Genomics

Functional clustering and lineage markers: Insights into cellular differentiation and gene function from large-scale microarray studies of purified primary cell populations.

Publication Date: 2010 Mar 5 PMID: 20211243
Authors: Hume, D. A. - Summers, K. M. - Raza, S. - Baillie, J. K. - Freeman, T. C.
Journal: Genomics

Very large microarray datasets showing gene expression across multiple tissues and cell populations potentially provide a window on the transcriptional networks that underpin the differences in functional activity between biological systems. Clusters of co-expressed genes potentially provide lineage markers, candidate regulators of cell function and, by applying the principle of guilt by association, candidate functions for genes of currently unknown function. We have analysed a dataset comprising pure cell populations from hemopoietic and non-hemopoietic cell types (http://biogps.gnf.org). Using a novel network visualisation and clustering approach, we demonstrate that it is possible to identify very tight expression signatures associated specifically with embryonic stem cells, mesenchymal cells and hematopoietic lineages. Selected examples validate the prediction that gene function can be inferred by co-expression. One expression cluster was enriched in phagocytes, which alongside endosome-lysosome constituents, contains genes that may make up a 'pathway' for phagocyte differentiation. Promoters of these genes are enriched for binding sites for the Ets/PU.1 and MITF family. Another cluster was associated with the production of a specific extracellular matrix, with high levels of gene expression shared by cells of mesenchymal origin (fibroblasts, adipocytes, osteoblasts and myoblasts). We discuss the limitations placed upon such data by the presence of alternative promoters with distinct tissue specificity within many protein-coding genes.

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Assembly algorithms for next-generation sequencing data.

Publication Date: 2010 Mar 5 PMID: 20211242
Authors: Miller, J. R. - Koren, S. - Sutton, G.
Journal: Genomics

The emergence of next-generation sequencing platforms led to resurgence of research in whole-genome shotgun assembly algorithms and software. DNA sequencing data from the Roche 454, Illumina/Solexa, and ABI SOLiD platforms typically present shorter read lengths, higher coverage, and different error profiles compared with Sanger sequencing data. Since 2005, several assembly software packages have been created or revised specifically for de novo assembly of next-generation sequencing data. This review summarizes and compares the published descriptions of packages named SSAKE, SHARCGS, VCAKE, Newbler, Celera Assembler, Euler, Velvet, ABySS, AllPaths, and SOAPdenovo. More generally, it compares the two standard methods known as the de Bruijn graph approach and the overlap/layout/consensus approach to assembly.

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Efficient discovery of ASCL1 regulatory sequences through transgene pooling.

Publication Date: 2010 Mar 3 PMID: 20206680
Authors: McGaughey, D. M. - McCallion, A. S.
Journal: Genomics

Zebrafish transgenesis is a powerful and increasingly common strategy to assay vertebrate transcriptional regulatory control. Several challenges remain, however, to the broader application of this technique; they include increasing the rate with which transgenes can be analyzed and maximizing the informational value of the data generated. Presently, many rely on the injection of individual constructs and the analysis of resulting reporter expression in mosaic G0 embryos. Here, we contrast these approaches, examining whether injecting pooled transgene constructs can increase the efficiency with which regulatory sequences can be assayed, restricting analysis to the offspring of germ line transmitting transgenic zebrafish in an effort to reduce potential subjectivity. We selected a 64kb interval encompassing the human ASCL1 locus as our model interval and report the analysis of 9 highly conserved putative enhancers therein. We identified 32 transgene-positive zebrafish, transmitting one or more independent constructs displaying ASCL1-like regulatory control. Through examination of embryos harboring one or more transgenes, we demonstrate that five of the nine sequences account for the observed control and describe their likely roles in ASCL1 regulation. These data demonstrate the utility of this approach and its potential for further adaptation and higher throughput application.

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The protein kinase Hal5p is the high-copy suppressor of lithium-sensitive mutations of genes involved in the sporulation and meiosis as well as the ergosterol biosynthesis in Saccharomyces cerevisiae.

Publication Date: 2010 Mar 3 PMID: 20206679
Authors: Zhao, J. - Lin, W. - Ma, X. - Lu, Q. - Ma, X. - Bian, G. - Jiang, L.
Journal: Genomics

From a genome-scale genetic screen, we have identified 114 lithium-sensitive and 6 lithium-tolerant gene mutations in Saccharomyces cerevisiae. Twenty-five of these identified lithium-sensitive mutations are of genes previously reported to be involved in sporulation and meiosis, whereas thirty-six of them are of genes involved in the vacuolar protein sorting (VPS) pathway, mainly functioning in the membrane docking and fusion. Accordingly, the lithium-sensitive phenotypes for one third of identified VPS mutants well correlate to their intracellular lithium contents in response to lithium stress. This indicates the integrity of the VPS pathway is critic for the ion homeostasis in yeast cells. The halotolerant protein kinase Hal5p, a regulator of the potassium transporter Trk1p, is shown to be the high-copy suppressor of nearly one third of identified lithium-sensitive mutations of genes involved in the sporulation and meiosis as well as in the biosynthesis of ergosterol. These results suggest that Hal5p-mediated ion homeostasis is important for these two biological processes.

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Genomic assessment of the evolution of the prion protein gene family in vertebrates.

Publication Date: 2010 Mar 2 PMID: 20206252
Authors: Harrison, P. M. - Kumar, M. - Khachane, A.
Journal: Genomics

Prion diseases are devastating neurological disorders caused by the propagation of particles containing an alternative beta-sheet-rich form of the prion protein (PrP). Genes paralogous to PrP, called Doppel and Shadoo, have been identified, that also have neuropathological relevance. To aid in the further functional characterization of PrP and its relatives, we annotated completely the PrP gene family (PrP-GF), in the genomes of 42 vertebrates, through combined strategic application of gene prediction programs and advanced remote homology detection techniques (such as HMMs, PSI-TBLASTN and pGenThreader). We have uncovered several previously undescribed paralogous genes and pseudogenes. We find that current high-quality genomic evidence indicates that the PrP relative Doppel, was likely present in the last common ancestor of present-day Tetrapoda, but was lost in the bird lineage, since its divergence from reptiles. Using the new gene annotations, we have defined the consensus of structural features that are characteristic of the PrP and Doppel structures, across diverse Tetrapoda clades. Furthermore, we describe in detail a transcribed pseudogene derived from Shadoo that is conserved across primates, and that overlaps the meiosis gene, SYCE1, thus possibly regulating its expression. In addition, we analysed the locus of Prnp/Prnd for significant conservation across the genomic DNA of eleven mammals, and determined the phylogenetic penetration of non-coding exons. The genomic evidence indicates that the second Prnp non-coding exon found in even-toed ungulates and rodents, is conserved in all high-coverage genome assemblies of primates (human, chimp, orang utan and macaque), and is, at least, likely to have fallen out of use during primate speciation. Furthermore, we have demonstrated that the Prnt gene (at the Prnp human locus) is conserved across at least sixteen mammals, and evolves like a long non-coding RNA, fashioned from fragments of ancient, long, interspersed elements. These annotations and evolutionary analyses will be of further use for functional characterisation of the PrP-GF, and will be updatable in a semi-automated fashion as more genomes accumulate.

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Polymorphisms in the human glutathione transferase Kappa (GSTK1) promoter alter gene expression.

Publication Date: 2010 Feb 26 PMID: 20193754
Authors: Shield, A. J. - Murray, T. P. - Cappello, J. Y. - Coggan, M. - Board, P. G.
Journal: Genomics

The level of glutathione transferase Kappa (GSTK1-1) has been correlated with obesity (Liu et.al. 2008 PNAS 105: 18302-7) and a polymorphism in the hGSTK1 promoter has been associated with insulin secretion and fat deposition (Gao et al 2009 Endocr J 56: 487-94). We searched for additional polymorphisms that may influence GSTK1-1 function or expression. Two SNPs were identified in the 5' non-coding region. A SNP at -1308 that occurs in Chinese subjects is predicted to eliminate a FXR/RXR transcription factor-binding site and causes a 55% increase in transcription rate in HepG2 cells and a 59% decrease in HEK293 cells. These data suggest that the impact of this polymorphism is complex and tissue specific. A SNP at -1032 alters a methylation site and represses transcription by 38%. These observations provide the first functional insight into genetic factors that regulate hGSTK1 expression and may directly influence insulin secretion and fat deposition.

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TSEG-1, a novel member of histone H2A variants, participates in spermatogenesis via promoting apoptosis of spermatogenic cells.

Publication Date: 2010 Feb 24 PMID: 20188161
Authors: Gu, C. - Tong, Q. - Zheng, L. - Liang, Z. - Pu, J. - Mei, H. - Hu, T. - Du, Z. - Tian, F. - Zeng, F.
Journal: Genomics

A novel variant of histone H2A, named as testis specific expressed gene 1 (TSEG-1), was identified from adult mouse testis. The TSEG-1 gene is 610-bp in length and consists of one exon. TSEG-1 transcript was robustly and exclusively expressed in adult mouse testis, mainly in spermatocytes. In developmental testis, the TSEG-1 transcript was robustly expressed since postnatal day (P) 21, peaked at P30, and gradually decreased in the testis of aging mouse. The surgical cryptorchidism mouse model showed an increase in the TSEG-1 expression, accompanied by enhanced apoptosis of spermatogenic cells. The EGFP-tagged TSEG-1 protein is located in the nuclei of cultured spermatocytes (GC-2spd cells). Transfection of TSEG-1 into GC-2spd cells resulted in suppressed cell viabilities, increased apoptosis, and decreased mitochondrial membrane potential. Intratesticular injection of TSEG-1 resulted in increased apoptosis of spermatogenic cells in vivo. These results suggest that TSEG-1 may participate in the spermatogenesis via regulating the apoptosis of spermatogenic cells.

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The construction of a tetraploid cotton genome wide comprehensive reference map.

Publication Date: 2010 Feb 18 PMID: 20171271
Authors: Yu, J. - Kohel, R. J. - Smith, C. W.
Journal: Genomics

Integration of multiple genomic maps provides a higher density of markers and greater genome coverage, which not only facilitates the identification and positioning of QTLs and candidate genes, but it also provides a basic structure for the genome sequence assembly. However, the diversity in markers and populations used in individual mapping studies limits the ability to fully integrate the available data. By concentrating on marker orders rather than marker distances, published map data could be used to produce a comprehensive reference map (CRM) that includes a majority of known markers with optimally estimated order of those markers across the genome. In this study, a tetraploid cotton genome-wide CRM was constructed from 28 public cotton genetic maps. The initial CRM contained 7,424 markers and represented over 93% of the combined mapping information from the 28 individual maps. The current output is stored and displayed through CottonDB (http://www.cottondb.org), the public cotton genome database.

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The consequence of natural selection on genetic variation in the mouse.

Publication Date: 2010 Feb 18 PMID: 20171270
Authors: Reuveni, E. - Birney, E. - Gross, C. T.
Journal: Genomics

Laboratory mouse strains are known to have emerged from recent interbreeding between individuals of Mus musculus isolated populations. As a result of this breeding history, the collection of polymorphisms observed between laboratory mouse strains is likely to harbor the effects of natural selection between reproductively isolated populations. Until now no study has systematically investigated the consequences of this breeding history on gene evolution. Here we have used a novel, unbiased evolutionary approach to predict the founder origin of laboratory mouse strains and to assess the balance between ancient and newly emerged mutations in the founder subspecies. Our results confirm a contribution from at least four distinct subspecies. Additionally, our method allowed us to identify regions of relaxed selective constraint among laboratory mouse strains. This unique structure of variation is likely to have significant consequences on the use of mouse to find genes underlying phenotypic variation.

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Identification of human specific gene duplications relative to other primates by array CGH and quantitative PCR.

Publication Date: 2010 Feb 11 PMID: 20153417
Authors: Armengol, G. - Knuutila, S. - Lozano, J. J. - Madrigal, I. - Caballin, M. R.
Journal: Genomics

In order to identify human lineage specific (HLS) copy number differences (CNDs) compared to other primates, we performed pair wise comparisons (human vs. chimpanzee, gorilla and orangutan) by using cDNA array comparative genomic hybridization (CGH). A set of 23 genes with HLS duplications were identified, as well as other lineage differences in gene copy number specific of chimpanzee, gorilla and orangutan. Each species has gained more copies of specific genes rather than losing gene copies. Eleven of the 23 genes have only been observed to have undergone HLS duplication in Fortna et al. (2004) and in the present study. Then, seven of these 11 genes were analyzed by quantitative PCR in chimpanzee, gorilla and orangutan, as well as in other six primate species (Hylobates lar, Cercopithecus aethiops, Papio hamadryas, Macaca mulatta, Lagothrix lagothricha, and Saimiri sciureus). Six genes confirmed array CGH data, and four of them appeared to have bona fide HLS duplications (ABCB10, E2F6, CDH12, and TDG genes). We propose that these gene duplications have a potential to contribute to specific human phenotypes.

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Knockdown of COPA, Identified by Loss-of-Function Screen, Induces Apoptosis and Suppresses Tumor Growth in Mesothelioma Mouse Model.

Publication Date: 2010 Feb 11 PMID: 20153416
Authors: Sudo, H. - Tsuji, A. B. - Sugyo, A. - Kohda, M. - Sogawa, C. - Yoshida, C. - Harada, Y. N. - Hino, O. - Saga, T.
Journal: Genomics

Malignant mesothelioma is a highly aggressive tumor arising from serosal surfaces of the pleura and is triggered by past exposure to asbestos. Currently, there is no widely accepted treatment for mesothelioma. Development of effective drug treatments for human cancers requires identification of therapeutic molecular targets. We therefore conducted a large-scale functional screening of mesothelioma cells using a genome-wide small interfering RNA library. We determined that knockdown of 39 genes suppressed mesothelioma cell proliferation. At least seven of the 39 genes-COPA, COPB2, EIF3S7, POLR2A, PSMA6, RBM8A, and RPL18A-would be involved in anti-apoptotic function. In particular, the COPA protein was highly expressed in some mesothelioma cell lines but not in a pleural mesothelial cell line. COPA knockdown induced apoptosis and suppressed tumor growth in a mesothelioma mouse model. Therefore, COPA may have the potential of a therapeutic target and a new diagnostic marker of mesothelioma.

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