and selective forces shaping its natural genetic diversity, and providing support for new breeding strategies. © 2015 John Wiley & Sons Ltd.

Novel genes identified in a high-density genome wide association study for nicotine dependence.

PubMed

Bierut, Laura Jean; Madden, Pamela A F; Breslau, Naomi; Johnson, Eric O; Hatsukami, Dorothy; Pomerleau, Ovide F; Swan, Gary E; Rutter, Joni; Bertelsen, Sarah; Fox, Louis; Fugman, Douglas; Goate, Alison M; Hinrichs, Anthony L; Konvicka, Karel; Martin, Nicholas G; Montgomery, Grant W; Saccone, Nancy L; Saccone, Scott F; Wang, Jen C; Chase, Gary A; Rice, John P; Ballinger, Dennis G

2007-01-01

Tobacco use is a leading contributor to disability and death worldwide, and genetic factors contribute in part to the development of nicotine dependence. To identify novel genes for which natural variation contributes to the development of nicotine dependence, we performed a comprehensive genome wide association study using nicotine dependent smokers as cases and non-dependent smokers as controls. To allow the efficient, rapid, and cost effective screen of the genome, the study was carried out using a two-stage design. In the first stage, genotyping of over 2.4 million single nucleotide polymorphisms (SNPs) was completed in case and control pools. In the second stage, we selected SNPs for individual genotyping based on the most significant allele frequency differences between cases and controls from the pooled results. Individual genotyping was performed in 1050 cases and 879 controls using 31 960 selected SNPs. The primary analysis, a logistic regression model with covariates of age, gender, genotype and gender by genotype interaction, identified 35 SNPs with P-values less than 10(-4) (minimum P-value 1.53 x 10(-6)). Although none of the individual findings is statistically significant after correcting for multiple tests, additional statistical analyses support the existence of true findings in this group. Our study nominates several novel genes, such as Neurexin 1 (NRXN1), in the development of nicotine dependence while also identifying a known candidate gene, the beta3 nicotinic cholinergic receptor. This work anticipates the future directions of large-scale genome wide association studies with state-of-the-art methodological approaches and sharing of data with the scientific community.

Molecular proxies for climate maladaptation in a long-lived tree (Pinus pinaster Aiton, Pinaceae).

PubMed

Jaramillo-Correa, Juan-Pablo; Rodríguez-Quilón, Isabel; Grivet, Delphine; Lepoittevin, Camille; Sebastiani, Federico; Heuertz, Myriam; Garnier-Géré, Pauline H; Alía, Ricardo; Plomion, Christophe; Vendramin, Giovanni G; González-Martínez, Santiago C

2015-03-01

Understanding adaptive genetic responses to climate change is a main challenge for preserving biological diversity. Successful predictive models for climate-driven range shifts of species depend on the integration of information on adaptation, including that derived from genomic studies. Long-lived forest trees can experience substantial environmental change across generations, which results in a much more prominent adaptation lag than in annual species. Here, we show that candidate-gene SNPs (single nucleotide polymorphisms) can be used as predictors of maladaptation to climate in maritime pine (Pinus pinaster Aiton), an outcrossing long-lived keystone tree. A set of 18 SNPs potentially associated with climate, 5 of them involving amino acid-changing variants, were retained after performing logistic regression, latent factor mixed models, and Bayesian analyses of SNP-climate correlations. These relationships identified temperature as an important adaptive driver in maritime pine and highlighted that selective forces are operating differentially in geographically discrete gene pools. The frequency of the locally advantageous alleles at these selected loci was strongly correlated with survival in a common garden under extreme (hot and dry) climate conditions, which suggests that candidate-gene SNPs can be used to forecast the likely destiny of natural forest ecosystems under climate change scenarios. Differential levels of forest decline are anticipated for distinct maritime pine gene pools. Geographically defined molecular proxies for climate adaptation will thus critically enhance the predictive power of range-shift models and help establish mitigation measures for long-lived keystone forest trees in the face of impending climate change. Copyright © 2015 by the Genetics Society of America.

Molecular Proxies for Climate Maladaptation in a Long-Lived Tree (Pinus pinaster Aiton, Pinaceae)

PubMed Central

Jaramillo-Correa, Juan-Pablo; Rodríguez-Quilón, Isabel; Grivet, Delphine; Lepoittevin, Camille; Sebastiani, Federico; Heuertz, Myriam; Garnier-Géré, Pauline H.; Alía, Ricardo; Plomion, Christophe; Vendramin, Giovanni G.; González-Martínez, Santiago C.

2015-01-01

Understanding adaptive genetic responses to climate change is a main challenge for preserving biological diversity. Successful predictive models for climate-driven range shifts of species depend on the integration of information on adaptation, including that derived from genomic studies. Long-lived forest trees can experience substantial environmental change across generations, which results in a much more prominent adaptation lag than in annual species. Here, we show that candidate-gene SNPs (single nucleotide polymorphisms) can be used as predictors of maladaptation to climate in maritime pine (Pinus pinaster Aiton), an outcrossing long-lived keystone tree. A set of 18 SNPs potentially associated with climate, 5 of them involving amino acid-changing variants, were retained after performing logistic regression, latent factor mixed models, and Bayesian analyses of SNP–climate correlations. These relationships identified temperature as an important adaptive driver in maritime pine and highlighted that selective forces are operating differentially in geographically discrete gene pools. The frequency of the locally advantageous alleles at these selected loci was strongly correlated with survival in a common garden under extreme (hot and dry) climate conditions, which suggests that candidate-gene SNPs can be used to forecast the likely destiny of natural forest ecosystems under climate change scenarios. Differential levels of forest decline are anticipated for distinct maritime pine gene pools. Geographically defined molecular proxies for climate adaptation will thus critically enhance the predictive power of range-shift models and help establish mitigation measures for long-lived keystone forest trees in the face of impending climate change. PMID:25549630

Draft Assembly of Elite Inbred Line PH207 Provides Insights into Genomic and Transcriptome Diversity in Maize

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

Hirsch, Candice N.; Hirsch, Cory D.; Brohammer, Alex B.

Intense artificial selection over the last 100 years has produced elite maize (Zea mays) inbred lines that combine to produce high-yielding hybrids. To further our understanding of how genome and transcriptome variation contribute to the production of high-yielding hybrids, we generated a draft genome assembly of the inbred line PH207 to complement and compare with the existing B73 reference sequence. B73 is a founder of the Stiff Stalk germplasm pool, while PH207 is a founder of Iodent germplasm, both of which have contributed substantially to the production of temperate commercial maize and are combined to make heterotic hybrids. Comparison ofmore » these two assemblies revealed over 2500 genes present in only one of the two genotypes and 136 gene families that have undergone extensive expansion or contraction. Transcriptome profiling revealed extensive expression variation, with as many as 10,564 differentially expressed transcripts and 7128 transcripts expressed in only one of the two genotypes in a single tissue. Genotype-specific genes were more likely to have tissue/condition-specific expression and lower transcript abundance. The availability of a high-quality genome assembly for the elite maize inbred PH207 expands our knowledge of the breadth of natural genome and transcriptome variation in elite maize inbred lines across heterotic pools.« less

Transient silencing of the KASII genes is feasible in Nicotiana benthamiana for metabolic engineering of wax ester composition

PubMed Central

Aslan, Selcuk; Hofvander, Per; Dutta, Paresh; Sitbon, Folke; Sun, Chuanxin

2015-01-01

The beta-ketoacyl-ACP synthase II (KASII) is an enzyme in fatty acid biosynthesis, catalyzing the elongation of 16:0-acyl carrier protein (ACP) to 18:0-ACP in plastids. Mutations in KASII genes in higher plants can lead to lethality, which makes it difficult to utilize the gene for lipid metabolic engineering. We demonstrated previously that transient expression of plastid-directed fatty acyl reductases and wax ester synthases could result in different compositions of wax esters. We hypothesized that changing the ratio between C16 (palmitoyl-compounds) and C18 (stearoyl-compounds) in the plastidic acyl-ACP pool by inhibition of KASII expression would change the yield and composition of wax esters via substrate preference of the introduced enzymes. Here, we report that transient inhibition of KASII expression by three different RNAi constructs in leaves of N. benthamiana results in almost complete inhibition of KASII expression. The transient RNAi approach led to a shift of carbon flux from a pool of C18 fatty acids to C16, which significantly increased wax ester production in AtFAR6-containing combinations. The results demonstrate that transient inhibition of KASII in vegetative tissues of higher plants enables metabolic studies towards industrial production of lipids such as wax esters with specific quality and composition. PMID:26063537

Draft Assembly of Elite Inbred Line PH207 Provides Insights into Genomic and Transcriptome Diversity in Maize

DOE PAGES

Hirsch, Candice N.; Hirsch, Cory D.; Brohammer, Alex B.; ...

2016-11-01

Intense artificial selection over the last 100 years has produced elite maize (Zea mays) inbred lines that combine to produce high-yielding hybrids. To further our understanding of how genome and transcriptome variation contribute to the production of high-yielding hybrids, we generated a draft genome assembly of the inbred line PH207 to complement and compare with the existing B73 reference sequence. B73 is a founder of the Stiff Stalk germplasm pool, while PH207 is a founder of Iodent germplasm, both of which have contributed substantially to the production of temperate commercial maize and are combined to make heterotic hybrids. Comparison ofmore » these two assemblies revealed over 2500 genes present in only one of the two genotypes and 136 gene families that have undergone extensive expansion or contraction. Transcriptome profiling revealed extensive expression variation, with as many as 10,564 differentially expressed transcripts and 7128 transcripts expressed in only one of the two genotypes in a single tissue. Genotype-specific genes were more likely to have tissue/condition-specific expression and lower transcript abundance. The availability of a high-quality genome assembly for the elite maize inbred PH207 expands our knowledge of the breadth of natural genome and transcriptome variation in elite maize inbred lines across heterotic pools.« less

Synchronized dynamics of bacterial niche-specific functions during biofilm development in a cold seep brine pool.

PubMed

Zhang, Weipeng; Wang, Yong; Bougouffa, Salim; Tian, Renmao; Cao, Huiluo; Li, Yongxin; Cai, Lin; Wong, Yue Him; Zhang, Gen; Zhou, Guowei; Zhang, Xixiang; Bajic, Vladimir B; Al-Suwailem, Abdulaziz; Qian, Pei-Yuan

2015-10-01

The biology of biofilm in deep-sea environments is barely being explored. Here, biofilms were developed at the brine pool (characterized by limited carbon sources) and the normal bottom water adjacent to Thuwal cold seeps. Comparative metagenomics based on 50 Gb datasets identified polysaccharide degradation, nitrate reduction and proteolysis as enriched functional categories for brine biofilms. The genomes of two dominant species: a novel Deltaproteobacterium and a novel Epsilonproteobacterium in the brine biofilms were reconstructed. Despite rather small genome sizes, the Deltaproteobacterium possessed enhanced polysaccharide fermentation pathways, whereas the Epsilonproteobacterium was a versatile nitrogen reactor possessing nar, nap and nif gene clusters. These metabolic functions, together with specific regulatory and hypersaline-tolerant genes, made the two bacteria unique compared with their close relatives, including those from hydrothermal vents. Moreover, these functions were regulated by biofilm development, as both the abundance and the expression level of key functional genes were higher in later stage biofilms, and co-occurrences between the two dominant bacteria were demonstrated. Collectively, unique mechanisms were revealed: (i) polysaccharides fermentation, proteolysis interacted with nitrogen cycling to form a complex chain for energy generation, and (ii) remarkably exploiting and organizing niche-specific functions would be an important strategy for biofilm-dependent adaptation to the extreme conditions. © 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.

Draft Assembly of Elite Inbred Line PH207 Provides Insights into Genomic and Transcriptome Diversity in Maize[OPEN

PubMed Central

Soifer, Ilya; Barad, Omer; Shem-Tov, Doron; Baruch, Kobi; Lu, Fei; Hernandez, Alvaro G.; Wright, Chris L.; Koehler, Klaus; Buell, C. Robin; de Leon, Natalia

2016-01-01

Intense artificial selection over the last 100 years has produced elite maize (Zea mays) inbred lines that combine to produce high-yielding hybrids. To further our understanding of how genome and transcriptome variation contribute to the production of high-yielding hybrids, we generated a draft genome assembly of the inbred line PH207 to complement and compare with the existing B73 reference sequence. B73 is a founder of the Stiff Stalk germplasm pool, while PH207 is a founder of Iodent germplasm, both of which have contributed substantially to the production of temperate commercial maize and are combined to make heterotic hybrids. Comparison of these two assemblies revealed over 2500 genes present in only one of the two genotypes and 136 gene families that have undergone extensive expansion or contraction. Transcriptome profiling revealed extensive expression variation, with as many as 10,564 differentially expressed transcripts and 7128 transcripts expressed in only one of the two genotypes in a single tissue. Genotype-specific genes were more likely to have tissue/condition-specific expression and lower transcript abundance. The availability of a high-quality genome assembly for the elite maize inbred PH207 expands our knowledge of the breadth of natural genome and transcriptome variation in elite maize inbred lines across heterotic pools. PMID:27803309

Rac1 modulates the formation of primordial follicles by facilitating STAT3-directed Jagged1, GDF9 and BMP15 transcription in mice

PubMed Central

Zhao, Lihua; Du, Xinhua; Huang, Kun; Zhang, Tuo; Teng, Zhen; Niu, Wanbao; Wang, Chao; Xia, Guoliang

2016-01-01

The size of the primordial follicle pool determines the reproductive potential of mammalian females, and establishment of the pool is highly dependent on specific genes expression. However, the molecular mechanisms by which the essential genes are regulated coordinately to ensure primordial follicle assembly remain a mystery. Here, we show that the small GTPase Rac1 plays an indispensable role in controlling the formation of primordial follicles in mouse ovary. Employing fetal mouse ovary organ culture system, we demonstrate that disruption of Rac1 retarded the breakdown of germline cell cysts while Rac1 overexpression accelerated the formation of primordial follicles. In addition, in vivo inhibitor injection resulted in the formation of multi-oocyte follicles. Subsequent investigation showed that Rac1 induced nuclear import of STAT3 by physical binding. In turn, nuclear STAT3 directly activated the transcription of essential oocyte-specific genes, including Jagged1, GDF9, BMP15 and Nobox. Further, GDF9 and BMP15 regulated the translation of Notch2 via mTORC1 activation in pregranulosa cells. Overexression or addition of Jagged1, GDF9 and BMP15 not only reversed the effect of Rac1 disruption, but also accelerated primordial follicle formation via Notch2 signaling activation. Collectively, these results indicate that Rac1 plays important roles as a key regulator in follicular assembly. PMID:27050391

Genome-wide transposon mutagenesis of Proteus mirabilis: Essential genes, fitness factors for catheter-associated urinary tract infection, and the impact of polymicrobial infection on fitness requirements.

PubMed

Armbruster, Chelsie E; Forsyth-DeOrnellas, Valerie; Johnson, Alexandra O; Smith, Sara N; Zhao, Lili; Wu, Weisheng; Mobley, Harry L T

2017-06-01

The Gram-negative bacterium Proteus mirabilis is a leading cause of catheter-associated urinary tract infections (CAUTIs), which are often polymicrobial. Numerous prior studies have uncovered virulence factors for P. mirabilis pathogenicity in a murine model of ascending UTI, but little is known concerning pathogenesis during CAUTI or polymicrobial infection. In this study, we utilized five pools of 10,000 transposon mutants each and transposon insertion-site sequencing (Tn-Seq) to identify the full arsenal of P. mirabilis HI4320 fitness factors for single-species versus polymicrobial CAUTI with Providencia stuartii BE2467. 436 genes in the input pools lacked transposon insertions and were therefore concluded to be essential for P. mirabilis growth in rich medium. 629 genes were identified as P. mirabilis fitness factors during single-species CAUTI. Tn-Seq from coinfection with P. stuartii revealed 217/629 (35%) of the same genes as identified by single-species Tn-Seq, and 1353 additional factors that specifically contribute to colonization during coinfection. Mutants were constructed in eight genes of interest to validate the initial screen: 7/8 (88%) mutants exhibited the expected phenotypes for single-species CAUTI, and 3/3 (100%) validated the expected phenotypes for polymicrobial CAUTI. This approach provided validation of numerous previously described P. mirabilis fitness determinants from an ascending model of UTI, the discovery of novel fitness determinants specifically for CAUTI, and a stringent assessment of how polymicrobial infection influences fitness requirements. For instance, we describe a requirement for branched-chain amino acid biosynthesis by P. mirabilis during coinfection due to high-affinity import of leucine by P. stuartii. Further investigation of genes and pathways that provide a competitive advantage during both single-species and polymicrobial CAUTI will likely provide robust targets for therapeutic intervention to reduce P. mirabilis CAUTI incidence and severity.

Genome-wide transposon mutagenesis of Proteus mirabilis: Essential genes, fitness factors for catheter-associated urinary tract infection, and the impact of polymicrobial infection on fitness requirements

PubMed Central

Smith, Sara N.; Zhao, Lili; Wu, Weisheng

2017-01-01

The Gram-negative bacterium Proteus mirabilis is a leading cause of catheter-associated urinary tract infections (CAUTIs), which are often polymicrobial. Numerous prior studies have uncovered virulence factors for P. mirabilis pathogenicity in a murine model of ascending UTI, but little is known concerning pathogenesis during CAUTI or polymicrobial infection. In this study, we utilized five pools of 10,000 transposon mutants each and transposon insertion-site sequencing (Tn-Seq) to identify the full arsenal of P. mirabilis HI4320 fitness factors for single-species versus polymicrobial CAUTI with Providencia stuartii BE2467. 436 genes in the input pools lacked transposon insertions and were therefore concluded to be essential for P. mirabilis growth in rich medium. 629 genes were identified as P. mirabilis fitness factors during single-species CAUTI. Tn-Seq from coinfection with P. stuartii revealed 217/629 (35%) of the same genes as identified by single-species Tn-Seq, and 1353 additional factors that specifically contribute to colonization during coinfection. Mutants were constructed in eight genes of interest to validate the initial screen: 7/8 (88%) mutants exhibited the expected phenotypes for single-species CAUTI, and 3/3 (100%) validated the expected phenotypes for polymicrobial CAUTI. This approach provided validation of numerous previously described P. mirabilis fitness determinants from an ascending model of UTI, the discovery of novel fitness determinants specifically for CAUTI, and a stringent assessment of how polymicrobial infection influences fitness requirements. For instance, we describe a requirement for branched-chain amino acid biosynthesis by P. mirabilis during coinfection due to high-affinity import of leucine by P. stuartii. Further investigation of genes and pathways that provide a competitive advantage during both single-species and polymicrobial CAUTI will likely provide robust targets for therapeutic intervention to reduce P. mirabilis CAUTI incidence and severity. PMID:28614382

Pooled Sequencing of 531 Genes in Inflammatory Bowel Disease Identifies an Associated Rare Variant in BTNL2 and Implicates Other Immune Related Genes

PubMed Central

Prescott, Natalie J.; Lehne, Benjamin; Stone, Kristina; Lee, James C.; Taylor, Kirstin; Knight, Jo; Papouli, Efterpi; Mirza, Muddassar M.; Simpson, Michael A.; Spain, Sarah L.; Lu, Grace; Fraternali, Franca; Bumpstead, Suzannah J.; Gray, Emma; Amar, Ariella; Bye, Hannah; Green, Peter; Chung-Faye, Guy; Hayee, Bu’Hussain; Pollok, Richard; Satsangi, Jack; Parkes, Miles; Barrett, Jeffrey C.; Mansfield, John C.; Sanderson, Jeremy; Lewis, Cathryn M.; Weale, Michael E.; Schlitt, Thomas; Mathew, Christopher G.

2015-01-01

The contribution of rare coding sequence variants to genetic susceptibility in complex disorders is an important but unresolved question. Most studies thus far have investigated a limited number of genes from regions which contain common disease associated variants. Here we investigate this in inflammatory bowel disease by sequencing the exons and proximal promoters of 531 genes selected from both genome-wide association studies and pathway analysis in pooled DNA panels from 474 cases of Crohn’s disease and 480 controls. 80 variants with evidence of association in the sequencing experiment or with potential functional significance were selected for follow up genotyping in 6,507 IBD cases and 3,064 population controls. The top 5 disease associated variants were genotyped in an extension panel of 3,662 IBD cases and 3,639 controls, and tested for association in a combined analysis of 10,147 IBD cases and 7,008 controls. A rare coding variant p.G454C in the BTNL2 gene within the major histocompatibility complex was significantly associated with increased risk for IBD (p = 9.65x10−10, OR = 2.3[95% CI = 1.75–3.04]), but was independent of the known common associated CD and UC variants at this locus. Rare (<1%) and low frequency (1–5%) variants in 3 additional genes showed suggestive association (p<0.005) with either an increased risk (ARIH2 c.338-6C>T) or decreased risk (IL12B p.V298F, and NICN p.H191R) of IBD. These results provide additional insights into the involvement of the inhibition of T cell activation in the development of both sub-phenotypes of inflammatory bowel disease. We suggest that although rare coding variants may make a modest overall contribution to complex disease susceptibility, they can inform our understanding of the molecular pathways that contribute to pathogenesis. PMID:25671699

Microbiological investigations on the water of a thermal bath at Budapest.

PubMed

Szuróczki, Sára; Kéki, Zsuzsa; Káli, Szandra; Lippai, Anett; Márialigeti, Károly; Tóth, Erika

2016-06-01

Thermal baths are unique aquatic environments combining a wide variety of natural and anthropogenic ecological factors, which also appear in their microbiological state. There is limited information on the microbiology of thermal baths in their complexity, tracking community shifts from the thermal wells to the pools. In the present study, the natural microbial community of well and pool waters in Gellért bath was studied in detail by cultivation-based techniques. To isolate bacteria, 10% R2A and minimal synthetic media (with "bath water") with agar-agar and gellan gum were used after prolonged incubation time; moreover, polyurethane blocks covered with media were also applied. Strains were identified by sequencing their 16S rRNA gene after grouping them by amplified rDNA restriction analysis. From each sample, the dominance of Alphaproteobacteria was characteristic though their diversity differed among samples. Members of Actinobacteria, Firmicutes, Beta- and Gamma-proteobacteria, Deinococcus-Thermus, and Bacteroidetes were also identified. Representatives of Deinococcus-Thermus phylum appeared only in the pool water. The largest groups in the pool water belonged to the Tistrella and Chelatococcus genera. The most dominant member in the well water was a new taxon, its similarity to Hartmannibacter diazotrophicus as closest relative was 93.93%.

"RCL-Pooling Assay": A Simplified Method for the Detection of Replication-Competent Lentiviruses in Vector Batches Using Sequential Pooling.

PubMed

Corre, Guillaume; Dessainte, Michel; Marteau, Jean-Brice; Dalle, Bruno; Fenard, David; Galy, Anne

2016-02-01

Nonreplicative recombinant HIV-1-derived lentiviral vectors (LV) are increasingly used in gene therapy of various genetic diseases, infectious diseases, and cancer. Before they are used in humans, preparations of LV must undergo extensive quality control testing. In particular, testing of LV must demonstrate the absence of replication-competent lentiviruses (RCL) with suitable methods, on representative fractions of vector batches. Current methods based on cell culture are challenging because high titers of vector batches translate into high volumes of cell culture to be tested in RCL assays. As vector batch size and titers are continuously increasing because of the improvement of production and purification methods, it became necessary for us to modify the current RCL assay based on the detection of p24 in cultures of indicator cells. Here, we propose a practical optimization of this method using a pairwise pooling strategy enabling easier testing of higher vector inoculum volumes. These modifications significantly decrease material handling and operator time, leading to a cost-effective method, while maintaining optimal sensibility of the RCL testing. This optimized "RCL-pooling assay" ameliorates the feasibility of the quality control of large-scale batches of clinical-grade LV while maintaining the same sensitivity.

Genotoxic Effects in Swimmers Exposed to Disinfection By-products in Indoor Swimming Pools

PubMed Central

Kogevinas, Manolis; Villanueva, Cristina M.; Font-Ribera, Laia; Liviac, Danae; Bustamante, Mariona; Espinoza, Felicidad; Nieuwenhuijsen, Mark J.; Espinosa, Aina; Fernandez, Pilar; DeMarini, David M.; Grimalt, Joan O.; Grummt, Tamara; Marcos, Ricard

2010-01-01

Background Exposure to disinfection by-products (DBPs) in drinking water has been associated with cancer risk. A recent study (Villanueva et al. 2007; Am J Epidemiol 165:148–156) found an increased bladder cancer risk among subjects attending swimming pools relative to those not attending. Objectives We evaluated adults who swam in chlorinated pools to determine whether exposure to DBPs in pool water is associated with biomarkers of genotoxicity. Methods We collected blood, urine, and exhaled air samples from 49 nonsmoking adult volunteers before and after they swam for 40 min in an indoor chlorinated pool. We estimated associations between the concentrations of four trihalomethanes (THMs) in exhaled breath and changes in micronuclei (MN) and DNA damage (comet assay) in peripheral blood lymphocytes before and 1 hr after swimming; urine mutagenicity (Ames assay) before and 2 hr after swimming; and MN in exfoliated urothelial cells before and 2 weeks after swimming. We also estimated associations and interactions with polymorphisms in genes related to DNA repair or to DBP metabolism. Results After swimming, the total concentration of the four THMs in exhaled breath was seven times higher than before swimming. The change in the frequency of micronucleated lymphocytes after swimming increased in association with higher exhaled concentrations of the brominated THMs (p = 0.03 for bromodichloromethane, p = 0.05 for chlorodibromomethane, p = 0.01 for bromoform) but not chloroform. Swimming was not associated with DNA damage detectable by the comet assay. Urine mutagenicity increased significantly after swimming, in association with the higher concentration of exhaled bromoform (p = 0.004). We found no significant associations with changes in micronucleated urothelial cells. Conclusions Our findings support potential genotoxic effects of exposure to DBPs from swimming pools. The positive health effects gained by swimming could be increased by reducing the potential health risks of pool water. PMID:20833606

West Nile virus, Anopheles flavivirus, a novel flavivirus as well as Merida-like rhabdovirus Turkey in field-collected mosquitoes from Thrace and Anatolia.

PubMed

Öncü, Ceren; Brinkmann, Annika; Günay, Filiz; Kar, Sırrı; Öter, Kerem; Sarıkaya, Yasemen; Nitsche, Andreas; Linton, Yvonne-Marie; Alten, Bülent; Ergünay, Koray

2018-01-01

Mosquitoes are involved in the transmission and maintenance of several viral diseases with significant health impact. Biosurveillance efforts have also revealed insect-specific viruses, observed to cocirculate with pathogenic strains. This report describes the findings of flavivirus and rhabdovirus screening, performed in eastern Thrace and Aegean region of Anatolia during 2016, including and expanding on locations with previously-documented virus activity. A mosquito cohort of 1545 individuals comprising 14 species were collected and screened in 108 pools via generic and specific amplification and direct metagenomics by next generation sequencing. Seven mosquito pools (6.4%) were positive in the flavivirus screening. West Nile virus lineage 1 clade 1a sequences were characterized in a pool Culex pipiens sensu lato specimens, providing the initial virus detection in Aegean region following 2010 outbreak. In an Anopheles maculipennis sensu lato pool, sequences closely-related to Anopheles flaviviruses were obtained, with similarities to several African and Australian strains of this new insect-specific flavivirus clade. In pools comprising Uranotaenia unguiculata (n=3), Cx. pipiens s.l. (n=1) and Aedes caspius (n=1) mosquitoes, sequences of a novel flavivirus, distantly-related to Flavivirus AV2011, identified previously in Spain and Turkey, were characterized. Moreover, DNA forms of the novel flavivirus were detected in two Ur. unguiculata pools. These sequences were highly-similar to the sequences amplified from viral RNA, with undisrupted reading frames, suggest the occurrence of viral DNA forms in natural conditions within mosquito hosts. Rhabdovirus screening revealed sequences of a recently-described novel virus, named the Merida-like virus Turkey (MERDLVT) in 5 Cx. pipiens s.l. pools (4.6%). Partial L and N gene sequences of MERDLVT were well-conserved among strains, with evidence for geographical clustering in phylogenetic analyses. Metagenomics provided the near-full genomic sequence in a specimen, revealing an identical genome organization and limited divergence from the prototype MERDLVT isolate. Copyright © 2017 Elsevier B.V. All rights reserved.

Ecological and population genetics of locally rare plants: A review

Treesearch

Simon A. Lei

2001-01-01

Plant species with limited dispersal ability, narrow geographical and physiological tolerance ranges, as well as with specific habitat and ecological requirements are likely to be rare. Small and isolated populations and species contain low levels of within-population genetic variation in many plant species. The gene pool of plants is a product of phenotype-environment...

Post-glacial evolution of Panicum virgatum: centers of diversity and gene pools revealed by SSR markers and cpDNA sequences

USDA-ARS?s Scientific Manuscript database

Switchgrass (Panicum virgatum) is native to the tallgrass prairie and associated ecosystems of the central and eastern USA. It is highly valued as a component in tallgrass prairie and savanna restoration and conservation projects and a potential bioenergy feedstock. The purpose of this study was to ...

Draft Genome Sequences for Five Strains of Trabulsiella odontotermitis, Isolated from Heterotermes sp. Termite Gut

PubMed Central

Olvera-García, Myrna; Fontes-Perez, Héctor; Chávez-Martínez, America; Ruiz Barrera, Oscar; Rodríguez-Almeida, Felipe A.

2015-01-01

Trabulsiella odontotermitis represents a novel species in the genus Trabulsiella with no complete genome reported yet. Here, we describe the draft genome sequences of five isolates from termites present in the north of Mexico, which have an interesting pool of genes related to cellulose degradation with biotechnological application. PMID:26543120

Phaseolus vulgaris endornavirus 1 and Phaseolus vulgaris endornavirus 2 infecting common bean Phaseolus vulgaris genotypes show differential infection patterns between gene pools

USDA-ARS?s Scientific Manuscript database

We investigated the occurrence of two plant endornaviruses, Phaseolus vulgaris endornavirus 1 (PvEV-1) and Phaseolus vulgaris endornavirus 2 (PvEV-2), in breeding-lines, cultivars, landraces, and wild genotypes of common bean (Phaseolus vulgaris) as well as other Phaseolus species collected from two...

Diversity of bacterial dimethylsulfoniopropionate degradation genes in surface seawater of Arctic Kongsfjorden.

PubMed

Zeng, Yin-Xin; Qiao, Zong-Yun; Yu, Yong; Li, Hui-Rong; Luo, Wei

2016-09-08

Dimethylsulfoniopropionate (DMSP), which is the major source of organic sulfur in the world's oceans, plays a significant role in the global sulfur cycle. This compound is rapidly degraded by marine bacteria either by cleavage to dimethylsulfide (DMS) or demethylation to 3-methylmercaptopropionate (MMPA). The diversity of genes encoding bacterial demethylation (dmdA) and DMS production (dddL and dddP) were measured in Arctic Kongsfjorden. Both dmdA and dddL genes were detected in all stations along a transect from the outer to the inner fjord, while dddP gene was only found in the outer and middle parts of the fjord. The dmdA gene was completely confined to the Roseobacter clade, while the dddL gene was confined to the genus Sulfitobacter. Although the dddP gene pool was also dominated by homologs from the Roseobacter clade, there were a few dddP genes showing close relationships to both Alphaproteobacter and Gammaproteobacter. The results of this study suggest that the Roseobacter clade may play an important role in DMSP catabolism via both demethylation and cleavage pathways in surface waters of Kongsfjorden during summer.

Diversity of bacterial dimethylsulfoniopropionate degradation genes in surface seawater of Arctic Kongsfjorden

NASA Astrophysics Data System (ADS)

Zeng, Yin-Xin; Qiao, Zong-Yun; Yu, Yong; Li, Hui-Rong; Luo, Wei

2016-09-01

Dimethylsulfoniopropionate (DMSP), which is the major source of organic sulfur in the world’s oceans, plays a significant role in the global sulfur cycle. This compound is rapidly degraded by marine bacteria either by cleavage to dimethylsulfide (DMS) or demethylation to 3-methylmercaptopropionate (MMPA). The diversity of genes encoding bacterial demethylation (dmdA) and DMS production (dddL and dddP) were measured in Arctic Kongsfjorden. Both dmdA and dddL genes were detected in all stations along a transect from the outer to the inner fjord, while dddP gene was only found in the outer and middle parts of the fjord. The dmdA gene was completely confined to the Roseobacter clade, while the dddL gene was confined to the genus Sulfitobacter. Although the dddP gene pool was also dominated by homologs from the Roseobacter clade, there were a few dddP genes showing close relationships to both Alphaproteobacter and Gammaproteobacter. The results of this study suggest that the Roseobacter clade may play an important role in DMSP catabolism via both demethylation and cleavage pathways in surface waters of Kongsfjorden during summer.

Food commensal microbes as a potentially important avenue in transmitting antibiotic resistance genes.

PubMed

Wang, Hua H; Manuzon, Michele; Lehman, Mark; Wan, Kai; Luo, Hongliang; Wittum, Thomas E; Yousef, Ahmed; Bakaletz, Lauren O

2006-01-01

The rapid emergence of antibiotic-resistant (ART) pathogens is a major threat to public health. While the surfacing of ART food-borne pathogens is alarming, the magnitude of the antibiotic resistance (AR) gene pool in food-borne commensal microbes is yet to be revealed. Incidence of ART commensals in selected retail food products was examined in this study. The presence of 10(2)-10(7) CFU of ART bacteria per gram of foods in many samples, particularly in ready-to-eat, 'healthy' food items, indicates that the ART bacteria are abundant in the food chain. AR-encoding genes were detected in ART isolates, and Streptococcus thermophilus was found to be a major host for AR genes in cheese microbiota. Lactococcus lactis and Leuconostoc sp. isolates were also found carrying AR genes. The data indicate that food could be an important avenue for ART bacterial evolution and dissemination. AR-encoding plasmids from several food-borne commensals were transmitted to Streptococcus mutans via natural gene transformation under laboratory conditions, suggesting the possible transfer of AR genes from food commensals to human residential bacteria via horizontal gene transfer.

Ngaingan virus, a macropod-associated rhabdovirus, contains a second glycoprotein gene and seven novel open reading frames.

PubMed

Gubala, Aneta; Davis, Steven; Weir, Richard; Melville, Lorna; Cowled, Chris; Walker, Peter; Boyle, David

2010-03-30

Ngaingan virus (NGAV) was isolated from a pool of biting midges that were collected in the tropics of northern Australia. Reported here is the full-length sequence of the NGAV genome, which, at over 15.7 kb, is the largest in any rhabdovirus described to date and contains 13 genes, the highest number of genes observed in any (-) ssRNA virus. Seven of these putative genes show no significant homology to known proteins. Like viruses in the genus Ephemerovirus, NGAV possesses a second glycoprotein gene (G(NS)). Phylogenetic analyses, however, place NGAV within the yet to be classified "Hart Park" group containing Wongabel and Flanders viruses, which do not contain a second glycoprotein gene. Screening of various animal sera from northern Australia has indicated that NGAV is currently circulating in macropods (wallabies, wallaroos and kangaroos), highlighting the need for further studies to determine its potential to cause disease in these species. Crown Copyright 2009. Published by Elsevier Inc. All rights reserved.

Mixture models for detecting differentially expressed genes in microarrays.

PubMed

Jones, Liat Ben-Tovim; Bean, Richard; McLachlan, Geoffrey J; Zhu, Justin Xi

2006-10-01

An important and common problem in microarray experiments is the detection of genes that are differentially expressed in a given number of classes. As this problem concerns the selection of significant genes from a large pool of candidate genes, it needs to be carried out within the framework of multiple hypothesis testing. In this paper, we focus on the use of mixture models to handle the multiplicity issue. With this approach, a measure of the local FDR (false discovery rate) is provided for each gene. An attractive feature of the mixture model approach is that it provides a framework for the estimation of the prior probability that a gene is not differentially expressed, and this probability can subsequently be used in forming a decision rule. The rule can also be formed to take the false negative rate into account. We apply this approach to a well-known publicly available data set on breast cancer, and discuss our findings with reference to other approaches.

Quantification of functional genes from procaryotes in soil by PCR.

PubMed

Sharma, Shilpi; Radl, Viviane; Hai, Brigitte; Kloos, Karin; Fuka, Mirna Mrkonjic; Engel, Marion; Schauss, Kristina; Schloter, Michael

2007-03-01

Controlling turnover processes and fluxes in soils and other environments requires information about the gene pool and possibilities for its in situ induction. Therefore in the recent years there has been a growing interest in genes and transcripts coding for metabolic enzymes. Besides questions addressing redundancy and diversity, more and more attention is given on the abundance of specific DNA and mRNA in the different habitats. This review will describe several PCR techniques that are suitable for quantification of functional genes and transcripts such as MPN-PCR, competitive PCR and real-time PCR. The advantages and disadvantages of the mentioned methods are discussed. In addition, the problems of quantitative extraction of nucleic acid and substances that inhibit polymerase are described. Finally, some examples from recent papers are given to demonstrate the applicability and usefulness of the different approaches.

Prevalence of parvovirus in Minnesota turkeys.

PubMed

Sharafeldin, T A; Singh, A; Abdel-Glil, M Y; Mor, S K; Porter, R E; Goyal, S M

2017-02-01

Poult enteritis syndrome (PES) is characterized by enteritis and decreased body weight gain in growing turkey poults between one d and 7 wk of age. Another syndrome called light turkey syndrome (LTS) causes a decrease in body weight of adult tom turkeys in Minnesota leading to huge economic losses. Reovirus, rotavirus, and astrovirus have been found in LTS and PES flocks in Minnesota. We tested 80 fecal pools collected from four LTS flocks and 35 fecal pools from non-LTS flocks for the presence of parvovirus. In addition, 116 fecal and meconium samples from turkeys submitted to the Minnesota Veterinary Diagnostic Laboratory (MVDL) also were tested. The samples were tested by PCR using primers for the non-structural 1 (NS1) gene of parvovirus. Of the 80 samples from LTS flocks, 41 were positive for parvovirus while 20 of 35 samples from non-LTS flocks were positive. The prevalence of parvovirus in fecal samples submitted to MVDL was relatively low; only five of the 116 pools were positive. The partial NS1 gene sequences from LTS and non-LTS samples showed 98 to 100% nt identity except for one divergent turkey parvovirus (TuPV) strain that revealed 90% identity and clustered with chicken-like parvoviruses. The presence of this divergent strain suggests circulation of a recombinant strain of TuPV in Minnesota turkeys. Our results indicate that TuPVs are circulating in both LTS and non-LTS flocks of turkeys in Minnesota, and further experimental studies are indicated to study the role of TuPV in LTS. © 2016 Poultry Science Association Inc.

Association of Malignancy Prevalence With Test Properties and Performance of the Gene Expression Classifier in Indeterminate Thyroid Nodules.

PubMed

Al-Qurayshi, Zaid; Deniwar, Ahmed; Thethi, Tina; Mallik, Tilak; Srivastav, Sudesh; Murad, Fadi; Bhatia, Parisha; Moroz, Krzysztof; Sholl, Andrew B; Kandil, Emad

2017-04-01

It is crucial for clinicians to know the malignancy prevalence within each indeterminate cytologic category to estimate the performance of the gene expression classifier (GEC). To examine the variability in the performance of the GEC. This retrospective cohort study of patients with Bethesda category III and IV thyroid nodules used single-institution data from January 1, 2013, through February 29, 2016. Expected negative predictive value (NPV) was calculated by adopting published sensitivity and specificity. Observed NPV was calculated based on the true-negative rate. Outcomes were compared with pooled data from 11 studies published January 1, 2010, to January 31, 2016. A total of 145 patients with 154 thyroid nodules were included in the study (mean [SD] age, 56.0 [16.2] years; 106 females [73.1%]). Malignancy prevalence was 45%. On the basis of this prevalence, the expected NPV is 85% and the observed NPV is 69%. If the prevalence is assumed to be 25%, the expected NPV would be 94%, whereas the observed NPV would be 85%. Pooled data analysis of 11 studies comprising 1303 participants revealed a malignancy prevalence of 31% (95% CI, 29%-34%) and a pooled NPV of 92% (95% CI, 87%-96%). In this study, variability in the performance of the GEC was not solely a function of malignancy prevalence and may have been attributable to intrinsic variability of the test sensitivity and specificity. The utility of the GEC in practice is elusive because of this variability. A better definition of the GEC's intrinsic properties is needed.

Large Deletions at the SHOX Locus in the Pseudoautosomal Region Are Associated with Skeletal Atavism in Shetland Ponies

PubMed Central

Rafati, Nima; Andersson, Lisa S.; Mikko, Sofia; Feng, Chungang; Raudsepp, Terje; Pettersson, Jessica; Janecka, Jan; Wattle, Ove; Ameur, Adam; Thyreen, Gunilla; Eberth, John; Huddleston, John; Malig, Maika; Bailey, Ernest; Eichler, Evan E.; Dalin, Göran; Chowdary, Bhanu; Andersson, Leif; Lindgren, Gabriella; Rubin, Carl-Johan

2016-01-01

Skeletal atavism in Shetland ponies is a heritable disorder characterized by abnormal growth of the ulna and fibula that extend the carpal and tarsal joints, respectively. This causes abnormal skeletal structure and impaired movements, and affected foals are usually killed. In order to identify the causal mutation we subjected six confirmed Swedish cases and a DNA pool consisting of 21 control individuals to whole genome resequencing. We screened for polymorphisms where the cases and the control pool were fixed for opposite alleles and observed this signature for only 25 SNPs, most of which were scattered on genome assembly unassigned scaffolds. Read depth analysis at these loci revealed homozygosity or compound heterozygosity for two partially overlapping large deletions in the pseudoautosomal region (PAR) of chromosome X/Y in cases but not in the control pool. One of these deletions removes the entire coding region of the SHOX gene and both deletions remove parts of the CRLF2 gene located downstream of SHOX. The horse reference assembly of the PAR is highly fragmented, and in order to characterize this region we sequenced bacterial artificial chromosome (BAC) clones by single-molecule real-time (SMRT) sequencing technology. This considerably improved the assembly and enabled size estimations of the two deletions to 160−180 kb and 60−80 kb, respectively. Complete association between the presence of these deletions and disease status was verified in eight other affected horses. The result of the present study is consistent with previous studies in humans showing crucial importance of SHOX for normal skeletal development. PMID:27207956

The Low Molecular Weight Protein PsaI Stabilizes the Light-Harvesting Complex II Docking Site of Photosystem I.

PubMed

Plöchinger, Magdalena; Torabi, Salar; Rantala, Marjaana; Tikkanen, Mikko; Suorsa, Marjaana; Jensen, Poul-Erik; Aro, Eva Mari; Meurer, Jörg

2016-09-01

PsaI represents one of three low molecular weight peptides of PSI. Targeted inactivation of the plastid PsaI gene in Nicotiana tabacum has no measurable effect on photosynthetic electron transport around PSI or on accumulation of proteins involved in photosynthesis. Instead, the lack of PsaI destabilizes the association of PsaL and PsaH to PSI, both forming the light-harvesting complex (LHC)II docking site of PSI. These alterations at the LHCII binding site surprisingly did not prevent state transition but led to an increased incidence of PSI-LHCII complexes, coinciding with an elevated phosphorylation level of the LHCII under normal growth light conditions. Remarkably, LHCII was rapidly phosphorylated in ΔpsaI in darkness even after illumination with far-red light. We found that this dark phosphorylation also occurs in previously described mutants impaired in PSI function or state transition. A prompt shift of the plastoquinone (PQ) pool into a more reduced redox state in the dark caused an enhanced LHCII phosphorylation in ΔpsaI Since the redox status of the PQ pool is functionally connected to a series of physiological, biochemical, and gene expression reactions, we propose that the shift of mutant plants into state 2 in darkness represents a compensatory and/or protective metabolic mechanism. This involves an increased reduction and/or reduced oxidation of the PQ pool, presumably to sustain a balanced excitation of both photosystems upon the onset of light. © 2016 American Society of Plant Biologists. All rights reserved.

Spatial and temporal dynamics of organohalide-respiring bacteria in a heterogeneous PCE-DNAPL source zone.

PubMed

Cápiro, Natalie L; Löffler, Frank E; Pennell, Kurt D

2015-11-01

Effective treatment of sites contaminated with dense non-aqueous phase liquids (DNAPLs) requires detailed understanding of the microbial community responses to changes in source zone strength and architecture. Changes in the spatial and temporal distributions of the organohalide-respiring Dehalococcoides mccartyi (Dhc) strains and Geobacter lovleyi strain SZ (GeoSZ) were examined in a heterogeneous tetrachloroethene- (PCE-) DNAPL source zone within a two-dimensional laboratory-scale aquifer flow cell. As part of a combined remedy approach, flushing with 2.3 pore volumes (PVs) of 4% (w/w) solution of the nonionic, biodegradable surfactant Tween® 80 removed 55% of the initial contaminant mass, and resulted in a PCE-DNAPL distribution that contained 51% discrete ganglia and 49% pools (ganglia-to-pool ratio of 1.06). Subsequent bioaugmentation with the PCE-to-ethene-dechlorinating consortium BDI-SZ resulted in cis-1,2-dichloroethene (cis-DCE) formation after 1 PV (ca. 7 days), while vinyl chloride (VC) and ethene were detected 10 PVs after bioaugmentation. Maximum ethene yields (ca. 90 μM) within DNAPL pool and ganglia regions coincided with the detection of the vcrA reductive dehalogenase (RDase) gene that exceeded the Dhc 16S rRNA genes by 2.0±1.3 and 4.0±1.7 fold in the pool and ganglia regions, respectively. Dhc and GeoSZ cell abundance increased by up to 4 orders-of-magnitude after 28 PVs of steady-state operation, with 1 to 2 orders-of-magnitude increases observed in close proximity to residual PCE-DNAPL. These observations suggest the involvement of these dechlorinators the in observed PCE dissolution enhancements of up to 2.3 and 6.0-fold within pool and ganglia regions, respectively. Analysis of the solid and aqueous samples at the conclusion of the experiment revealed that the highest VC (≥155 μM) and ethene (≥65 μM) concentrations were measured in zones where Dhc and GeoSZ were predominately attached to the solids. These findings demonstrate dynamic responses of organohalide-respiring bacteria in a heterogeneous DNAPL source zone, and emphasize the influence of source zone architecture on bioremediation performance. Copyright © 2015 Elsevier B.V. All rights reserved.

Homozygous diploid deletion strains of Saccharomyces cerevisiae that determine lag phase and dehydration tolerance.

PubMed

D'Elia, Riccardo; Allen, Patricia L; Johanson, Kelly; Nickerson, Cheryl A; Hammond, Timothy G

2005-06-01

This study identifies genes that determine length of lag phase, using the model eukaryotic organism, Saccharomyces cerevisiae. We report growth of a yeast deletion series following variations in the lag phase induced by variable storage times after drying-down yeast on filters. Using a homozygous diploid deletion pool, lag times ranging from 0 h to 90 h were associated with increased drop-out of mitochondrial genes and increased survival of nuclear genes. Simple linear regression (R2 analysis) shows that there are over 500 genes for which > 70% of the variation can be explained by lag alone. In the genes with a positive correlation, such that the gene abundance increases with lag and hence the deletion strain is suitable for survival during prolonged storage, there is a strong predominance of nucleonic genes. In the genes with a negative correlation, such that the gene abundance decreases with lag and hence the strain may be critical for getting yeast out of the lag phase, there is a strong predominance of glycoproteins and transmembrane proteins. This study identifies yeast deletion strains with survival advantage on prolonged storage and amplifies our understanding of the genes critical for getting out of the lag phase.

Homozygous diploid deletion strains of Saccharomyces cerevisiae that determine lag phase and dehydration tolerance

NASA Technical Reports Server (NTRS)

D'Elia, Riccardo; Allen, Patricia L.; Johanson, Kelly; Nickerson, Cheryl A.; Hammond, Timothy G.

2005-01-01

This study identifies genes that determine length of lag phase, using the model eukaryotic organism, Saccharomyces cerevisiae. We report growth of a yeast deletion series following variations in the lag phase induced by variable storage times after drying-down yeast on filters. Using a homozygous diploid deletion pool, lag times ranging from 0 h to 90 h were associated with increased drop-out of mitochondrial genes and increased survival of nuclear genes. Simple linear regression (R2 analysis) shows that there are over 500 genes for which > 70% of the variation can be explained by lag alone. In the genes with a positive correlation, such that the gene abundance increases with lag and hence the deletion strain is suitable for survival during prolonged storage, there is a strong predominance of nucleonic genes. In the genes with a negative correlation, such that the gene abundance decreases with lag and hence the strain may be critical for getting yeast out of the lag phase, there is a strong predominance of glycoproteins and transmembrane proteins. This study identifies yeast deletion strains with survival advantage on prolonged storage and amplifies our understanding of the genes critical for getting out of the lag phase.

Patient-Level DNA Damage and Repair Pathway Profiles and Prognosis After Prostatectomy for High-Risk Prostate Cancer

PubMed Central

Evans, Joseph R.; Zhao, Shuang G.; Chang, S. Laura; Tomlins, Scott A.; Erho, Nicholas; Sboner, Andrea; Schiewer, Matthew J.; Spratt, Daniel E.; Kothari, Vishal; Klein, Eric A.; Den, Robert B.; Dicker, Adam P.; Karnes, R. Jeffrey; Yu, Xiaochun; Nguyen, Paul L.; Rubin, Mark A.; de Bono, Johann; Knudsen, Karen E.; Davicioni, Elai; Feng, Felix Y.

2017-01-01

IMPORTANCE A substantial number of patients diagnosed with high-risk prostate cancer are at risk for metastatic progression after primary treatment. Better biomarkers are needed to identify patients at the highest risk to guide therapy intensification. OBJECTIVE To create a DNA damage and repair (DDR) pathway profiling method for use as a prognostic signature biomarker in high-risk prostate cancer. DESIGN, SETTING, AND PARTICIPANTS A cohort of 1090 patients with high-risk prostate cancer who underwent prostatectomy and were treated at 3 different academic institutions were divided into a training cohort (n = 545) and 3 pooled validation cohorts (n = 232, 130, and 183) assembled for case-control or case-cohort studies. Profiling of 9 DDR pathways using 17 gene sets for GSEA (Gene Set Enrichment Analysis) of high-density microarray gene expression data from formalin-fixed paraffin-embedded prostatectomy samples with median 10.3 years follow-up was performed. Prognostic signature development from DDR pathway profiles was studied, and DDR pathway gene mutation in published cohorts was analyzed. MAIN OUTCOMES AND MEASURES Biochemical recurrence-free, metastasis-free, and overall survival. RESULTS Across the training cohort and pooled validation cohorts, 1090 men were studied; mean (SD) age at diagnosis was 65.3 (6.4) years. We found that there are distinct clusters of DDR pathways within the cohort, and DDR pathway enrichment is only weakly correlated with clinical variables such as age (Spearman ρ [ρ], range, −0.07 to 0.24), Gleason score (ρ, range, 0.03 to 0.20), prostate-specific antigen level (ρ, range, −0.07 to 0.10), while 13 of 17 DDR gene sets are strongly correlated with androgen receptor pathway enrichment (ρ, range, 0.33 to 0.82). In published cohorts, DDR pathway genes are rarely mutated. A DDR pathway profile prognostic signature built in the training cohort was significantly associated with biochemical recurrence-free, metastasis-free, and overall survival in the pooled validation cohorts independent of standard clinicopathological variables. The prognostic performance of the signature for metastasis-free survival appears to be stronger in the younger patients (HR, 1.67; 95%CI, 1.12–2.50) than in the older patients (HR, 0.77; 95%CI, 0.29–2.07) on multivariate Cox analysis. CONCLUSIONS AND RELEVANCE DNA damage and repair pathway profiling revealed patient-level variations and the DDR pathways are rarely affected by mutation. A DDR pathway signature showed strong prognostic performance with the long-term outcomes of metastasis-free and overall survival that may be useful for risk stratification of high-risk prostate cancer patients. PMID:26746117

Population-based rare variant detection via pooled exome or custom hybridization capture with or without individual indexing.

PubMed

Ramos, Enrique; Levinson, Benjamin T; Chasnoff, Sara; Hughes, Andrew; Young, Andrew L; Thornton, Katherine; Li, Allie; Vallania, Francesco L M; Province, Michael; Druley, Todd E

2012-12-06

Rare genetic variation in the human population is a major source of pathophysiological variability and has been implicated in a host of complex phenotypes and diseases. Finding disease-related genes harboring disparate functional rare variants requires sequencing of many individuals across many genomic regions and comparing against unaffected cohorts. However, despite persistent declines in sequencing costs, population-based rare variant detection across large genomic target regions remains cost prohibitive for most investigators. In addition, DNA samples are often precious and hybridization methods typically require large amounts of input DNA. Pooled sample DNA sequencing is a cost and time-efficient strategy for surveying populations of individuals for rare variants. We set out to 1) create a scalable, multiplexing method for custom capture with or without individual DNA indexing that was amenable to low amounts of input DNA and 2) expand the functionality of the SPLINTER algorithm for calling substitutions, insertions and deletions across either candidate genes or the entire exome by integrating the variant calling algorithm with the dynamic programming aligner, Novoalign. We report methodology for pooled hybridization capture with pre-enrichment, indexed multiplexing of up to 48 individuals or non-indexed pooled sequencing of up to 92 individuals with as little as 70 ng of DNA per person. Modified solid phase reversible immobilization bead purification strategies enable no sample transfers from sonication in 96-well plates through adapter ligation, resulting in 50% less library preparation reagent consumption. Custom Y-shaped adapters containing novel 7 base pair index sequences with a Hamming distance of ≥2 were directly ligated onto fragmented source DNA eliminating the need for PCR to incorporate indexes, and was followed by a custom blocking strategy using a single oligonucleotide regardless of index sequence. These results were obtained aligning raw reads against the entire genome using Novoalign followed by variant calling of non-indexed pools using SPLINTER or SAMtools for indexed samples. With these pipelines, we find sensitivity and specificity of 99.4% and 99.7% for pooled exome sequencing. Sensitivity, and to a lesser degree specificity, proved to be a function of coverage. For rare variants (≤2% minor allele frequency), we achieved sensitivity and specificity of ≥94.9% and ≥99.99% for custom capture of 2.5 Mb in multiplexed libraries of 22-48 individuals with only ≥5-fold coverage/chromosome, but these parameters improved to ≥98.7 and 100% with 20-fold coverage/chromosome. This highly scalable methodology enables accurate rare variant detection, with or without individual DNA sample indexing, while reducing the amount of required source DNA and total costs through less hybridization reagent consumption, multi-sample sonication in a standard PCR plate, multiplexed pre-enrichment pooling with a single hybridization and lesser sequencing coverage required to obtain high sensitivity.

AbMiner: a bioinformatic resource on available monoclonal antibodies and corresponding gene identifiers for genomic, proteomic, and immunologic studies.

PubMed

Major, Sylvia M; Nishizuka, Satoshi; Morita, Daisaku; Rowland, Rick; Sunshine, Margot; Shankavaram, Uma; Washburn, Frank; Asin, Daniel; Kouros-Mehr, Hosein; Kane, David; Weinstein, John N

2006-04-06

Monoclonal antibodies are used extensively throughout the biomedical sciences for detection of antigens, either in vitro or in vivo. We, for example, have used them for quantitation of proteins on "reverse-phase" protein lysate arrays. For those studies, we quality-controlled > 600 available monoclonal antibodies and also needed to develop precise information on the genes that encode their antigens. Translation among the various protein and gene identifier types proved non-trivial because of one-to-many and many-to-one relationships. To organize the antibody, protein, and gene information, we initially developed a relational database in Filemaker for our own use. When it became apparent that the information would be useful to many other researchers faced with the need to choose or characterize antibodies, we developed it further as AbMiner, a fully relational web-based database under MySQL, programmed in Java. AbMiner is a user-friendly, web-based relational database of information on > 600 commercially available antibodies that we validated by Western blot for protein microarray studies. It includes many types of information on the antibody, the immunogen, the vendor, the antigen, and the antigen's gene. Multiple gene and protein identifier types provide links to corresponding entries in a variety of other public databases, including resources for phosphorylation-specific antibodies. AbMiner also includes our quality-control data against a pool of 60 diverse cancer cell types (the NCI-60) and also protein expression levels for the NCI-60 cells measured using our high-density "reverse-phase" protein lysate microarrays for a selection of the listed antibodies. Some other available database resources give information on antibody specificity for one or a couple of cell types. In contrast, the data in AbMiner indicate specificity with respect to the antigens in a pool of 60 diverse cell types from nine different tissues of origin. AbMiner is a relational database that provides extensive information from our own laboratory and other sources on more than 600 available antibodies and the genes that encode the antibodies' antigens. The data will be made freely available at http://discover.nci.nih.gov/abminer.

Next generation haplotyping to decipher nuclear genomic interspecific admixture in Citrus species: analysis of chromosome 2.

PubMed

Curk, Franck; Ancillo, Gema; Garcia-Lor, Andres; Luro, François; Perrier, Xavier; Jacquemoud-Collet, Jean-Pierre; Navarro, Luis; Ollitrault, Patrick

2014-12-29

The most economically important Citrus species originated by natural interspecific hybridization between four ancestral taxa (Citrus reticulata, Citrus maxima, Citrus medica, and Citrus micrantha) and from limited subsequent interspecific recombination as a result of apomixis and vegetative propagation. Such reticulate evolution coupled with vegetative propagation results in mosaic genomes with large chromosome fragments from the basic taxa in frequent interspecific heterozygosity. Modern breeding of these species is hampered by their complex heterozygous genomic structures that determine species phenotype and are broken by sexual hybridisation. Nevertheless, a large amount of diversity is present in the citrus gene pool, and breeding to allow inclusion of desirable traits is of paramount importance. However, the efficient mobilization of citrus biodiversity in innovative breeding schemes requires previous understanding of Citrus origins and genomic structures. Haplotyping of multiple gene fragments along the whole genome is a powerful approach to reveal the admixture genomic structure of current species and to resolve the evolutionary history of the gene pools. In this study, the efficiency of parallel sequencing with 454 methodology to decipher the hybrid structure of modern citrus species was assessed by analysis of 16 gene fragments on chromosome 2. 454 amplicon libraries were established using the Fluidigm array system for 48 genotypes and 16 gene fragments from chromosome 2. Haplotypes were established from the reads of each accession and phylogenetic analyses were performed using the haplotypic data for each gene fragment. The length of 454 reads and the level of differentiation between the ancestral taxa of modern citrus allowed efficient haplotype phylogenetic assignations for 12 of the 16 gene fragments. The analysis of the mixed genomic structure of modern species and cultivars (i) revealed C. maxima introgressions in modern mandarins, (ii) was consistent with previous hypotheses regarding the origin of secondary species, and (iii) provided a new picture of the evolution of chromosome 2. 454 sequencing was an efficient strategy to establish haplotypes with significant phylogenetic assignations in Citrus, providing a new picture of the mixed structure on chromosome 2 in 48 citrus genotypes.

Association between estrogen receptora gene (ESR1) PvuII (T/C) and XbaI (A/G) polymorphisms and premature ovarian failure risk: evidence from a meta-analysis.

PubMed

He, Meirong; Shu, Jingcheng; Huang, Xing; Tang, Hui

2015-02-01

Genetic factors are important in the pathogenesis of Premature ovarian failure (POF). Notably, estrogen receptor-a (ESR1) has been suggested as a possible candidate gene for POF; however, published studies of ESR1 gene polymorphisms have been hampered by small sample sizes and inconclusive or ambiguous results. The aim of this meta analysis is to investigate the associations between two novel common ESR1 polymorphisms (intron 1 polymorphisms PvuII-rs2234693: T.C and XbaI-rs9340799: A.G) and POF. A comprehensive search was conducted to identify all studies on the association of ESR1 gene polymorphisms with POF up to August 2014. Pooled odds ratio (OR) and corresponding 95 % confidence interval (CI) were calculated using fixed-or random-effects model in the meta-analysis. Three studies covering 1396 subjects were identified. Pooled data showed significant association between ESR1 gene PvuII polymorphism and risk of POF: [allele model: Cvs. T, OR = 0.735, 95%CI: 0.624 ~ 0.865, p = 0.001; co-dominant models: CCvs.TT, OR = 0.540, 95%CI: 0.382 ~ 0.764, p = 0.001, CTvs.TT, OR = 0.735, 95%CI: 0.555 ~ 0.972, p = 0.031; dominant model: CT + CCvs.TT, OR = 0.618, 95%CI: 0.396 ~ 0.966, p = 0.035; recessive model: CCvs.TT + CT, OR = 0.659, 95%CI: 0.502 ~ 0.864, p = 0.003]. Subgroup analyses showed a significant association in all models in Asian population, but no significant association in any model in European population. For the XbaI polymorphism, overall, no significant association was observed under any genetic models. However, under dominant model, ESR1 gene XbaI polymorphism is significantly association with risk of POF in Asian population. The present meta-analysis suggests that ESR1gene PvuII polymorphism is significantly associated with an increased risk of POF. And ESR1gene XbaI polymorphism is not association with risk of POF overall. However, under dominant model, ESR1gene XbaI polymorphism is significantly association with risk of POF in Asian population. Further large and well-designed studies are needed to confirm the association.

A 3,000-loci transcription map of chromosome 3B unravels the structural and functional features of gene islands in hexaploid wheat.

PubMed

Rustenholz, Camille; Choulet, Frédéric; Laugier, Christel; Safár, Jan; Simková, Hana; Dolezel, Jaroslav; Magni, Federica; Scalabrin, Simone; Cattonaro, Federica; Vautrin, Sonia; Bellec, Arnaud; Bergès, Hélène; Feuillet, Catherine; Paux, Etienne

2011-12-01

To improve our understanding of the organization and regulation of the wheat (Triticum aestivum) gene space, we established a transcription map of a wheat chromosome (3B) by hybridizing a newly developed wheat expression microarray with bacterial artificial chromosome pools from a new version of the 3B physical map as well as with cDNA probes derived from 15 RNA samples. Mapping data for almost 3,000 genes showed that the gene space spans the whole chromosome 3B with a 2-fold increase of gene density toward the telomeres due to an increase in the number of genes in islands. Comparative analyses with rice (Oryza sativa) and Brachypodium distachyon revealed that these gene islands are composed mainly of genes likely originating from interchromosomal gene duplications. Gene Ontology and expression profile analyses for the 3,000 genes located along the chromosome revealed that the gene islands are enriched significantly in genes sharing the same function or expression profile, thereby suggesting that genes in islands acquired shared regulation during evolution. Only a small fraction of these clusters of cofunctional and coexpressed genes was conserved with rice and B. distachyon, indicating a recent origin. Finally, genes with the same expression profiles in remote islands (coregulation islands) were identified suggesting long-distance regulation of gene expression along the chromosomes in wheat.

Neo-Darwinism, the Modern Synthesis and selfish genes: are they of use in physiology?

PubMed Central

Noble, Denis

2011-01-01

This article argues that the gene-centric interpretations of evolution, and more particularly the selfish gene expression of those interpretations, form barriers to the integration of physiological science with evolutionary theory. A gene-centred approach analyses the relationships between genotypes and phenotypes in terms of differences (change the genotype and observe changes in phenotype). We now know that, most frequently, this does not correctly reveal the relationships because of extensive buffering by robust networks of interactions. By contrast, understanding biological function through physiological analysis requires an integrative approach in which the activity of the proteins and RNAs formed from each DNA template is analysed in networks of interactions. These networks also include components that are not specified by nuclear DNA. Inheritance is not through DNA sequences alone. The selfish gene idea is not useful in the physiological sciences, since selfishness cannot be defined as an intrinsic property of nucleotide sequences independently of gene frequency, i.e. the ‘success’ in the gene pool that is supposed to be attributable to the ‘selfish’ property. It is not a physiologically testable hypothesis. PMID:21135048

Neo-Darwinism, the modern synthesis and selfish genes: are they of use in physiology?

PubMed

Noble, Denis

2011-03-01

This article argues that the gene-centric interpretations of evolution, and more particularly the selfish gene expression of those interpretations, form barriers to the integration of physiological science with evolutionary theory. A gene-centred approach analyses the relationships between genotypes and phenotypes in terms of differences (change the genotype and observe changes in phenotype). We now know that, most frequently, this does not correctly reveal the relationships because of extensive buffering by robust networks of interactions. By contrast, understanding biological function through physiological analysis requires an integrative approach in which the activity of the proteins and RNAs formed from each DNA template is analysed in networks of interactions. These networks also include components that are not specified by nuclear DNA. Inheritance is not through DNA sequences alone. The selfish gene idea is not useful in the physiological sciences, since selfishness cannot be defined as an intrinsic property of nucleotide sequences independently of gene frequency, i.e. the 'success' in the gene pool that is supposed to be attributable to the 'selfish' property. It is not a physiologically testable hypothesis.

Genome-wide analysis of the structural genes regulating defense phenylpropanoid metabolism in Populus

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

Tschaplinski, Timothy J; Tsai, Chung-Jui; Harding, Scott A

Salicin-based phenolic glycosides, hydroxycinnamate derivatives and flavonoid-derived condensed tannins comprise up to one-third of Populus leaf dry mass. Genes regulating the abundance and chemical diversity of these substances have not been comprehensively analysed in tree species exhibiting this metabolically demanding level of phenolic metabolism. Here, shikimate-phenylpropanoid pathway genes thought to give rise to these phenolic products were annotated from the Populus genome, their expression assessed by semiquantitative or quantitative reverse transcription polymerase chain reaction (PCR), and metabolic evidence for function presented. Unlike Arabidopsis, Populus leaves accumulate an array of hydroxycinnamoyl-quinate esters, which is consistent with broadened function of the expandedmore » hydroxycinnamoyl-CoA transferase gene family. Greater flavonoid pathway diversity is also represented, and flavonoid gene families are larger. Consistent with expanded pathway function, most of these genes were upregulated during wound-stimulated condensed tannin synthesis in leaves. The suite of Populus genes regulating phenylpropanoid product accumulation should have important application in managing phenolic carbon pools in relation to climate change and global carbon cycling.« less

Differentially-Expressed Pseudogenes in HIV-1 Infection

PubMed Central

Gupta, Aditi; Brown, C. Titus; Zheng, Yong-Hui; Adami, Christoph

2015-01-01

Not all pseudogenes are transcriptionally silent as previously thought. Pseudogene transcripts, although not translated, contribute to the non-coding RNA pool of the cell that regulates the expression of other genes. Pseudogene transcripts can also directly compete with the parent gene transcripts for mRNA stability and other cell factors, modulating their expression levels. Tissue-specific and cancer-specific differential expression of these “functional” pseudogenes has been reported. To ascertain potential pseudogene:gene interactions in HIV-1 infection, we analyzed transcriptomes from infected and uninfected T-cells and found that 21 pseudogenes are differentially expressed in HIV-1 infection. This is interesting because parent genes of one-third of these differentially-expressed pseudogenes are implicated in HIV-1 life cycle, and parent genes of half of these pseudogenes are involved in different viral infections. Our bioinformatics analysis identifies candidate pseudogene:gene interactions that may be of significance in HIV-1 infection. Experimental validation of these interactions would establish that retroviruses exploit this newly-discovered layer of host gene expression regulation for their own benefit. PMID:26426037

IL-7–dependent STAT1 activation limits homeostatic CD4+ T cell expansion

PubMed Central

Le Saout, Cecile; Luckey, Megan A.; Villarino, Alejandro V.; Smith, Mindy; Hasley, Rebecca B.; Myers, Timothy G.; Imamichi, Hiromi; Park, Jung-Hyun; O’Shea, John J.; Lane, H. Clifford

2017-01-01

IL-7 regulates homeostatic mechanisms that maintain the overall size of the T cell pool throughout life. We show that, under steady-state conditions, IL-7 signaling is principally mediated by activation of signal transducers and activators of transcription 5 (STAT5). In contrast, under lymphopenic conditions, there is a modulation of STAT1 expression resulting in an IL-7–dependent STAT1 and STAT5 activation. Consequently, the IL-7–induced transcriptome is altered with enrichment of IFN-stimulated genes (ISGs). Moreover, STAT1 overexpression was associated with reduced survival in CD4+ T cells undergoing lymphopenia-induced proliferation (LIP). We propose a model in which T cells undergoing LIP upregulate STAT1 protein, “switching on” an alternate IL-7–dependent program. This mechanism could be a physiological process to regulate the expansion and size of the CD4+ T cell pool. During HIV infection, the virus could exploit this pathway, leading to the homeostatic dysregulation of the T cell pools observed in these patients. PMID:29202461

Habitat heterogeneity and connectivity shape microbial communities in South American peatlands

PubMed Central

Oloo, Felix; Valverde, Angel; Quiroga, María Victoria; Vikram, Surendra; Cowan, Don; Mataloni, Gabriela

2016-01-01

Bacteria play critical roles in peatland ecosystems. However, very little is known of how habitat heterogeneity affects the structure of the bacterial communities in these ecosystems. Here, we used amplicon sequencing of the 16S rRNA and nifH genes to investigate phylogenetic diversity and bacterial community composition in three different sub-Antarctic peat bog aquatic habitats: Sphagnum magellanicum interstitial water, and water from vegetated and non-vegetated pools. Total and putative nitrogen-fixing bacterial communities from Sphagnum interstitial water differed significantly from vegetated and non-vegetated pool communities (which were colonized by the same bacterial populations), probably as a result of differences in water chemistry and biotic interactions. Total bacterial communities from pools contained typically aquatic taxa, and were more dissimilar in composition and less species rich than those from Sphagnum interstitial waters (which were enriched in taxa typically from soils), probably reflecting the reduced connectivity between the former habitats. These results show that bacterial communities in peatland water habitats are highly diverse and structured by multiple concurrent factors. PMID:27162086

High-nitrogen fixation rates in the particulate and dissolved pools in the Western Tropical Pacific (Solomon and Bismarck Seas)

NASA Astrophysics Data System (ADS)

Berthelot, H.; Benavides, M.; Moisander, P. H.; Grosso, O.; Bonnet, S.

2017-08-01

Dinitrogen (N2) fixation rates were investigated in the euphotic layer of the Bismarck and Solomon Seas using 15N2 incubation assays taking into account both the particulate and the dissolved pools. Average depth-integrated particulate N2 fixation rates were 203 (range 43-399) and 1396 (range 176-3132) μmol N m-2 d-1 in the Bismarck and Solomon Seas, respectively. In both seas, N2 fixation measured in the dissolved pool was similar to particulate N2 fixation, highlighting the potentially substantial underestimation of N2 fixation in oceanic budgets when only particulate N2 fixation is considered. Among the diazotroph phylotypes targeted using quantitative polymerase chain reaction amplification of nifH genes, Trichodesmium was the most abundant. Regression analyses suggest that it accounted for the major proportion of N2 fixation. However, unicellular cyanobacterial and non-cyanobacterial diazotrophs were also occasionally abundant. This study reports high pelagic N2 fixation rates and confirms that the Western Tropical South Pacific is a hot spot for marine N2 fixation.

Overview of recurrent chromosomal losses in retinoblastoma detected by low coverage next generation sequencing

PubMed Central

García-Chequer, A.J.; Méndez-Tenorio, A.; Olguín-Ruiz, G.; Sánchez-Vallejo, C.; Isa, P.; Arias, C.F.; Torres, J.; Hernández-Angeles, A.; Ramírez-Ortiz, M.A.; Lara, C.; Cabrera-Muñoz, M.L.; Sadowinski-Pine, S.; Bravo-Ortiz, J.C.; Ramón-García, G.; Diegopérez-Ramírez, J.; Ramírez-Reyes, G.; Casarrubias-Islas, R.; Ramírez, J.; Orjuela, M.A.; Ponce-Castañeda, M.V.

2016-01-01

Genes are frequently lost or gained in malignant tumors and the analysis of these changes can be informative about the underlying tumor biology. Retinoblastoma is a pediatric intraocular malignancy, and since deletions in chromosome 13 have been described in this tumor, we performed genome wide sequencing with the Illumina platform to test whether recurrent losses could be detected in low coverage data from DNA pools of Rb cases. An in silico reference profile for each pool was created from the human genome sequence GRCh37p5; a chromosome integrity score and a graphics 40 Kb window analysis approach, allowed us to identify with high resolution previously reported non random recurrent losses in all chromosomes of these tumors. We also found a pattern of gains and losses associated to clear and dark cytogenetic bands respectively. We further analyze a pool of medulloblastoma and found a more stable genomic profile and previously reported losses in this tumor. This approach facilitates identification of recurrent deletions from many patients that may be biological relevant for tumor development. PMID:26883451

Adult height is associated with increased risk of ovarian cancer: a Mendelian randomisation study.

PubMed

Dixon-Suen, Suzanne C; Nagle, Christina M; Thrift, Aaron P; Pharoah, Paul D P; Ewing, Ailith; Pearce, Celeste Leigh; Zheng, Wei; Chenevix-Trench, Georgia; Fasching, Peter A; Beckmann, Matthias W; Lambrechts, Diether; Vergote, Ignace; Lambrechts, Sandrina; Van Nieuwenhuysen, Els; Rossing, Mary Anne; Doherty, Jennifer A; Wicklund, Kristine G; Chang-Claude, Jenny; Jung, Audrey Y; Moysich, Kirsten B; Odunsi, Kunle; Goodman, Marc T; Wilkens, Lynne R; Thompson, Pamela J; Shvetsov, Yurii B; Dörk, Thilo; Park-Simon, Tjoung-Won; Hillemanns, Peter; Bogdanova, Natalia; Butzow, Ralf; Nevanlinna, Heli; Pelttari, Liisa M; Leminen, Arto; Modugno, Francesmary; Ness, Roberta B; Edwards, Robert P; Kelley, Joseph L; Heitz, Florian; du Bois, Andreas; Harter, Philipp; Schwaab, Ira; Karlan, Beth Y; Lester, Jenny; Orsulic, Sandra; Rimel, Bobbie J; Kjær, Susanne K; Høgdall, Estrid; Jensen, Allan; Goode, Ellen L; Fridley, Brooke L; Cunningham, Julie M; Winham, Stacey J; Giles, Graham G; Bruinsma, Fiona; Milne, Roger L; Southey, Melissa C; Hildebrandt, Michelle A T; Wu, Xifeng; Lu, Karen H; Liang, Dong; Levine, Douglas A; Bisogna, Maria; Schildkraut, Joellen M; Berchuck, Andrew; Cramer, Daniel W; Terry, Kathryn L; Bandera, Elisa V; Olson, Sara H; Salvesen, Helga B; Thomsen, Liv Cecilie Vestrheim; Kopperud, Reidun K; Bjorge, Line; Kiemeney, Lambertus A; Massuger, Leon F A G; Pejovic, Tanja; Bruegl, Amanda; Cook, Linda S; Le, Nhu D; Swenerton, Kenneth D; Brooks-Wilson, Angela; Kelemen, Linda E; Lubiński, Jan; Huzarski, Tomasz; Gronwald, Jacek; Menkiszak, Janusz; Wentzensen, Nicolas; Brinton, Louise; Yang, Hannah; Lissowska, Jolanta; Høgdall, Claus K; Lundvall, Lene; Song, Honglin; Tyrer, Jonathan P; Campbell, Ian; Eccles, Diana; Paul, James; Glasspool, Rosalind; Siddiqui, Nadeem; Whittemore, Alice S; Sieh, Weiva; McGuire, Valerie; Rothstein, Joseph H; Narod, Steven A; Phelan, Catherine; Risch, Harvey A; McLaughlin, John R; Anton-Culver, Hoda; Ziogas, Argyrios; Menon, Usha; Gayther, Simon A; Ramus, Susan J; Gentry-Maharaj, Aleksandra; Wu, Anna H; Pike, Malcolm C; Tseng, Chiu-Chen; Kupryjanczyk, Jolanta; Dansonka-Mieszkowska, Agnieszka; Budzilowska, Agnieszka; Rzepecka, Iwona K; Webb, Penelope M

2018-04-01

Observational studies suggest greater height is associated with increased ovarian cancer risk, but cannot exclude bias and/or confounding as explanations for this. Mendelian randomisation (MR) can provide evidence which may be less prone to bias. We pooled data from 39 Ovarian Cancer Association Consortium studies (16,395 cases; 23,003 controls). We applied two-stage predictor-substitution MR, using a weighted genetic risk score combining 609 single-nucleotide polymorphisms. Study-specific odds ratios (OR) and 95% confidence intervals (CI) for the association between genetically predicted height and risk were pooled using random-effects meta-analysis. Greater genetically predicted height was associated with increased ovarian cancer risk overall (pooled-OR (pOR) = 1.06; 95% CI: 1.01-1.11 per 5 cm increase in height), and separately for invasive (pOR = 1.06; 95% CI: 1.01-1.11) and borderline (pOR = 1.15; 95% CI: 1.02-1.29) tumours. Women with a genetic propensity to being taller have increased risk of ovarian cancer. This suggests genes influencing height are involved in pathways promoting ovarian carcinogenesis.

Failure of bacterial screening to detect Staphylococcus aureus: the English experience of donor follow-up.

PubMed

Brailsford, S R; Tossell, J; Morrison, R; McDonald, C P; Pitt, T L

2018-05-24

Between February 2011 and December 2016, over 1·6 million platelet units, 36% pooled platelets, underwent bacterial screening prior to issue. Contamination rates for apheresis and pooled platelets were 0·02% and 0·07%, respectively. Staphylococcus aureus accounted for 21 contaminations, including four pooled platelets, one confirmed transfusion-transmitted infection (TTI) and three 'near-miss' incidents detected on visual inspection which were negative on screening. We describe follow-up investigations of 16 donors for skin carriage of S. aureus and molecular characterisation of donor and pack isolates. Units were screened by the BacT/ALERT 3D detection system. Contributing donors were interviewed and consent requested for skin and nasal swabbing. S. aureus isolates were referred for spa gene type and DNA macrorestriction profile to determine identity between carriage strains and packs. Donors of 10 apheresis and two pooled packs screen positive for S. aureus were confirmed as the source of contamination; seven had a history of skin conditions, predominantly eczema; 11 were nasal carriers. The 'near-miss' incidents were associated with apheresis donors, two donors harboured strains indistinguishable from the pack strain. The TTI was due to a screen-negative pooled unit, and a nasal isolate of one donor was indistinguishable from that in the unit. Staphylococcus aureus contamination is rare but potentially harmful in platelet units. Donor isolates showed almost universal correspondence in molecular type with pack isolates, thus confirming the source of contamination. The importance of visual inspection of packs prior to transfusion is underlined by the 'near-miss' incidents. © 2018 International Society of Blood Transfusion.

Mitochondrial Genome Analysis of Wild Rice (Oryza minuta) and Its Comparison with Other Related Species.

PubMed

Asaf, Sajjad; Khan, Abdul Latif; Khan, Abdur Rahim; Waqas, Muhammad; Kang, Sang-Mo; Khan, Muhammad Aaqil; Shahzad, Raheem; Seo, Chang-Woo; Shin, Jae-Ho; Lee, In-Jung

2016-01-01

Oryza minuta (Poaceae family) is a tetraploid wild relative of cultivated rice with a BBCC genome. O. minuta has the potential to resist against various pathogenic diseases such as bacterial blight (BB), white backed planthopper (WBPH) and brown plant hopper (BPH). Here, we sequenced and annotated the complete mitochondrial genome of O. minuta. The mtDNA genome is 515,022 bp, containing 60 protein coding genes, 31 tRNA genes and two rRNA genes. The mitochondrial genome organization and the gene content at the nucleotide level are highly similar (89%) to that of O. rufipogon. Comparison with other related species revealed that most of the genes with known function are conserved among the Poaceae members. Similarly, O. minuta mt genome shared 24 protein-coding genes, 15 tRNA genes and 1 ribosomal RNA gene with other rice species (indica and japonica). The evolutionary relationship and phylogenetic analysis revealed that O. minuta is more closely related to O. rufipogon than to any other related species. Such studies are essential to understand the evolutionary divergence among species and analyze common gene pools to combat risks in the current scenario of a changing environment.

New genes from non-coding sequence: the role of de novo protein-coding genes in eukaryotic evolutionary innovation.

PubMed

McLysaght, Aoife; Guerzoni, Daniele

2015-09-26

The origin of novel protein-coding genes de novo was once considered so improbable as to be impossible. In less than a decade, and especially in the last five years, this view has been overturned by extensive evidence from diverse eukaryotic lineages. There is now evidence that this mechanism has contributed a significant number of genes to genomes of organisms as diverse as Saccharomyces, Drosophila, Plasmodium, Arabidopisis and human. From simple beginnings, these genes have in some instances acquired complex structure, regulated expression and important functional roles. New genes are often thought of as dispensable late additions; however, some recent de novo genes in human can play a role in disease. Rather than an extremely rare occurrence, it is now evident that there is a relatively constant trickle of proto-genes released into the testing ground of natural selection. It is currently unknown whether de novo genes arise primarily through an 'RNA-first' or 'ORF-first' pathway. Either way, evolutionary tinkering with this pool of genetic potential may have been a significant player in the origins of lineage-specific traits and adaptations. © 2015 The Authors.

Gametocidal genes of Aegilops: segregation distorters in wheat-Aegilops wide hybridization.

PubMed

Niranjana, M

2017-08-01

Aegilops is a genus belonging to the family Poaceace, which have played an indispensible role in the evolution of bread wheat and continues to do so by transferring genes by wide hybridization. Being the secondary gene pool of wheat, gene transfer from Aegilops poses difficulties and segregation distortion is common. Gametocidal genes are the most well characterized class of segregation distorters reported in interspecific crosses of wheat with Aegilops. These "selfish" genetic elements ensure their preferential transmission to progeny at the cost of gametes lacking them without providing any phenotypic benefits to the plant, thereby causing a proportional reduction in fertility. Gametocidal genes (Gc) have been reported in different species of Aegilops belonging to the sections Aegilops (Ae. geniculata and Ae. triuncialis), Cylindropyrum (Ae. caudata and Ae. cylindrica), and Sitopsis (Ae. longissima, Ae. sharonensis, and Ae. speltoides). Gametocidal activity is mostly confined to 2, 3, and 4 homeologous groups of C, S, S 1 , S sh , and M g genomes. Removal of such genes is necessary for successful alien gene introgression and can be achieved by mutagenesis or allosyndetic pairing. However, there are some instances where Gc genes are constructively utilized for development of deletion stocks in wheat, improving genetic variability and chromosome engineering.

Identification of SNPs associated with muscle yield and quality traits using allelic-imbalance analysis analyses of pooled RNA-Seq samples in rainbow trout

USDA-ARS?s Scientific Manuscript database

Coding/functional SNPs change the biological function of a gene and, therefore, could serve as “large-effect” genetic markers. In this study, we used two bioinformatics pipelines, GATK and SAMtools, for discovering coding/functional SNPs with allelic-imbalances associated with total body weight, mus...

Sequencing of a QTL-rich region of the Theobroma cacao genome using pooled BACs and the identification of trait specific candidate genes

USDA-ARS?s Scientific Manuscript database

Background: BAC-based physical maps provide for sequencing across an entire genome or selected sub-genome regions of biological interest. Using the minimum tiling path as a guide, it is possible to select specific BAC clones from prioritized genome sections such as a genetically defined QTL interv...

Characterization and genome-wide association mapping of resistance to leaf rust, stem rust and stripe rust in a geographically diverse collection of spring wheat landraces

USDA-ARS?s Scientific Manuscript database

The challenge posed by rapidly changing wheat rust pathogens, both in virulence and in environmental adaptation, calls for the development and application of new techniques to accelerate the process of breeding for durable resistance. To expand the wheat resistance gene pool available for germplasm ...

An Ehrlichia strain from a llama (Lama glama) and Llama-associated ticks (Ixodes pacificus).

PubMed Central

Barlough, J E; Madigan, J E; Turoff, D R; Clover, J R; Shelly, S M; Dumler, J S

1997-01-01

An ehrlichia was identified in the blood of a diseased llama (lama glama). Sequencing of its 16S rRNA gene showed the ehrlichia to be closely related to members of the Ehrlichia phagocytophila genogroup. The agent was also found in a pool of ticks (Ixodes pacificus) collected at the llama site. PMID:9157118

A Neo-Darwinian View of Learning and Its Value for Science and Science Education.

ERIC Educational Resources Information Center

Schaverien, Lynette; Cosgrove, Mark

The modern history of biology shows how Darwin's selectionist theory has replaced instructionist theories in explaining the operations of living things: first with inheritance through the gene pool of the 1850s, and second with the replacement of a template theory of immune system function in the 1960s. Today scholars in several disciplines…

Sirt1 Protects Stressed Adult Hematopoietic Stem Cells | Center for Cancer Research

Cancer.gov

The immune system relies on a stable pool of hematopoietic stem and progenitor cells (HSPCs) to respond properly to injury or stress. Maintaining genomic integrity and appropriate gene expression is essential for HSPC homeostasis, and dysregulation can result in myeloproliferative disorders or loss of immune function. Sirt1 is a histone deacetylase that can protect embryonic

ROOT TRAITS AND NODULATION OF RECOMBINANT INBRED BEAN LINES FROM A ‘JAMAPA × CALIMA’ POPULATION INOCULATED WITH TWO STRAINS OF RHIZOBIUM

USDA-ARS?s Scientific Manuscript database

Bean cultivars of Andean and Middle American origin often have contrasting above-ground traits. Less is known, however, of possible differences in root traits of beans from different gene pools. Recombinant inbred lines (RIL) derived from a cross between the Andean cultivar ‘Calima’ and the Middle A...

Whole-tree silvic identifications and the microsatellite genetic structure of a red oak species complex in an Indiana old-growth forest

Treesearch

Preston R. Aldrich; George R. Parker; Charles H. Michler; Jeanne Romero-Severson

2003-01-01

The red oaks (Quercus section Lobatae) include important timber species, but we know little about their gene pools. Red oak species can be difficult to identify, possibly because of extensive interspecific hybridization, although most evidence of this is morphological. We used 15 microsatellite loci to examine the genetic...

Genetic structure of the invasive tree Ailanthus altissima in eastern United States cities

Treesearch

Preston R. Aldrich; Joseph S. Briguglio; Shyam N. Kapadia; Minesh U. Morker; Ankit Rawal; Preeti Kalra; Cynthia D. Huebner; Gary K. Greer

2010-01-01

Ailanthus altissima is an invasive tree from Asia. It now occurs in most US states, and although primarily an urban weed, it has become a problem in forested areas especially in the eastern states. Little is known about its genetic structure. We explore its naturalized gene pool from 28 populations, mostly of the eastern US where infestations are...

In Search of Genes Associated with Risk for Psychopathic Tendencies in Children: A Two-Stage Genome-Wide Association Study of Pooled DNA

ERIC Educational Resources Information Center

Viding, Essi; Hanscombe, Ken B.; Curtis, Charles J. C.; Davis, Oliver S. P.; Meaburn, Emma L.; Plomin, Robert

2010-01-01

Background: Quantitative genetic data from our group indicates that antisocial behaviour (AB) is strongly heritable when coupled with psychopathic, callous-unemotional (CU) personality traits. We have also demonstrated that the genetic influences for AB and CU overlap considerably. We conducted a genome-wide association scan that capitalises on…

The role of fire in Research Natural Areas in the Northern Rockies and Pacific Northwest

Treesearch

Sarah E. Greene; Angela Evenden

1996-01-01

Forest Service Research Natural Areas are established to preserve examples of all significant natural ecosystems for comparison with those influenced andlor managed by humans, to provide educational and research areas for ecological and environmental studies, and to preserve gene pools for typical and rare and endangered species. The Research Natural Area program in...

A modified mass selection scheme for creating winter-hardy faba bean (Vicia faba L.) lines with a broad genetic base

USDA-ARS?s Scientific Manuscript database

Winter-hardy faba bean (Vicia faba L.) from northern Europe is represented by a rather narrow gene pool. Limited selection gains for overwintering beyond a maximum of -25°C have restricted the adoption of this crop. Therefore, the faba bean collection maintained by the USDA-ARS National Plant Germpl...

Comparative proteomic analysis of pituitary glands from Huoyan geese between pre-laying and laying periods using an iTRAQ-based approach

PubMed Central

Xing, Zhe; Liu, Mei; Gao, Ming; Meng, Bo; Fan, Ruiming

2017-01-01

In this study, we performed a comprehensive evaluation of the proteomic profile of the pituitary gland of the Huoyan goose during the laying period compared to the pre-laying period using an iTRAQ-based approach. Protein samples were prepared from pituitary gland tissues of nine pre-laying period and nine laying period geese. Then the protein samples from three randomly selected geese within each period were pooled in equal amounts to generate one biological sample pool. We identified 684 differentially expressed proteins, including 418 up-regulated and 266 down-regulated proteins. GO annotation and KEGG pathway analyses of these proteins were conducted. Some of these proteins were found to be associated with hormone and neurotransmitter secretion and transport, neuropeptide signalling and GnRH signalling pathways, among others. Subsequently, the modification of the abundance of three proteins (prolactin, chromogranin-A and ITPR3) was verified using western blotting. Our results will provide a new source for mining genes and gene products related to the egg-laying performance of Huoyan geese, and may provide important information for the conservation and utilization of local goose breeds. PMID:28945779

Evidence for a genetic discontinuity between Neandertals and 24,000-year-old anatomically modern Europeans.

PubMed

Caramelli, David; Lalueza-Fox, Carles; Vernesi, Cristiano; Lari, Martina; Casoli, Antonella; Mallegni, Francesco; Chiarelli, Brunetto; Dupanloup, Isabelle; Bertranpetit, Jaume; Barbujani, Guido; Bertorelle, Giorgio

2003-05-27

During the late Pleistocene, early anatomically modern humans coexisted in Europe with the anatomically archaic Neandertals for some thousand years. Under the recent variants of the multiregional model of human evolution, modern and archaic forms were different but related populations within a single evolving species, and both have contributed to the gene pool of current humans. Conversely, the Out-of-Africa model considers the transition between Neandertals and anatomically modern humans as the result of a demographic replacement, and hence it predicts a genetic discontinuity between them. Following the most stringent current standards for validation of ancient DNA sequences, we typed the mtDNA hypervariable region I of two anatomically modern Homo sapiens sapiens individuals of the Cro-Magnon type dated at about 23 and 25 thousand years ago. Here we show that the mtDNAs of these individuals fall well within the range of variation of today's humans, but differ sharply from the available sequences of the chronologically closer Neandertals. This discontinuity is difficult to reconcile with the hypothesis that both Neandertals and early anatomically modern humans contributed to the current European gene pool.

Proteomics of the organohalide-respiring Epsilonproteobacterium Sulfurospirillum multivorans adapted to tetrachloroethene and other energy substrates

PubMed Central

Goris, Tobias; Schiffmann, Christian L.; Gadkari, Jennifer; Schubert, Torsten; Seifert, Jana; Jehmlich, Nico; von Bergen, Martin; Diekert, Gabriele

2015-01-01

Organohalide respiration is an environmentally important but poorly characterized type of anaerobic respiration. We compared the global proteome of the versatile organohalide-respiring Epsilonproteobacterium Sulfurospirillum multivorans grown with different electron acceptors (fumarate, nitrate, or tetrachloroethene [PCE]). The most significant differences in protein abundance were found for gene products of the organohalide respiration region. This genomic region encodes the corrinoid and FeS cluster containing PCE reductive dehalogenase PceA and other proteins putatively involved in PCE metabolism such as those involved in corrinoid biosynthesis. The latter gene products as well as PceA and a putative quinol dehydrogenase were almost exclusively detected in cells grown with PCE. This finding suggests an electron flow from the electron donor such as formate or pyruvate via the quinone pool and a quinol dehydrogenase to PceA and the terminal electron acceptor PCE. Two putative accessory proteins, an IscU-like protein and a peroxidase-like protein, were detected with PCE only and might be involved in PceA maturation. The proteome of cells grown with pyruvate instead of formate as electron donor indicates a route of electrons from reduced ferredoxin via an Epsilonproteobacterial complex I and the quinone pool to PCE. PMID:26387727

Aiding pest control management of long-tailed macaques (Macaca fascicularis fascicularis) in Malaysia by using molecular markers of mitochondrial DNA

NASA Astrophysics Data System (ADS)

Abdul-Latiff, M. A. B.; Abdul-Patah, P.; Yaakop, S.; Md-Zain, B. M.

2017-10-01

The long-tailed macaques (Macaca fascicularis fascicularis) has been the center of human wildlife conflict in Malaysia since 1970s. This well-adapted and opportunistic primates have been dominating wide range of habitat in Malaysia such as primary and secondary forest, mangrove, as well as human settlements. The conventional practices of translocation by the authorities are threatening the uniqueness of gene pool for this species and ironically contradicting with the ultimate purpose of genetic conservation of this species. The objectives of this study is to determine the level of genetic separation between populations of long-tailed macaques, primarily focusing on populations distributed in northern Peninsular Malaysia. A total of 954 base pairs of control regions mtDNA was sequenced and analyzed from 27 samples of M. fascicularis. The results exhibited a highly homogenous state of populations for long-tailed macaques genetically and this ultimately indicate unsuitable management and planning in terms of pest control management of the species. Authorities are suggested to translocate the species at least within the state boundaries to avoid homogeneity of gene pools for the particular species.

THE ROLES OF METAL IONS IN REGULATION BY RIBOSWITCHES

PubMed Central

2012-01-01

Metal ions are required by all organisms in order to execute an array of essential molecular functions. They play a critical role in many catalytic mechanisms and structural properties. Proper homeostasis of ions is critical; levels that are aberrantly low or high are deleterious to cellular physiology. To maintain stable intracellular pools, metal ion-sensing regulatory (metalloregulatory) proteins couple metal ion concentration fluctuations with expression of genes encoding for cation transport or sequestration. However, these transcriptional-based regulatory strategies are not the only mechanisms by which organisms coordinate metal ions with gene expression. Intriguingly, a few classes of signal-responsive RNA elements have also been discovered to function as metalloregulatory agents. This suggests that RNA-based regulatory strategies can be precisely tuned to intracellular metal ion pools, functionally akin to metalloregulatory proteins. In addition to these metal-sensing regulatory RNAs, there is a yet broader role for metal ions in directly assisting the structural integrity of other signal-responsive regulatory RNA elements. In this chapter, we discuss how the intimate physicochemical relationship between metal ions and nucleic acids is important for the structure and function of metal ion- and metabolite-sensing regulatory RNAs. PMID:22010271

The emotion system promotes diversity and evolvability

PubMed Central

Giske, Jarl; Eliassen, Sigrunn; Fiksen, Øyvind; Jakobsen, Per J.; Aksnes, Dag L.; Mangel, Marc; Jørgensen, Christian

2014-01-01

Studies on the relationship between the optimal phenotype and its environment have had limited focus on genotype-to-phenotype pathways and their evolutionary consequences. Here, we study how multi-layered trait architecture and its associated constraints prescribe diversity. Using an idealized model of the emotion system in fish, we find that trait architecture yields genetic and phenotypic diversity even in absence of frequency-dependent selection or environmental variation. That is, for a given environment, phenotype frequency distributions are predictable while gene pools are not. The conservation of phenotypic traits among these genetically different populations is due to the multi-layered trait architecture, in which one adaptation at a higher architectural level can be achieved by several different adaptations at a lower level. Our results emphasize the role of convergent evolution and the organismal level of selection. While trait architecture makes individuals more constrained than what has been assumed in optimization theory, the resulting populations are genetically more diverse and adaptable. The emotion system in animals may thus have evolved by natural selection because it simultaneously enhances three important functions, the behavioural robustness of individuals, the evolvability of gene pools and the rate of evolutionary innovation at several architectural levels. PMID:25100697

The emotion system promotes diversity and evolvability.

PubMed

Giske, Jarl; Eliassen, Sigrunn; Fiksen, Øyvind; Jakobsen, Per J; Aksnes, Dag L; Mangel, Marc; Jørgensen, Christian

2014-09-22

Studies on the relationship between the optimal phenotype and its environment have had limited focus on genotype-to-phenotype pathways and their evolutionary consequences. Here, we study how multi-layered trait architecture and its associated constraints prescribe diversity. Using an idealized model of the emotion system in fish, we find that trait architecture yields genetic and phenotypic diversity even in absence of frequency-dependent selection or environmental variation. That is, for a given environment, phenotype frequency distributions are predictable while gene pools are not. The conservation of phenotypic traits among these genetically different populations is due to the multi-layered trait architecture, in which one adaptation at a higher architectural level can be achieved by several different adaptations at a lower level. Our results emphasize the role of convergent evolution and the organismal level of selection. While trait architecture makes individuals more constrained than what has been assumed in optimization theory, the resulting populations are genetically more diverse and adaptable. The emotion system in animals may thus have evolved by natural selection because it simultaneously enhances three important functions, the behavioural robustness of individuals, the evolvability of gene pools and the rate of evolutionary innovation at several architectural levels.

Draft Genome of Scalindua rubra, Obtained from the Interface Above the Discovery Deep Brine in the Red Sea, Sheds Light on Potential Salt Adaptation Strategies in Anammox Bacteria.

PubMed

Speth, Daan R; Lagkouvardos, Ilias; Wang, Yong; Qian, Pei-Yuan; Dutilh, Bas E; Jetten, Mike S M

2017-07-01

Several recent studies have indicated that members of the phylum Planctomycetes are abundantly present at the brine-seawater interface (BSI) above multiple brine pools in the Red Sea. Planctomycetes include bacteria capable of anaerobic ammonium oxidation (anammox). Here, we investigated the possibility of anammox at BSI sites using metagenomic shotgun sequencing of DNA obtained from the BSI above the Discovery Deep brine pool. Analysis of sequencing reads matching the 16S rRNA and hzsA genes confirmed presence of anammox bacteria of the genus Scalindua. Phylogenetic analysis of the 16S rRNA gene indicated that this Scalindua sp. belongs to a distinct group, separate from the anammox bacteria in the seawater column, that contains mostly sequences retrieved from high-salt environments. Using coverage- and composition-based binning, we extracted and assembled the draft genome of the dominant anammox bacterium. Comparative genomic analysis indicated that this Scalindua species uses compatible solutes for osmoadaptation, in contrast to other marine anammox bacteria that likely use a salt-in strategy. We propose the name Candidatus Scalindua rubra for this novel species, alluding to its discovery in the Red Sea.

Epidemiology, ecology and gene pool of influenza A virus in Egypt: Will Egypt be the epicentre of the next influenza pandemic?

PubMed Central

Abdelwhab, EM; Abdel-Moneim, Ahmed S

2015-01-01

Outside Asia, Egypt is considered to be an influenza H5N1 epicentre and presents a far greater pandemic risk than other countries. The long-term endemicity of H5N1 and the recent emergence of H9N2 in poultry call attention to the need for unravelling the epidemiology, ecology and highly diverse gene pool of influenza A virus (IAV) in Egypt which is the aim of this review. Isolation of a considerable number of IAV subtypes from several avian and mammalian hosts was described. Co-infections of poultry with H5N1 and H9N2 and subclinical infections of pigs and humans with H1N1 and H5N1 may raise the potential for the reassortment of these viruses. Moreover, the adjustment of IAV genomes, particularly H5N1, to optimize their evolution toward efficient transmission in human is progressing in Egypt. Understanding the present situation of influenza viruses in Egypt will help in the control of the disease and can potentially prevent a possible pandemic. PMID:25635701

Meta-analysis of promoter methylation in eight tumor-suppressor genes and its association with the risk of thyroid cancer.

PubMed

Khatami, Fatemeh; Larijani, Bagher; Heshmat, Ramin; Keshtkar, Abbasali; Mohammadamoli, Mahsa; Teimoori-Toolabi, Ladan; Nasiri, Shirzad; Tavangar, Seyed Mohammad

2017-01-01

Promoter methylation in a number of tumor-suppressor genes (TSGs) can play crucial roles in the development of thyroid carcinogenesis. The focus of the current meta-analysis was to determine the impact of promoter methylation of eight selected candidate TSGs on thyroid cancer and to identify the most important molecules in this carcinogenesis pathway. A comprehensive search was performed using Pub Med, Scopus, and ISI Web of Knowledge databases, and eligible studies were included. The methodological quality of the included studies was evaluated according to the Newcastle Ottawa scale table and pooled odds ratios (ORs); 95% confidence intervals (CIs) were used to estimate the strength of the associations with Stata 12.0 software. Egger's and Begg's tests were applied to detect publication bias, in addition to the "Metatrim" method. A total of 55 articles were selected, and 135 genes with altered promoter methylation were found. Finally, we included eight TSGs that were found in more than four studies (RASSF1, TSHR, PTEN, SLC5A, DAPK, P16, RARβ2, and CDH1). The order of the pooled ORs for these eight TSGs from more to less significant was CDH1 (OR = 6.73), SLC5 (OR = 6.15), RASSF1 (OR = 4.16), PTEN (OR = 3.61), DAPK (OR = 3.51), P16 (OR = 3.31), TSHR (OR = 2.93), and RARβ2 (OR = 1.50). Analyses of publication bias and sensitivity confirmed that there was very little bias. Thus, our findings showed that CDH1 and SCL5A8 genes were associated with the risk of thyroid tumor genesis.

Using DGGE and 16S rRNA gene sequence analysis to evaluate changes in oral bacterial composition.

PubMed

Chen, Zhou; Trivedi, Harsh M; Chhun, Nok; Barnes, Virginia M; Saxena, Deepak; Xu, Tao; Li, Yihong

2011-01-01

To investigate whether a standard dental prophylaxis followed by tooth brushing with an antibacterial dentifrice will affect the oral bacterial community, as determined by denaturing gradient gel electrophoresis (DGGE) combined with 16S rRNA gene sequence analysis. Twenty-four healthy adults were instructed to brush their teeth using commercial dentifrice for 1 week during a washout period. An initial set of pooled supragingival plaque samples was collected from each participant at baseline (0 h) before prophylaxis treatment. The subjects were given a clinical examination and dental prophylaxis and asked to brush for 1 min with a dentifrice containing 0.3% triclosan, 2.0% PVM/MA copolymer and 0.243% sodium fluoride (Colgate Total). On the following day, a second set of pooled supragingival plaque samples (24 h) was collected. Total bacterial genomic DNA was isolated from the samples. Differences in the microbial composition before and after the prophylactic procedure and tooth brushing were assessed by comparing the DGGE profiles and 16S rRNA gene segments sequence analysis. Two distinct clusters of DGGE profiles were found, suggesting that a shift in the microbial composition had occurred 24 h after the prophylaxis and brushing. A detailed sequencing analysis of 16S rRNA gene segments further identified 6 phyla and 29 genera, including known and unknown bacterial species. Importantly, an increase in bacterial diversity was observed after 24 h, including members of the Streptococcaceae family, Prevotella, Corynebacterium, TM7 and other commensal bacteria. The results suggest that the use of a standard prophylaxis followed by the use of the dentifrice containing 0.3% triclosan, 2.0% PVM/MA copolymer and 0.243% sodium fluoride may promote a healthier composition within the oral bacterial community.

Domestication to Crop Improvement: Genetic Resources for Sorghum and Saccharum (Andropogoneae)

PubMed Central

Dillon, Sally L.; Shapter, Frances M.; Henry, Robert J.; Cordeiro, Giovanni; Izquierdo, Liz; Lee, L. Slade

2007-01-01

Background Both sorghum (Sorghum bicolor) and sugarcane (Saccharum officinarum) are members of the Andropogoneae tribe in the Poaceae and are each other's closest relatives amongst cultivated plants. Both are relatively recent domesticates and comparatively little of the genetic potential of these taxa and their wild relatives has been captured by breeding programmes to date. This review assesses the genetic gains made by plant breeders since domestication and the progress in the characterization of genetic resources and their utilization in crop improvement for these two related species. Genetic Resources The genome of sorghum has recently been sequenced providing a great boost to our knowledge of the evolution of grass genomes and the wealth of diversity within S. bicolor taxa. Molecular analysis of the Sorghum genus has identified close relatives of S. bicolor with novel traits, endosperm structure and composition that may be used to expand the cultivated gene pool. Mutant populations (including TILLING populations) provide a useful addition to genetic resources for this species. Sugarcane is a complex polyploid with a large and variable number of copies of each gene. The wild relatives of sugarcane represent a reservoir of genetic diversity for use in sugarcane improvement. Techniques for quantitative molecular analysis of gene or allele copy number in this genetically complex crop have been developed. SNP discovery and mapping in sugarcane has been advanced by the development of high-throughput techniques for ecoTILLING in sugarcane. Genetic linkage maps of the sugarcane genome are being improved for use in breeding selection. The improvement of both sorghum and sugarcane will be accelerated by the incorporation of more diverse germplasm into the domesticated gene pools using molecular tools and the improved knowledge of these genomes. PMID:17766842

De novo transcriptomic analysis and development of EST-SSR markers in the Siberian tiger (Panthera tigris altaica).

PubMed

Lu, Taofeng; Sun, Yujiao; Ma, Qin; Zhu, Minghao; Liu, Dan; Ma, Jianzhang; Ma, Yuehui; Chen, Hongyan; Guan, Weijun

2016-12-01

The Siberian tiger, Panthera tigris altaica, is an endangered species, and much more work is needed to protect this species, which is still vulnerable to extinction. Conservation efforts may be supported by the genetic assessment of wild populations, for which highly specific microsatellite markers are required. However, only a limited amount of genetic sequence data is available for this species. To identify the genes involved in the lung transcriptome and to develop additional simple sequence repeat (SSR) markers for the Siberian tiger, we used high-throughput RNA-Seq to characterize the Siberian tiger transcriptome in lung tissue (designated 'PTA-lung') and a pooled tissue sample (designated 'PTA'). Approximately 47.5 % (33,187/69,836) of the lung transcriptome was annotated in four public databases (Nr, Swiss-Prot, KEGG, and COG). The annotated genes formed a potential pool for gene identification in the tiger. An analysis of the genes differentially expressed in the PTA lung, and PTA samples revealed that the tiger may have suffered a series of diseases before death. In total, 1062 non-redundant SSRs were identified in the Siberian tiger transcriptome. Forty-three primer pairs were randomly selected for amplification reactions, and 26 of the 43 pairs were also used to evaluate the levels of genetic polymorphism. Fourteen primer pairs (32.56 %) amplified products that were polymorphic in size in P. tigris altaica. In conclusion, the transcriptome sequences will provide a valuable genomic resource for genetic research, and these new SSR markers comprise a reasonable number of loci for the genetic analysis of wild and captive populations of P. tigris altaica.

Plasmids of Carotenoid-Producing Paracoccus spp. (Alphaproteobacteria) - Structure, Diversity and Evolution

PubMed Central

Maj, Anna; Dziewit, Lukasz; Czarnecki, Jakub; Wlodarczyk, Miroslawa; Baj, Jadwiga; Skrzypczyk, Grazyna; Giersz, Dorota; Bartosik, Dariusz

2013-01-01

Plasmids are components of many bacterial genomes. They enable the spread of a large pool of genetic information via lateral gene transfer. Many bacterial strains contain mega-sized replicons and these are particularly common in Alphaproteobacteria. Considerably less is known about smaller alphaproteobacterial plasmids. We analyzed the genomes of 14 such plasmids residing in 4 multireplicon carotenoid-producing strains of the genus Paracoccus (Alphaproteobacteria): P. aestuarii DSM 19484, P. haeundaensis LG P-21903, P. marcusii DSM 11574 and P. marcusii OS22. Comparative analyses revealed mosaic structures of the plasmids and recombinational shuffling of diverse genetic modules involved in (i) plasmid replication, (ii) stabilization (including toxin-antitoxin systems of the relBE/parDE, tad-ata, higBA, mazEF and toxBA families) and (iii) mobilization for conjugal transfer (encoding relaxases of the MobQ, MobP or MobV families). A common feature of the majority of the plasmids is the presence of AT-rich sequence islets (located downstream of exc1-like genes) containing genes, whose homologs are conserved in the chromosomes of many bacteria (encoding e.g. RelA/SpoT, SMC-like proteins and a retron-type reverse transcriptase). The results of this study have provided insight into the diversity and plasticity of plasmids of Paracoccus spp., and of the entire Alphaproteobacteria. Some of the identified plasmids contain replication systems not described previously in this class of bacteria. The composition of the plasmid genomes revealed frequent transfer of chromosomal genes into plasmids, which significantly enriches the pool of mobile DNA that can participate in lateral transfer. Many strains of Paracoccus spp. have great biotechnological potential, and the plasmid vectors constructed in this study will facilitate genetic studies of these bacteria. PMID:24260361

The Val/Met functional polymorphism in COMT confers susceptibility to bipolar disorder: evidence from an association study and a meta-analysis.

PubMed

Zhang, Zhao; Lindpaintner, Klaus; Che, Ronglin; He, Zangdong; Wang, Peng; Yang, Ping; Feng, Guoyin; He, Lin; Shi, Yongyong

2009-10-01

The COMT gene is considered as one of the prominent candidate genes for susceptibility to BP, and most studies focused a functional polymorphism in the gene: the Val/Met polymorphism (rs4680). However, results from these studies are sometimes contradictory, due to small sample size or heterogeneity. In this study, we first investigate the possible association between the Val/Met polymorphism in COMT and bipolar disorder in the Han population, which has never been done before. Then a systematic meta-analysis was conducted to determine if the low-activity allele (Met) increases the risk of BP in different ethnic groups. A total of 478 BP patients and 469 healthy subjects were recruited in our case/control study. MIX software package was employed to perform the meta-analysis on 19 studies after careful search and selection. We observed statistically-significant differences in allele (p = 0.00060) and genotype (p = 0.00203) frequencies between patients and controls in our samples. The meta-analysis also provided a significant pooled OR for association of the Met allele in rs4680 with BP in the total population (p = 0.0223) and in the Asian population (p = 0.0232). Although a significant pooled OR was also found for the Caucasian population (p = 0.0409) after one of the studies as discussed below was removed, the role for Val/Met polymorphism in BP in Caucasian ethnicity was not yet to be confirmed. In conclusion, the low-activity allele (Met) of rs4680 in COMT gene possibly confers risk for bipolar disorder in the Han population, while it needs further evidence for concluding its association with BP in the Caucasian population.

GlnR-Mediated Regulation of ectABCD Transcription Expands the Role of the GlnR Regulon to Osmotic Stress Management

PubMed Central

Shao, ZhiHui; Deng, WanXin; Li, ShiYuan; He, JuanMei; Ren, ShuangXi; Huang, WeiRen; Lu, YinHua; Zhao, GuoPing

2015-01-01

ABSTRACT Ectoine and hydroxyectoine are excellent compatible solutes for bacteria to deal with environmental osmotic stress and temperature damages. The biosynthesis cluster of ectoine and hydroxyectoine is widespread among microorganisms, and its expression is activated by high salinity and temperature changes. So far, little is known about the mechanism of the regulation of the transcription of ect genes and only two MarR family regulators (EctR1 in methylobacteria and the EctR1-related regulator CosR in Vibrio cholerae) have been found to negatively regulate the expression of ect genes. Here, we characterize GlnR, the global regulator for nitrogen metabolism in actinomycetes, as a negative regulator for the transcription of ectoine/hydroxyectoine biosynthetic genes (ect operon) in Streptomyces coelicolor. The physiological role of this transcriptional repression by GlnR is proposed to protect the intracellular glutamate pool, which acts as a key nitrogen donor for both the nitrogen metabolism and the ectoine/hydroxyectoine biosynthesis. IMPORTANCE High salinity is deleterious, and cells must evolve sophisticated mechanisms to cope with this osmotic stress. Although production of ectoine and hydroxyectoine is one of the most frequently adopted strategies, the in-depth mechanism of regulation of their biosynthesis is less understood. So far, only two MarR family negative regulators, EctR1 and CosR, have been identified in methylobacteria and Vibrio, respectively. Here, our work demonstrates that GlnR, the global regulator for nitrogen metabolism, is a negative transcriptional regulator for ect genes in Streptomyces coelicolor. Moreover, a close relationship is found between nitrogen metabolism and osmotic resistance, and GlnR-mediated regulation of ect transcription is proposed to protect the intracellular glutamate pool. Meanwhile, the work reveals the multiple roles of GlnR in bacterial physiology. PMID:26170409

A High Resolution Genome-Wide Scan for Significant Selective Sweeps: An Application to Pooled Sequence Data in Laying Chickens

PubMed Central

Qanbari, Saber; Strom, Tim M.; Haberer, Georg; Weigend, Steffen; Gheyas, Almas A.; Turner, Frances; Burt, David W.; Preisinger, Rudolf; Gianola, Daniel; Simianer, Henner

2012-01-01

In most studies aimed at localizing footprints of past selection, outliers at tails of the empirical distribution of a given test statistic are assumed to reflect locus-specific selective forces. Significance cutoffs are subjectively determined, rather than being related to a clear set of hypotheses. Here, we define an empirical p-value for the summary statistic by means of a permutation method that uses the observed SNP structure in the real data. To illustrate the methodology, we applied our approach to a panel of 2.9 million autosomal SNPs identified from re-sequencing a pool of 15 individuals from a brown egg layer line. We scanned the genome for local reductions in heterozygosity, suggestive of selective sweeps. We also employed a modified sliding window approach that accounts for gaps in the sequence and increases scanning resolution by moving the overlapping windows by steps of one SNP only, and suggest to call this a “creeping window” strategy. The approach confirmed selective sweeps in the region of previously described candidate genes, i.e. TSHR, PRL, PRLHR, INSR, LEPR, IGF1, and NRAMP1 when used as positive controls. The genome scan revealed 82 distinct regions with strong evidence of selection (genome-wide p-value<0.001), including genes known to be associated with eggshell structure and immune system such as CALB1 and GAL cluster, respectively. A substantial proportion of signals was found in poor gene content regions including the most extreme signal on chromosome 1. The observation of multiple signals in a highly selected layer line of chicken is consistent with the hypothesis that egg production is a complex trait controlled by many genes. PMID:23209582

Ancient Migratory Events in the Middle East: New Clues from the Y-Chromosome Variation of Modern Iranians

PubMed Central

Grugni, Viola; Battaglia, Vincenza; Hooshiar Kashani, Baharak; Parolo, Silvia; Al-Zahery, Nadia; Achilli, Alessandro; Olivieri, Anna; Gandini, Francesca; Houshmand, Massoud; Sanati, Mohammad Hossein; Torroni, Antonio; Semino, Ornella

2012-01-01

Knowledge of high resolution Y-chromosome haplogroup diversification within Iran provides important geographic context regarding the spread and compartmentalization of male lineages in the Middle East and southwestern Asia. At present, the Iranian population is characterized by an extraordinary mix of different ethnic groups speaking a variety of Indo-Iranian, Semitic and Turkic languages. Despite these features, only few studies have investigated the multiethnic components of the Iranian gene pool. In this survey 938 Iranian male DNAs belonging to 15 ethnic groups from 14 Iranian provinces were analyzed for 84 Y-chromosome biallelic markers and 10 STRs. The results show an autochthonous but non-homogeneous ancient background mainly composed by J2a sub-clades with different external contributions. The phylogeography of the main haplogroups allowed identifying post-glacial and Neolithic expansions toward western Eurasia but also recent movements towards the Iranian region from western Eurasia (R1b-L23), Central Asia (Q-M25), Asia Minor (J2a-M92) and southern Mesopotamia (J1-Page08). In spite of the presence of important geographic barriers (Zagros and Alborz mountain ranges, and the Dasht-e Kavir and Dash-e Lut deserts) which may have limited gene flow, AMOVA analysis revealed that language, in addition to geography, has played an important role in shaping the nowadays Iranian gene pool. Overall, this study provides a portrait of the Y-chromosomal variation in Iran, useful for depicting a more comprehensive history of the peoples of this area as well as for reconstructing ancient migration routes. In addition, our results evidence the important role of the Iranian plateau as source and recipient of gene flow between culturally and genetically distinct populations. PMID:22815981

Cellular stress created by intermediary metabolite imbalances.

PubMed

Lee, Sang Jun; Trostel, Andrei; Le, Phuoc; Harinarayanan, Rajendran; Fitzgerald, Peter C; Adhya, Sankar

2009-11-17

Small molecules generally activate or inhibit gene transcription as externally added substrates or as internally accumulated end-products, respectively. Rarely has a connection been made that links an intracellular intermediary metabolite as a signal of gene expression. We report that a perturbation in the critical step of a metabolic pathway--the D-galactose amphibolic pathway--changes the dynamics of the pathways leading to accumulation of the intermediary metabolite UDP-galactose. This accumulation causes cell stress and transduces signals that alter gene expression so as to cope with the stress by restoring balance in the metabolite pool. This underscores the importance of studying the global effects of alterations in the level of intermediary metabolites in causing stress and coping with it by transducing signals to genes to reach a stable state of equilibrium (homeostasis). Such studies are an essential component in the integration of metabolomics, proteomics, and transcriptomics.

Metagenomics as a Tool for Enzyme Discovery: Hydrolytic Enzymes from Marine-Related Metagenomes.

PubMed

Popovic, Ana; Tchigvintsev, Anatoly; Tran, Hai; Chernikova, Tatyana N; Golyshina, Olga V; Yakimov, Michail M; Golyshin, Peter N; Yakunin, Alexander F

2015-01-01

This chapter discusses metagenomics and its application for enzyme discovery, with a focus on hydrolytic enzymes from marine metagenomic libraries. With less than one percent of culturable microorganisms in the environment, metagenomics, or the collective study of community genetics, has opened up a rich pool of uncharacterized metabolic pathways, enzymes, and adaptations. This great untapped pool of genes provides the particularly exciting potential to mine for new biochemical activities or novel enzymes with activities tailored to peculiar sets of environmental conditions. Metagenomes also represent a huge reservoir of novel enzymes for applications in biocatalysis, biofuels, and bioremediation. Here we present the results of enzyme discovery for four enzyme activities, of particular industrial or environmental interest, including esterase/lipase, glycosyl hydrolase, protease and dehalogenase.

Combining experimental evolution with next-generation sequencing: a powerful tool to study adaptation from standing genetic variation.

PubMed

Schlötterer, C; Kofler, R; Versace, E; Tobler, R; Franssen, S U

2015-05-01

Evolve and resequence (E&R) is a new approach to investigate the genomic responses to selection during experimental evolution. By using whole genome sequencing of pools of individuals (Pool-Seq), this method can identify selected variants in controlled and replicable experimental settings. Reviewing the current state of the field, we show that E&R can be powerful enough to identify causative genes and possibly even single-nucleotide polymorphisms. We also discuss how the experimental design and the complexity of the trait could result in a large number of false positive candidates. We suggest experimental and analytical strategies to maximize the power of E&R to uncover the genotype-phenotype link and serve as an important research tool for a broad range of evolutionary questions.

Sequencing of Australian wild rice genomes reveals ancestral relationships with domesticated rice.

PubMed

Brozynska, Marta; Copetti, Dario; Furtado, Agnelo; Wing, Rod A; Crayn, Darren; Fox, Glen; Ishikawa, Ryuji; Henry, Robert J

2017-06-01

The related A genome species of the Oryza genus are the effective gene pool for rice. Here, we report draft genomes for two Australian wild A genome taxa: O. rufipogon-like population, referred to as Taxon A, and O. meridionalis-like population, referred to as Taxon B. These two taxa were sequenced and assembled by integration of short- and long-read next-generation sequencing (NGS) data to create a genomic platform for a wider rice gene pool. Here, we report that, despite the distinct chloroplast genome, the nuclear genome of the Australian Taxon A has a sequence that is much closer to that of domesticated rice (O. sativa) than to the other Australian wild populations. Analysis of 4643 genes in the A genome clade showed that the Australian annual, O. meridionalis, and related perennial taxa have the most divergent (around 3 million years) genome sequences relative to domesticated rice. A test for admixture showed possible introgression into the Australian Taxon A (diverged around 1.6 million years ago) especially from the wild indica/O. nivara clade in Asia. These results demonstrate that northern Australia may be the centre of diversity of the A genome Oryza and suggest the possibility that this might also be the centre of origin of this group and represent an important resource for rice improvement. © 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

Postexposure protection of guinea pigs against a lethal ebola virus challenge is conferred by RNA interference.

PubMed

Geisbert, Thomas W; Hensley, Lisa E; Kagan, Elliott; Yu, Erik Zhaoying; Geisbert, Joan B; Daddario-DiCaprio, Kathleen; Fritz, Elizabeth A; Jahrling, Peter B; McClintock, Kevin; Phelps, Janet R; Lee, Amy C H; Judge, Adam; Jeffs, Lloyd B; MacLachlan, Ian

2006-06-15

Ebola virus (EBOV) infection causes a frequently fatal hemorrhagic fever (HF) that is refractory to treatment with currently available antiviral therapeutics. RNA interference represents a powerful, naturally occurring biological strategy for the inhibition of gene expression and has demonstrated utility in the inhibition of viral replication. Here, we describe the development of a potential therapy for EBOV infection that is based on small interfering RNAs (siRNAs). Four siRNAs targeting the polymerase (L) gene of the Zaire species of EBOV (ZEBOV) were either complexed with polyethylenimine (PEI) or formulated in stable nucleic acid-lipid particles (SNALPs). Guinea pigs were treated with these siRNAs either before or after lethal ZEBOV challenge. Treatment of guinea pigs with a pool of the L gene-specific siRNAs delivered by PEI polyplexes reduced plasma viremia levels and partially protected the animals from death when administered shortly before the ZEBOV challenge. Evaluation of the same pool of siRNAs delivered using SNALPs proved that this system was more efficacious, as it completely protected guinea pigs against viremia and death when administered shortly after the ZEBOV challenge. Additional experiments showed that 1 of the 4 siRNAs alone could completely protect guinea pigs from a lethal ZEBOV challenge. Further development of this technology has the potential to yield effective treatments for EBOV HF as well as for diseases caused by other agents that are considered to be biological threats.

Gene expression deregulation in postnatal skeletal muscle of TK2 deficient mice reveals a lower pool of proliferating myogenic progenitor cells.

PubMed

Paredes, João A; Zhou, Xiaoshan; Höglund, Stefan; Karlsson, Anna

2013-01-01

Loss of thymidine kinase 2 (TK2) causes a heterogeneous myopathic form of mitochondrial DNA (mtDNA) depletion syndrome (MDS) in humans that predominantly affects skeletal muscle tissue. In mice, TK2 deficiency also affects several tissues in addition to skeletal muscle, including brain, heart, adipose tissue, kidneys and causes death about 3 weeks after birth. We analysed skeletal muscle and heart muscle tissues of Tk2 knockout mice at postnatal development phase and observed that TK2 deficient pups grew slower and their skeletal muscles appeared significantly underdeveloped, whereas heart was close to normal in size. Both tissues showed mtDNA depletion and mitochondria with altered ultrastructure, as revealed by transmission electron microscopy. Gene expression microarray analysis showed a strong down-regulation of genes involved in cell cycle and cell proliferation in both tissues, suggesting a lower pool of undifferentiated proliferating cells. Analysis of isolated primary myoblasts from Tk2 knockout mice showed slow proliferation, less ability to differentiate and signs of premature senescence, even in absence of mtDNA depletion. Our data demonstrate that TK2 deficiency disturbs myogenic progenitor cells function in postnatal skeletal muscle and we propose this as one of the causes of underdeveloped phenotype and myopathic characteristic of the TK2 deficient mice, in addition to the progressive mtDNA depletion, mitochondrial damage and respiratory chain deficiency in post-mitotic differentiated tissue.

Gene Expression Deregulation in Postnatal Skeletal Muscle of TK2 Deficient Mice Reveals a Lower Pool of Proliferating Myogenic Progenitor Cells

PubMed Central

Paredes, João A.; Zhou, Xiaoshan; Höglund, Stefan; Karlsson, Anna

2013-01-01

Loss of thymidine kinase 2 (TK2) causes a heterogeneous myopathic form of mitochondrial DNA (mtDNA) depletion syndrome (MDS) in humans that predominantly affects skeletal muscle tissue. In mice, TK2 deficiency also affects several tissues in addition to skeletal muscle, including brain, heart, adipose tissue, kidneys and causes death about 3 weeks after birth. We analysed skeletal muscle and heart muscle tissues of Tk2 knockout mice at postnatal development phase and observed that TK2 deficient pups grew slower and their skeletal muscles appeared significantly underdeveloped, whereas heart was close to normal in size. Both tissues showed mtDNA depletion and mitochondria with altered ultrastructure, as revealed by transmission electron microscopy. Gene expression microarray analysis showed a strong down-regulation of genes involved in cell cycle and cell proliferation in both tissues, suggesting a lower pool of undifferentiated proliferating cells. Analysis of isolated primary myoblasts from Tk2 knockout mice showed slow proliferation, less ability to differentiate and signs of premature senescence, even in absence of mtDNA depletion. Our data demonstrate that TK2 deficiency disturbs myogenic progenitor cells function in postnatal skeletal muscle and we propose this as one of the causes of underdeveloped phenotype and myopathic characteristic of the TK2 deficient mice, in addition to the progressive mtDNA depletion, mitochondrial damage and respiratory chain deficiency in post-mitotic differentiated tissue. PMID:23341978

Evaluation of reference genes for gene expression studies in radish (Raphanus sativus L.) using quantitative real-time PCR.

PubMed

Xu, Yuanyuan; Zhu, Xianwen; Gong, Yiqin; Xu, Liang; Wang, Yan; Liu, Liwang

2012-08-03

Real-time quantitative reverse transcription PCR (RT-qPCR) is a rapid and reliable method for gene expression studies. Normalization based on reference genes can increase the reliability of this technique; however, recent studies have shown that almost no single reference gene is universal for all possible experimental conditions. In this study, eight frequently used reference genes were investigated, including Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Actin2/7 (ACT), Tubulin alpha-5 (TUA), Tubulin beta-1 (TUB), 18S ribosomal RNA (18SrRNA), RNA polymerase-II transcription factor (RPII), Elongation factor 1-b (EF-1b) and Translation elongation factor 2 (TEF2). Expression stability of candidate reference genes was examined across 27 radish samples, representing a range of tissue types, cultivars, photoperiodic and vernalization treatments, and developmental stages. The eight genes in these sample pools displayed a wide range of Ct values and were variably expressed. Two statistical software packages, geNorm and NormFinder showed that TEF2, RPII and ACT appeared to be relatively stable and therefore the most suitable for use as reference genes. These results facilitate selection of desirable reference genes for accurate gene expression studies in radish. Copyright © 2012 Elsevier Inc. All rights reserved.

Estimation of distribution overlap of urn models.

PubMed

Hampton, Jerrad; Lladser, Manuel E

2012-01-01

A classical problem in statistics is estimating the expected coverage of a sample, which has had applications in gene expression, microbial ecology, optimization, and even numismatics. Here we consider a related extension of this problem to random samples of two discrete distributions. Specifically, we estimate what we call the dissimilarity probability of a sample, i.e., the probability of a draw from one distribution not being observed in [Formula: see text] draws from another distribution. We show our estimator of dissimilarity to be a [Formula: see text]-statistic and a uniformly minimum variance unbiased estimator of dissimilarity over the largest appropriate range of [Formula: see text]. Furthermore, despite the non-Markovian nature of our estimator when applied sequentially over [Formula: see text], we show it converges uniformly in probability to the dissimilarity parameter, and we present criteria when it is approximately normally distributed and admits a consistent jackknife estimator of its variance. As proof of concept, we analyze V35 16S rRNA data to discern between various microbial environments. Other potential applications concern any situation where dissimilarity of two discrete distributions may be of interest. For instance, in SELEX experiments, each urn could represent a random RNA pool and each draw a possible solution to a particular binding site problem over that pool. The dissimilarity of these pools is then related to the probability of finding binding site solutions in one pool that are absent in the other.

Generation of an arrayed CRISPR-Cas9 library targeting epigenetic regulators: from high-content screens to in vivo assays

PubMed Central

2017-01-01

ABSTRACT The CRISPR-Cas9 system has revolutionized genome engineering, allowing precise modification of DNA in various organisms. The most popular method for conducting CRISPR-based functional screens involves the use of pooled lentiviral libraries in selection screens coupled with next-generation sequencing. Screens employing genome-scale pooled small guide RNA (sgRNA) libraries are demanding, particularly when complex assays are used. Furthermore, pooled libraries are not suitable for microscopy-based high-content screens or for systematic interrogation of protein function. To overcome these limitations and exploit CRISPR-based technologies to comprehensively investigate epigenetic mechanisms, we have generated a focused sgRNA library targeting 450 epigenetic regulators with multiple sgRNAs in human cells. The lentiviral library is available both in an arrayed and pooled format and allows temporally-controlled induction of gene knock-out. Characterization of the library showed high editing activity of most sgRNAs and efficient knock-out at the protein level in polyclonal populations. The sgRNA library can be used for both selection and high-content screens, as well as for targeted investigation of selected proteins without requiring isolation of knock-out clones. Using a variety of functional assays we show that the library is suitable for both in vitro and in vivo applications, representing a unique resource to study epigenetic mechanisms in physiological and pathological conditions. PMID:29327641

Insights into the environmental resistance gene pool from the genome sequence of the multidrug-resistant environmental isolate Escherichia coli SMS-3-5.

PubMed

Fricke, W Florian; Wright, Meredith S; Lindell, Angela H; Harkins, Derek M; Baker-Austin, Craig; Ravel, Jacques; Stepanauskas, Ramunas

2008-10-01

The increasing occurrence of multidrug-resistant pathogens of clinical and agricultural importance is a global public health concern. While antimicrobial use in human and veterinary medicine is known to contribute to the dissemination of antimicrobial resistance, the impact of microbial communities and mobile resistance genes from the environment in this process is not well understood. Isolated from an industrially polluted aquatic environment, Escherichia coli SMS-3-5 is resistant to a record number of antimicrobial compounds from all major classes, including two front-line fluoroquinolones (ciprofloxacin and moxifloxacin), and in many cases at record-high concentrations. To gain insights into antimicrobial resistance in environmental bacterial populations, the genome of E. coli SMS-3-5 was sequenced and compared to the genome sequences of other E. coli strains. In addition, selected genetic loci from E. coli SMS-3-5 predicted to be involved in antimicrobial resistance were phenotypically characterized. Using recombinant vector clones from shotgun sequencing libraries, resistance to tetracycline, streptomycin, and sulfonamide/trimethoprim was assigned to a single mosaic region on a 130-kb plasmid (pSMS35_130). The remaining plasmid backbone showed similarity to virulence plasmids from avian-pathogenic E. coli (APEC) strains. Individual resistance gene cassettes from pSMS35_130 are conserved among resistant bacterial isolates from multiple phylogenetic and geographic sources. Resistance to quinolones was assigned to several chromosomal loci, mostly encoding transport systems that are also present in susceptible E. coli isolates. Antimicrobial resistance in E. coli SMS-3-5 is therefore dependent both on determinants acquired from a mobile gene pool that is likely available to clinical and agricultural pathogens, as well, and on specifically adapted multidrug efflux systems. The association of antimicrobial resistance with APEC virulence genes on pSMS35_130 highlights the risk of promoting the spread of virulence through the extensive use of antibiotics.