Showing papers by "George M. Weinstock published in 2003"
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Baylor College of Medicine1, Chinese Academy of Sciences2, Chinese National Human Genome Center3, University of Hong Kong4, The Chinese University of Hong Kong5, Hong Kong University of Science and Technology6, Illumina7, McGill University8, Washington University in St. Louis9, University of California, San Francisco10, Wellcome Trust Sanger Institute11, Beijing Normal University12, Health Sciences University of Hokkaido13, Shinshu University14, University of Tsukuba15, Howard University16, University of Ibadan17, Case Western Reserve University18, University of Utah19, Cold Spring Harbor Laboratory20, Johns Hopkins University21, University of Oxford22, North Carolina State University23, National Institutes of Health24, Massachusetts Institute of Technology25, Chinese Academy of Social Sciences26, Kyoto University27, Nagasaki University28, Wellcome Trust29, Genome Canada30, Foundation for the National Institutes of Health31, University of Maryland, Baltimore32, Vanderbilt University33, Stanford University34, New York University35, University of California, Berkeley36, University of Oklahoma37, University of New Mexico38, Université de Montréal39, University of California, Los Angeles40, University of Michigan41, University of Wisconsin-Madison42, London School of Economics and Political Science43, Genetic Alliance44, GlaxoSmithKline45, University of Washington46, Harvard University47, University of Chicago48, Fred Hutchinson Cancer Research Center49, University of Tokyo50
TL;DR: The HapMap will allow the discovery of sequence variants that affect common disease, will facilitate development of diagnostic tools, and will enhance the ability to choose targets for therapeutic intervention.
Abstract: The goal of the International HapMap Project is to determine the common patterns of DNA sequence variation in the human genome and to make this information freely available in the public domain. An international consortium is developing a map of these patterns across the genome by determining the genotypes of one million or more sequence variants, their frequencies and the degree of association between them, in DNA samples from populations with ancestry from parts of Africa, Asia and Europe. The HapMap will allow the discovery of sequence variants that affect common disease, will facilitate development of diagnostic tools, and will enhance our ability to choose targets for therapeutic intervention.
5,926 citations
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TL;DR: The results demonstrate that acm, which encodes a potential virulence factor, is functional only in certain infection‐derived clinical isolates of E. faecium, and suggest that Acm is the primary adhesin responsible for the ability of E.'s faecia to bind collagen.
Abstract: A collagen-binding adhesin of Enterococcus faecium, Acm, was identified. Acm shows 62% similarity to the Staphylococcus aureus collagen adhesin Cna over the entire protein and is more similar to Cna (60% and 75% similarity with Cna A and B domains respectively) than to the Enterococcus faecalis collagen-binding adhesin, Ace, which shares homology with Acm only in the A domain. Despite the detection of acm in 32 out of 32 E. faecium isolates, only 11 of these (all clinical isolates, including four vancomycin-resistant endocarditis isolates and seven other isolates) exhibited binding to collagen type I (CI). Although acm from three CI-binding vancomycin-resistant E. faecium clinical isolates showed 100% identity, analysis of acm genes and their promoter regions from six non-CI-binding strains identified deletions or mutations that introduced stop codons and/or IS elements within the gene or the promoter region in five out of six strains, suggesting that the presence of an intact functional acm gene is necessary for binding of E. faecium strains to CI. Recombinant Acm A domain showed specific and concentration-dependent binding to collagen, and this protein competed with E. faecium binding to immobilized CI. Consistent with the adherence phenotype and sequence data, probing with Acm-specific IgGs purified from anti-recombinant Acm A polyclonal rabbit serum confirmed the surface expression of Acm in three out of three collagen-binding clinical isolates of E. faecium tested, but in none of the strains with a non-functional pseudo acm gene. Introduction of a functional acm gene into two non-CI-binding natural acm mutant strains conferred a CI-binding phenotype, further confirming that native Acm is sufficient for the binding of E. faecium to CI. These results demonstrate that acm, which encodes a potential virulence factor, is functional only in certain infection-derived clinical isolates of E. faecium, and suggest that Acm is the primary adhesin responsible for the ability of E. faecium to bind collagen.
179 citations
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TL;DR: The extracelluar E. faecium SagA protein is apparently essential for growth, shows broad-spectrum binding to ECM proteins, forms oligomers, and is antigenic during infection.
Abstract: A gene encoding a major secreted antigen, SagA, was identified in Enterococcus faecium by screening an E. faecium genomic expression library with sera from patients with E. faecium-associated endocarditis. Recombinant SagA protein showed broad-spectrum binding to extracellular matrix (ECM) proteins, including fibrinogen, collagen type I, collagen type IV, fibronectin, and laminin. A fibrinogen-binding protein, purified from culture supernatants of an E. faecium clinical isolate, was found to match the N-terminal sequence of the predicted SagA protein and to react with the anti-SagA antibody, confirming that it was the SagA protein; this protein appeared as an 80- to 90-kDa smear on a Western blot that was sensitive to proteinase K and resistant to periodate treatment and glycoprotein staining. When overexpressed in E. faecium and Escherichia coli, the native and recombinant SagA proteins formed stable oligomers, apparently via their C-terminal domains. The SagA protein is composed of three domains: (i) a putative coiled-coil N-terminal domain that shows homology to the N-terminal domain of Streptococcus mutans SagA protein (42% similarity), previously shown to be involved in cell wall integrity and cell shape maintenance, and to the P45 protein of Listeria monocytogenes (41% similarity); (ii) a central domain containing direct repeats; and (iii) a C-terminal domain that is similar to that found in various proteins, including P45 (50% similarity) and P60 (52% similarity) of L. monocytogenes. The P45 and P60 proteins both have cell wall hydrolase activity, and the latter has also been shown to be involved in virulence, whereas cell wall hydrolase activity was not detected for SagA protein. The E. faecium sagA gene, like the S. mutans homologue, is located in a cluster of genes encoding proteins that appear to be involved in cell wall metabolism and could not be disrupted unless it was first transcomplemented, suggesting that the sagA gene is essential for E. faecium growth and may be involved in cell wall metabolism. In conclusion, the extracelluar E. faecium SagA protein is apparently essential for growth, shows broad-spectrum binding to ECM proteins, forms oligomers, and is antigenic during infection.
86 citations
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TL;DR: In this paper, DNA microarray technology was utilized to study gene expression by the syphilis spirochete Treponema pallidum (Nichols) during infection of rabbits.
Abstract: DNA microarray technology was utilized to study gene expression
by the syphilis spirochete Treponema pallidum subsp. pallidum
(Nichols) during infection of rabbits. Microarrays containing
all 1039 annotated ORFs of Treponema pallidum subspecies
pallidum (Nichols) were printed on glass slides. For 1034 ORFs
(out of 1039), signals higher than the threshold (average of
negative control spots + 3 SDs) were detected for both RNA and
DNA probes. Total RNA from T. pallidum isolated from rabbit
testes 10 days post infection was labeled and standardized by
cohybridization of the same arrays with treponemal chromosomal
DNA labeled with a different fluorescent marker. This internal
standardization technique proved to be highly reproducible and
to decrease the impact of variables such as host nucleic acid
contamination or variable target DNA lengths. The most highly
transcribed genes were found to correlate with the most
conspicuous spots identified by two dimensional gel
electrophoresis, indicating that the transcript levels
generally corresponded to the relative protein concentrations.
Genes with high transcript concentrations included those
encoding flagellar filament and cytoplasmic filament proteins,
prominent lipoproteins and membrane proteins, chaperonins,
proteins involved in red-ox balance, chemotaxis regulatory
proteins, a V-ATPase operon, and certain metabolic enzymes such
as glycolytic pathway enzymes. Independent quantitation of the
expression of 84 T. pallidum genes using real-time RT-PCR
approach yielded a high degree of correlation (r = 0.94).
Characterization of the T. pallidum transcriptome during
experimental infection provides further insight into the
importance of gene expression levels in the survival and
pathogenesis of this bacterium in the mammalian host.
53 citations
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TL;DR: The potential of the T. pallidum clone set for antigen discovery and, more generally, for advancing the biology of this enigmatic spirochete is demonstrated.
Abstract: A topoisomerase-based method was used to clone PCR products encoding 991 of the 1041 open reading frames identified in the genome sequence of the bacterium that causes syphilis, Treponema pallidum subsp. pallidum. Cloning the open reading frames into the univector plasmid system permitted the rapid conversion of the original clone set to other functional vectors containing a variety of promoters or tag sequences. A computational prediction of signal sequences identified 248 T. pallidum proteins that are potentially secreted from the cell. These clones were systematically converted into vectors designed to express the encoded proteins as glutathione-S-transferase fusion proteins. To test the potential of the clone set for novel antigen discovery, 85 of these fusion proteins were expressed from Escherichia coli, partially purified, and tested for antigenicity by using sera from rabbits infected with T. pallidum. Twelve of the 85 proteins bound significant levels of antibody. Of these 12 proteins, seven had previously been identified as T. pallidum antigens, and the remaining five represent novel antigens. These results demonstrate the potential of the T. pallidum clone set for antigen discovery and, more generally, for advancing the biology of this enigmatic spirochete.
40 citations
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TL;DR: A comparison of 8 cyanobacterial genomes reveals that there are 181 shared genes that do not have obvious orthologs in other bacteria, which suggests there may be regulatory processes that have been preserved throughout the long history of the cyanob bacterial phenotype.
Abstract: A comparison of 8 cyanobacterial genomes reveals that there are 181 shared genes that do not have obvious orthologs in other bacteria. These signature genes define aspects of the genotype that are uniquely cyanobacterial. Approximately 25% of these genes have been associated with some function. These signature genes may or may not be involved in photosynthesis but likely they will be in many cases. In addition, several examples of widely conserved gene order involving two or more signature genes were observed. This suggests there may be regulatory processes that have been preserved throughout the long history of the cyanobacterial phenotype. The results presented here will be especially useful because they identify which of the many genes of unassigned function are likely to be of the greatest interest.
23 citations
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9 citations
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TL;DR: Three strains ofEscherichia fergusonii (EF873, EF1496, EF939) of 50 strains tested produced the hydroxamate siderophore aerobactin, suggesting that E. f Fergusonii iucABCD iutA genes are a portion of a larger segment of DNA similar to pathogenicity islands of other bacteria.
Abstract: Three strains ofEscherichia fergusonii (EF873, EF1496, EF939) of 50 strains tested produced the hydroxamate siderophore aerobactin. Screening of a cosmid library of the strain EF873 chromosomal DNA (in aerobactin nonproducingEscherichia coli VCS257) for aerobactin production identifiediucABCD andiutA gene orthologues. The predicted IucABCD and IutA proteins showed 59–65% identity to the corresponding proteins ofShigella flexneri andE. coli. Aerobactin molecules synthesized byE. fergusonii andE. coli strains stimulated growth of aerobactin indicator strains harboring eitherE. coli orE. fergusonii iutA genes. In the 12 kb upstream and 17 kb downstream regions of theiuc andiut genes, 20 additional ORFs were identified. Their gene products showed homology to proteins fromE. coli, S. flexneri, Klebsiella aerogenes, Pseudomonas aeruginosa andVibrio cholerae. Probes recognizing DNA sequences from a region of more than 25 kb, which included theiucABCD andiutA genes, hybridized with chromosomal DNA of two aerobactin-producing strains (EF873 and EF939), but not with other nonproducingE. fergusonii strains tested. These data, together with the genetic organization of this region, suggest thatE. fergusonii iucABCD iutA genes are a portion of a larger segment of DNA similar to pathogenicity islands of other bacteria.
5 citations
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TL;DR: This article focuses on the following two key innovations in mapping and sequencing: first, shotgun sequencing of clone pools to combine the benefits of whole-genome shotgun and clone-by-clone strategies, and the leveraging of newly available assembled genomic sequences to improve the effectiveness of new sequencing projects through comparative mapping and comparative sequence assembly.
Abstract: Comparative genomic sequencing and analysis offers new wealth of information for target selection and the development of therapeutics. This article focuses on the following two key innovations in mapping and sequencing: first, shotgun sequencing of clone pools to combine the benefits of whole-genome shotgun and clone-by-clone strategies, and second, the leveraging of newly available assembled genomic sequences to improve the effectiveness of new sequencing projects through comparative mapping and comparative sequence assembly. The following specific sequencing and mapping methods are discussed in detail: clone-array pooled shotgun sequencing (CAPSS); transversal shotgun pooling designs; clone-array pooled shotgun mapping (CAPS-MAP); pooled genomic indexing (PGI); short-tag pooled genomic indexing (ST-PGI); and comparative sequence assembly (the CSA™ method). The methods can be implemented with only modest modifications of current large-scale sequencing pipelines and are highly synergistic with the next generation of sequencing technologies.
5 citations