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PubMed2005

Genome-wide associations of gene expression variation in humans.

The exploration of quantitative variation in human populations has become one of the major priorities for medical genetics. The successful identification of variants that contribute to complex traits is highly dependent on reliable assays and genetic maps. We have performed a genome-wide quantitative trait analysis of 630 genes in 60 unrelated Utah residents with ancestry from Northern and Western Europe using the publicly available phase I data of the International HapMap project. The genes are located in regions of the human genome with elevated functional annotation and disease interest including the ENCODE regions spanning 1% of the genome, Chromosome 21 and Chromosome 20q12-13.2. We apply three different methods of multiple test correction, including Bonferroni, false discovery rate, and permutations. For the 374 expressed genes, we find many regions with statistically significant association of single nucleotide polymorphisms (SNPs) with expression variation in lymphoblastoid cell lines after correcting for multiple tests. Based on our analyses, the signal proximal (cis-) to the genes of interest is more abundant and more stable than distal and trans across statistical methodologies. Our results suggest that regulatory polymorphism is widespread in the human genome and show that the 5-kb (phase I) HapMap has sufficient density to enable linkage disequilibrium mapping in humans. Such studies will significantly enhance our ability to annotate the non-coding part of the genome and interpret functional variation. In addition, we demonstrate that the HapMap cell lines themselves may serve as a useful resource for quantitative measurements at the cellular level.

Chromosome Mapping↗
PubMed2004

A single bifunctional UDP-GlcNAc/Glc 4-epimerase supports the synthesis of three cell surface glycoconjugates in Campylobacter jejuni.

The major cell-surface carbohydrates (lipooligosaccharide, capsule, and glycoprotein N-linked heptasaccharide) of Campylobacter jejuni NCTC 11168 contain Gal and/or GalNAc residues. GalE is the sole annotated UDP-glucose 4-epimerase in this bacterium. The presence of GalNAc residues in these carbohydrates suggested that GalE might be a UDP-GlcNAc 4-epimerase. GalE was shown to epimerize UDP-Glc and UDP-GlcNAc in coupled assays with C. jejuni glycosyltransferases and in sugar nucleotide epimerization equilibria studies. Thus, GalE possesses UDP-GlcNAc 4-epimerase activity and was renamed Gne. The Km(app) values of a purified MalE-Gne fusion protein for UDP-GlcNAc and UDP-GalNAc are 1087 and 1070 microm, whereas those for UDP-Glc and UDP-Gal are 780 and 784 microm. The kcat and kcat/Km(app) values were three to four times higher for UDP-GalNAc and UDP-Gal than for UDP-GlcNAc and UDP-Glc. The comparison of the kinetic parameters of MalE-Gne to those of other characterized bacterial UDP-GlcNAc 4-epimerases indicated that Gne is a bifunctional UDP-GlcNAc/Glc 4-epimerase. The UDP sugar-binding site of Gne was modeled by using the structure of the UDP-GlcNAc 4-epimerase WbpP from Pseudomonas aeruginosa. Small differences were noted, and these may explain the bifunctional character of the C. jejuni Gne. In a gne mutant of C. jejuni, the lipooligosaccharide was shown by capillary electrophoresis-mass spectrometry to be truncated by at least five sugars. Furthermore, both the glycoprotein N-linked heptasaccharide and capsule were no longer detectable by high resolution magic angle spinning NMR. These data indicate that Gne is the enzyme providing Gal and GalNAc residues with the synthesis of all three cell-surface carbohydrates in C. jejuni NCTC 11168.

Amino Acid Motifs↗
PubMed2026

Proteome analyses reveal endoplasmic reticulum stress-induced changes in protein abundance associated with Ube2j2 deficiency in human cell culture.

The unfolded protein response (UPR) helps reinstate cellular proteostasis upon an accumulation of misfolded proteins in the endoplasmic reticulum (ER), in part through ER-associated degradation (ERAD). Ube2j2 is an ER-localized E2 ubiquitin-conjugating enzyme that participates in ERAD. We used mass spectrometry analysis of cultured U2OS cells to investigate how the loss of Ube2j2 affects the cellular proteome in response to tunicamycin-induced ER stress. We constructed a network of twelve statistically distinct modules of protein abundance profiles across conditions. We describe the gene ontology annotations for each module along with the "hub gene" proteins whose abundance levels most closely adhere to each module's protein abundance profile. Our analysis identifies known Ube2j2-associated pathways (eg the UPR and ERAD) and cellular functions that were previously unassociated with Ube2j2 (eg RNA metabolism, ER-Golgi transport, and cell-cycle progression). These data are available via ProteomeXchange with identifier PXD076153 and provide avenues for further investigation into the cellular functions of Ube2j2 under basal and ER-stressed conditions.

Humans↗
PubMed2005

Targeted overexpression of the Escherichia coli MinC protein in higher plants results in abnormal chloroplasts.

Higher plant chloroplast division involves some of the same types of proteins that are required in prokaryotic cell division. These include two of the three Min proteins, MinD and MinE, encoded by the min operon in bacteria. Noticeably absent from annotated sequences from higher plants is a MinC homologue. A higher plant functional MinC homologue that would interfere with FtsZ polymerization, has yet to be identified. We sought to determine whether expression of the bacterial MinC in higher plants could affect chloroplast division. The Escherichia coli minC (EcMinC) gene was isolated and inserted behind the Arabidopsis thaliana RbcS transit peptide sequence for chloroplast targeting. This TP-EcMinC gene driven by the CaMV 35S(2) constitutive promoter was then transformed into tobacco (Nicotiana tabacum L.). Abnormally large chloroplasts were observed in the transgenic plants suggesting that overexpression of the E. coli MinC perturbed higher plant chloroplast division.

Chloroplasts↗
PubMed2004

Prediction and functional analysis of native disorder in proteins from the three kingdoms of life.

An automatic method for recognizing natively disordered regions from amino acid sequence is described and benchmarked against predictors that were assessed at the latest critical assessment of techniques for protein structure prediction (CASP) experiment. The method attains a Wilcoxon score of 90.0, which represents a statistically significant improvement on the methods evaluated on the same targets at CASP. The classifier, DISOPRED2, was used to estimate the frequency of native disorder in several representative genomes from the three kingdoms of life. Putative, long (>30 residue) disordered segments are found to occur in 2.0% of archaean, 4.2% of eubacterial and 33.0% of eukaryotic proteins. The function of proteins with long predicted regions of disorder was investigated using the gene ontology annotations supplied with the Saccharomyces genome database. The analysis of the yeast proteome suggests that proteins containing disorder are often located in the cell nucleus and are involved in the regulation of transcription and cell signalling. The results also indicate that native disorder is associated with the molecular functions of kinase activity and nucleic acid binding.

Databases, Genetic↗
PubMed2003

Drug-induced alterations in gene expression of the asexual blood forms of Plasmodium falciparum.

The complete genome sequence of the malarial parasite, Plasmodium falciparum [Gardner, M.J., Hall, N., Fung, E., White, O., Berriman, M., and Hyman, R.W. (2002) Nature 419: 498-510; Hyman, R.W., Fung, E., Conway, A., Kurdi, O., Mao, J., Miranda, M. et al. (2002) Nature 419: 534-537], has provided researchers with the informational base for establishing genomic [Volkman, S.K., Hartl, D.L., Wirth, D.F., Nielsen, K.M., Choi, M., Batalov, S., et al. (2002) Science 298: 216-218], proteomic [Florens, L., Washburn, M.P.J.D.R., Anthony, R.M., Grainger, M., Haynes, J.D., et al. (2002) Nature 419: 520-526; Lasonder, E., Ishihama, Y., Anderson, J.S., Vermunt, A.M.W., Pain, A., Sauerwein, R.W., et al. (2002) Nature 419: 537-542] and genome-wide RNA expression [Ben Mamoun, C., Gluzman, I.Y., Hott, C., MacMillan, S.K., Amarakone, A.S., Anderson, D.L., et al. (2001) Mol Microbiol 39: 26-36; Hayward, R.E., Derisi, J.L., Alfadhli, S., Kaslow, D.C., Brown, P.O., and Rathod, P.K. (2000) Mol Microbiol 35: 6-14] analyses in this system. In fact, we have previously utilized SAGE (serial analysis of gene expression) to identify abundant loci that probably constitute part of the active metabolome [Patankar, S., Munasinghe, A., Shoaibi, A., Cummings, L.M., and Wirth, D.F. (2001) Mol Biol Cell 12: 3114-3125], as well as to characterize antisense transcription on a global scale in Plasmodium. In the present study, the comprehensive annotation of SAGE libraries derived from an asexual stage population exposed to drug and its matched control was used to assess the modulation of gene expression by chloroquine. Here, we observed a constellation of changes, with the differential regulation of over 100 transcripts, and have confirmed the data by alternate methods. A few responsive loci, including PfMDR1, have previously been implicated in the mechanism of chloroquine action/resistance. Several others, however, were derived from unexpected categories, including a large number of unknown open reading frames (ORFs), whose induction after drug exposure may provide first hints to their possible function.

Animals↗
PubMed2004

Protein interaction mapping on a functional shotgun sequence of Rickettsia sibirica.

Protein interaction maps can reveal novel pathways and functional complexes, allowing 'guilt by association' annotation of uncharacterized proteins. To address the need for large-scale protein interaction analyses, a bacterial two-hybrid system was coupled with a whole genome shotgun sequencing approach for microbial genome analysis. We report the first large-scale proteomics study using this system, integrating de novo genome sequencing with functional interaction mapping and annotation in a high-throughput format. We apply the approach by shotgun sequencing and annotating the genome of Rickettsia sibirica strain 246, an obligate intracellular human pathogen among the Spotted Fever Group rickettsiae. The bacteria invade endothelial cells and cause lysis after large amounts of progeny have accumulated. Little is known about specific Rickettsial virulence factors and their mode of pathogenicity. Analysis of the combined genomic sequence and protein-protein interaction data for a set of virulence related Type IV secretion system (T4SS) proteins revealed over 250 interactions and will provide insight into the mechanism of Rickettsial pathogenicity.

Bacterial Proteins↗
PubMed1991

The X-inactivation imprinting model of the fragile-X syndrome: annotated references, 1990.

The X-inactivation imprinting model of the fragile-X syndrome (1), the inference from this model and from pedigree data that human oogonia are derived from two progenitor cells present after the initial event that leads to chromosome imprinting (13), and the general model of fragile sites (8), provide a theoretical framework that is consistent with data on the fragile-X syndrome. This framework has led to novel predictions, some of which have been tested. Results of most tests have been consistent with predictions (10, 13, 17, 18, 27, 28). More direct tests are necessary to explore the underlying mechanisms of chromosome change, here termed imprinting, of the molecular basis of the change, and of the mutation itself.

Dosage Compensation, Genetic↗
PubMed2006

Fine specificity and sequence of antibodies directed against the ectodomain of matrix protein 2 of influenza A virus.

The ectodomain of matrix protein 2 (M2e) has remained remarkably conserved amongst human influenza A viruses and is a target for Abs with protective activity. For these reasons, M2e is being investigated for its potential as a broadly protective influenza A virus vaccine. Here, we report on the fine specificity and sequence of seven M2e-specific mAbs isolated from three BALB/c mice after different immunization protocols. The mAbs recognized epitopes comprised within a 13aa long peptide corresponding to M2e(4-16). They originated from 4 distinct precursor B cells and showed a highly restricted variable (V) gene usage, in that their heavy chain V regions were all formed by the same V(H), D and J(H) gene segments and their light chain V regions made use of only two distinct Vkappa genes (Vkappa19-15/IGKV6-15 and Vkappa8-30/IGKV8-30; NCBI/IMGT annotation, respectively). The consensus sequence of the expressed V(H) genes belongs to the J558/HV1 family. It showed 96% identity with the BALB/c germline gene J558.n/IGHV1S137 and 100% identity with a V(H) gene expressed by several BALB/c B-1 B cells. This suggests that the consensus sequence is that of a functional BALB/c germline V(H) gene. The genetic restriction of this response may in part underlie the generally poor M2e-specific Ab response induced by infection.

Amino Acid Sequence↗
PubMed2002

Global analysis of a "simple" proteome: Methanococcus jannaschii.

The completed genome of Methanococcus jannaschii, including the main chromosome and two extra-chromosomal elements, predicts a proteome comprised of 1783 proteins. How many of those proteins are expressed at any given time and the relative abundance of the expressed proteins, however, cannot be predicted solely from the genome sequence. Two-dimensional gel electrophoresis coupled with peptide mass spectrometry is being used to identify the proteins expressed by M. jannaschii cells grown under different conditions as part of an effort to correlate protein expression with regulatory mechanisms. Here we describe the identification of 170 of the most abundant proteins found in total lysates of M. jannaschii grown under optimal fermentation conditions. To optimize the number of proteins detected, two different protein specific stains (Coomassie Blue R250 or silver nitrate) and two different first dimension separation methods (isoelectric focusing or nonequilibrium pH gradient electrophoresis) were used. Thirty-two percent of the proteins identified are annotated as hypothetical (21% conserved hypothetical and 11% hypothetical), 21% are enzymes involved in energy metabolism, 12% are proteins required for protein synthesis, and the remainder include proteins necessary for intermediary metabolism, cell division, and cell structure. Evidence of post-translational modification of numerous M. jannaschii proteins has been found, as well as indications of incomplete dissociation of protein-protein complexes. These results demonstrate the complexity of proteome analysis even when dealing with a relatively simple genome.

Archaeal Proteins↗
PubMed2006

Engineering embryonic stem cells with recombinase systems.

The combined use of site-specific recombination and gene targeting or trapping in embryonic stem cells (ESCs) has resulted in the emergence of technologies that enable the induction of mouse mutations in a prespecified temporal and spatially restricted manner. Their large-scale implementation by several international mouse mutagenesis programs will lead to the assembly of a library of ES cell lines harboring conditional mutations in every single gene of the mouse genome. In anticipation of this unprecedented resource, this chapter will focus on site-specific recombination strategies and issues pertinent to ESCs and mice. The upcoming ESC resource and the increasing sophistication of site-specific recombination technologies will greatly assist the functional annotation of the human genome and the animal modeling of human disease.

Animals↗
PubMed2002

DNA microarray analysis of cortical gene expression during early recirculation after focal brain ischemia in rat.

Focal brain ischemia is followed by changes in gene expression as reflected by altered mRNA levels. DNA microarray analysis can be used to survey thousands of genes for differential expression triggered by ischemic metabolic stress. In this study, Sprague-Dawley rats were subjected to 2 h of middle cerebral artery occlusion (MCAO) using an intravascular poly-L-lysine-coated filament, and brains were removed after 3 h of recirculation for mRNA isolation. A differential measurement of mRNAs from post-ischemic and sham control animals was performed using the Mouse UniGene 1 microarray. Established values for differential expression were used (> or =1.7 or < or =-1.7 fold), and hits (n=2-3 arrays) divided into known 'ischemia-hypoxia response' genes and 'newly connected' annotated genes. n=28 ischemia-hypoxia response genes were up-regulated and n=6 were down-regulated. Regulated genes comprised immediate early genes, heat shock proteins, anti-oxidative enzymes, trophic factors, and genes involved in RNA metabolism, inflammation and cell signaling. Based on the ability of the microarray to replicate known changes in gene expression, n=35 newly connected genes were found up-regulated and n=41 down-regulated. DNA microarray analysis allows one to develop novel working hypotheses for responses to brain ischemia based on the regulation of annotated genes.

Animals↗
PubMed2005

Transcriptome analysis of human gastric cancer.

To elucidate the genetic events associated with gastric cancer, 124,704 cDNA clones were collected from 37 human gastric cDNA libraries, including 20 full-length enriched cDNA libraries of gastric cancer cell lines and tissues from Korean patients. An analysis of the collected ESTs revealed that 97,930 high-quality ESTs coalesced into 13,001 clusters, of which 11,135 clusters (85.6%) were annotated to known ESTs. The analysis of the full-length cDNAs also revealed that 4862 clusters (51.7%) contained at least one putative full-length cDNA clone with an initiation codon, with the average length of the 5' UTR of 140 bp. A large number appear to have a diverse transcription start site (TSS). An examination of the TSS of some genes, such as TEGT and GAPD, using 5' RACE revealed that the predicted TSSs are actually found in human gastric cancer cells and that several TSSs differ depending on the specific gastric cell line. Furthermore, of the human gastric ESTs, 766 genes (9.5%) were present as putative alternatively spliced variants. Confirmation of the predicted spliced isoforms using RT-PCR showed that the predicted isoforms exist in gastric cancer cells and some isoforms coexist in gastric cell lines. These results provide potentially useful information for elucidating the molecular mechanisms associated with gastric oncogenesis.

5' Untranslated Regions↗
PubMed2006

Components of the antigen processing and presentation pathway revealed by gene expression microarray analysis following B cell antigen receptor (BCR) stimulation.

BACKGROUND: Activation of naïve B lymphocytes by extracellular ligands, e.g. antigen, lipopolysaccharide (LPS) and CD40 ligand, induces a combination of common and ligand-specific phenotypic changes through complex signal transduction pathways. For example, although all three of these ligands induce proliferation, only stimulation through the B cell antigen receptor (BCR) induces apoptosis in resting splenic B cells. In order to define the common and unique biological responses to ligand stimulation, we compared the gene expression changes induced in normal primary B cells by a panel of ligands using cDNA microarrays and a statistical approach, CLASSIFI (Cluster Assignment for Biological Inference), which identifies significant co-clustering of genes with similar Gene Ontology annotation. RESULTS: CLASSIFI analysis revealed an overrepresentation of genes involved in ion and vesicle transport, including multiple components of the proton pump, in the BCR-specific gene cluster, suggesting that activation of antigen processing and presentation pathways is a major biological response to antigen receptor stimulation. Proton pump components that were not included in the initial microarray data set were also upregulated in response to BCR stimulation in follow up experiments. MHC Class II expression was found to be maintained specifically in response to BCR stimulation. Furthermore, ligand-specific internalization of the BCR, a first step in B cell antigen processing and presentation, was demonstrated. CONCLUSION: These observations provide experimental validation of the computational approach implemented in CLASSIFI, demonstrating that CLASSIFI-based gene expression cluster analysis is an effective data mining tool to identify biological processes that correlate with the experimental conditional variables. Furthermore, this analysis has identified at least thirty-eight candidate components of the B cell antigen processing and presentation pathway and sets the stage for future studies focused on a better understanding of the components involved in and unique to B cell antigen processing and presentation.

Algorithms↗
PubMed2004

The Centre for Modeling Human Disease Gene Trap resource.

Gene trap mutagenesis of mouse embryonic stem cells generates random loss-of-function mutations, which can be identified by a sequence tag and can often report the endogenous expression of the mutated gene. The Centre for Modeling Human Disease is performing expression- and sequence-based screens of gene trap insertions to generate new mouse mutations as a resource for the scientific community. The gene trap insertions are screened using multiplexed in vitro differentiation and induction assays, and sequence tags are generated to complement expression profiles. Researchers may search for insertions in genes expressed in target cell lineages, under specific in vitro conditions, or based upon sequence identity via an online searchable database (http://www.cmhd.ca/sub/genetrap.asp). The clones are available as a resource to researchers worldwide to help to functionally annotate the mammalian genome and will serve as a source to test candidate loci identified by phenotype-driven mutagenesis screens.

Animals↗
PubMed2001

Proteome study of colorectal carcinogenesis.

Development of cancer is a complex process involving multiple changes in gene expression. To unravel these alterations, a proteome approach aimed at the identification of qualitative and quantitative changes in protein composition, including their post-translational modifications, attracts great attention. Our study was focused on the identification of proteins whose amount is altered in the course of malignant transformation of colon mucosa. Proteins extracted from tissue specimens or cell lysates were separated by two-dimensional gel electrophoresis (2-DE). Comparative analyses of 2-DE protein patterns were done using computerized image analysis. Selected proteins exhibiting statistically significant abundance alterations comparing healthy and diseased tissues were identified by mass spectrometry. Globally, we have found 57 proteins that exhibited either a significant decrease or increase in amount in pathological tissues, and 18 of these were annotated by mass spectrometry. The alterations in the expression of nine proteins were common for both precancerous and neoplastic tissues suggesting their role in colon tumorigenesis. The epithelial origin of all identified spots was checked in two cell lines Caco-2 and DLD-1 originating from well-differentiated and poorly differentiated colon carcinoma, respectively.

Adenocarcinoma↗
PubMed2006

Definition and spatial annotation of the dynamic secretome during early kidney development.

The term "secretome" has been defined as a set of secreted proteins (Grimmond et al. [2003] Genome Res 13:1350-1359). The term "secreted protein" encompasses all proteins exported from the cell including growth factors, extracellular proteinases, morphogens, and extracellular matrix molecules. Defining the genes encoding secreted proteins that change in expression during organogenesis, the dynamic secretome, is likely to point to key drivers of morphogenesis. Such secreted proteins are involved in the reciprocal interactions between the ureteric bud (UB) and the metanephric mesenchyme (MM) that occur during organogenesis of the metanephros. Some key metanephric secreted proteins have been identified, but many remain to be determined. In this study, microarray expression profiling of E10.5, E11.5, and E13.5 kidney and consensus bioinformatic analysis were used to define a dynamic secretome of early metanephric development. In situ hybridisation was used to confirm microarray results and clarify spatial expression patterns for these genes. Forty-one secreted factors were dynamically expressed between the E10.5 and E13.5 timeframe profiled, and 25 of these factors had not previously been implicated in kidney development. A text-based anatomical ontology was used to spatially annotate the expression pattern of these genes in cultured metanephric explants.

Animals↗
PubMed2006

Bovine T cell receptor gamma variable and constant genes: combinatorial usage by circulating gammadelta T cells.

Studies here describe expression and sequence of several new bovine T cell receptor gamma (TRG) genes to yield a total of 11 TRG variable (TRGV) genes (in eight subgroups) and six TRG constant (TRGC) genes. Publicly available genomic sequences were annotated to show their placement. Homologous TRG genes in cattle and sheep were assigned, using four accepted criteria. New genes described here include the bovine TRGC6, TRGV2, and TRGV4, homologues of ovine TRGC4, TRGV2, and TRGV4, respectively. The bovine Vgamma7 and BTGV1 clones (previously TRGV4 and TRGV2, respectively) were reassigned to new subgroups TRGV7 and TRGV8, respectively, with approval by the IMGT Nomenclature Committee. Three TRGV subgroups (TRGV5, TRGV6, and TRGV8) were further designated as TRGV5-1 and TRGV5-2, TRGV6-1 and TRGV6-2, and TRGV8-1 and TRGV8-2 because each subgroup is comprised of two mapped genes. The complete sequence of bovine TRGC5 is also reported, for which a limited number of nucleotides was previously available, and shown to be most closely related to ovine TRGC5. Analysis of circulating gammadelta T cells revealed that rearrangement of TRGV genes with TRGC genes is largely dictated by their proximity within one of the six genomic V-J-C cassettes, with all TRG genes expressed by bovine peripheral blood gammadelta T cells. Cattle are useful models for gammadelta T cell biology because they have gammadelta T cells that respond to isopentenylpyrophosphate (IPP) antigens, while mice do not, and some bovine TRGV genes cluster closely with human genes.

Amino Acid Sequence↗