RESUMO
Comparative analysis of multiple genomes in a phylogenetic framework dramatically improves the precision and sensitivity of evolutionary inference, producing more robust results than single-genome analyses can provide. The genomes of 12 Drosophila species, ten of which are presented here for the first time (sechellia, simulans, yakuba, erecta, ananassae, persimilis, willistoni, mojavensis, virilis and grimshawi), illustrate how rates and patterns of sequence divergence across taxa can illuminate evolutionary processes on a genomic scale. These genome sequences augment the formidable genetic tools that have made Drosophila melanogaster a pre-eminent model for animal genetics, and will further catalyse fundamental research on mechanisms of development, cell biology, genetics, disease, neurobiology, behaviour, physiology and evolution. Despite remarkable similarities among these Drosophila species, we identified many putatively non-neutral changes in protein-coding genes, non-coding RNA genes, and cis-regulatory regions. These may prove to underlie differences in the ecology and behaviour of these diverse species.
Assuntos
Drosophila/classificação , Drosophila/genética , Evolução Molecular , Genes de Insetos/genética , Genoma de Inseto/genética , Genômica , Filogenia , Animais , Códon/genética , Elementos de DNA Transponíveis/genética , Drosophila/imunologia , Drosophila/metabolismo , Proteínas de Drosophila/genética , Ordem dos Genes/genética , Genoma Mitocondrial/genética , Imunidade/genética , Família Multigênica/genética , RNA não Traduzido/genética , Reprodução/genética , Alinhamento de Sequência , Análise de Sequência de DNA , Sintenia/genéticaRESUMO
Systematic mapping of protein-protein interactions, or 'interactome' mapping, was initiated in model organisms, starting with defined biological processes and then expanding to the scale of the proteome. Although far from complete, such maps have revealed global topological and dynamic features of interactome networks that relate to known biological properties, suggesting that a human interactome map will provide insight into development and disease mechanisms at a systems level. Here we describe an initial version of a proteome-scale map of human binary protein-protein interactions. Using a stringent, high-throughput yeast two-hybrid system, we tested pairwise interactions among the products of approximately 8,100 currently available Gateway-cloned open reading frames and detected approximately 2,800 interactions. This data set, called CCSB-HI1, has a verification rate of approximately 78% as revealed by an independent co-affinity purification assay, and correlates significantly with other biological attributes. The CCSB-HI1 data set increases by approximately 70% the set of available binary interactions within the tested space and reveals more than 300 new connections to over 100 disease-associated proteins. This work represents an important step towards a systematic and comprehensive human interactome project.
Assuntos
Proteoma/metabolismo , Clonagem Molecular , Humanos , Fases de Leitura Aberta/genética , Ligação Proteica , Proteoma/genética , RNA/genética , RNA/metabolismo , Saccharomyces cerevisiae/genética , Técnicas do Sistema de Duplo-HíbridoRESUMO
Tetraodon nigroviridis is a freshwater puffer fish with the smallest known vertebrate genome. Here, we report a draft genome sequence with long-range linkage and substantial anchoring to the 21 Tetraodon chromosomes. Genome analysis provides a greatly improved fish gene catalogue, including identifying key genes previously thought to be absent in fish. Comparison with other vertebrates and a urochordate indicates that fish proteins have diverged markedly faster than their mammalian homologues. Comparison with the human genome suggests approximately 900 previously unannotated human genes. Analysis of the Tetraodon and human genomes shows that whole-genome duplication occurred in the teleost fish lineage, subsequent to its divergence from mammals. The analysis also makes it possible to infer the basic structure of the ancestral bony vertebrate genome, which was composed of 12 chromosomes, and to reconstruct much of the evolutionary history of ancient and recent chromosome rearrangements leading to the modern human karyotype.
Assuntos
Cromossomos/genética , Peixes/genética , Duplicação Gênica , Genoma , Vertebrados/genética , Animais , Composição de Bases , Cromossomos Humanos/genética , Sequência Conservada/genética , Evolução Molecular , Genes/genética , Humanos , Cariotipagem , Mamíferos/genética , Modelos Genéticos , Dados de Sequência Molecular , Mapeamento Físico do Cromossomo , Proteoma , Análise de Sequência de DNA , Sintenia/genética , Urocordados/genéticaRESUMO
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.
Assuntos
Proteínas de Bactérias/metabolismo , Mapeamento de Interação de Proteínas/métodos , Rickettsia/genética , Proteínas de Bactérias/genética , Sequência de Bases , Genoma Bacteriano , Biblioteca Genômica , Rickettsia/metabolismo , Rickettsia/patogenicidadeRESUMO
The first version of the Caenorhabditis elegans ORFeome cloning project, based on release WS9 of Wormbase (August 1999), provided experimental verifications for approximately 55% of predicted protein-encoding open reading frames (ORFs). The remaining 45% of predicted ORFs could not be cloned, possibly as a result of mispredicted gene boundaries. Since the release of WS9, gene predictions have improved continuously. To test the accuracy of evolving predictions, we attempted to PCR-amplify from a highly representative worm cDNA library and Gateway-clone approximately 4200 ORFs missed earlier and for which new predictions are available in WS100 (May 2003). In this set we successfully cloned 63% of ORFs with supporting experimental data ("touched" ORFs), and 42% of ORFs with no supporting experimental evidence ("untouched" ORFs). Approximately 2000 full-length ORFs were cloned in-frame, 13% of which were corrected in their exon/intron structure relative to WS100 predictions. In total, approximately 12,500 C. elegans ORFs are now available as Gateway Entry clones for various reverse proteomics (ORFeome v3.1). This work illustrates why the cloning of a complete C. elegans ORFeome, and likely the ORFeomes of other multicellular organisms, needs to be an iterative process that requires multiple rounds of experimental validation together with gradually improving gene predictions.
Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Biologia Computacional/métodos , Genes de Helmintos/genética , Genoma , Fases de Leitura Aberta/genética , Animais , Proteínas de Caenorhabditis elegans/metabolismo , Clonagem Molecular , DNA Complementar/genética , Bases de Dados Genéticas , Éxons , Etiquetas de Sequências Expressas , Expressão Gênica , Genômica , Íntrons , Proteoma , Proteômica , SoftwareRESUMO
An important aspect of the development of systems biology approaches in metazoans is the characterization of expression patterns of nearly all genes predicted from genome sequences. Such "localizome" maps should provide information on where (in what cells or tissues) and when (at what stage of development or under what conditions) genes are expressed. They should also indicate in what cellular compartments the corresponding proteins are localized. Caenorhabditis elegans is particularly suited for the development of a localizome map since all its 959 adult somatic cells can be visualized by microscopy, and its cell lineage has been completely described. Here we address one of the challenges of C. elegans localizome mapping projects: that of obtaining a genome-wide resource of C. elegans promoters needed to generate transgenic animals expressing localization markers such as the green fluorescent protein (GFP). To ensure high flexibility for future uses, we utilized the newly developed MultiSite Gateway system. We generated and validated "version 1.1" of the Promoterome: a resource of approximately 6000 C. elegans promoters. These promoters can be transferred easily into various Gateway Destination vectors to drive expression of markers such as GFP, alone (promoter::GFP constructs), or in fusion with protein-encoding open reading frames available in ORFeome resources (promoter::ORF::GFP).
Assuntos
Caenorhabditis elegans/genética , Genes de Helmintos , Fases de Leitura Aberta/fisiologia , Regiões Promotoras Genéticas/genética , Fatores de Transcrição/fisiologia , Animais , Animais Geneticamente Modificados , Clonagem Molecular , Expressão Gênica , Proteínas de Fluorescência Verde , Proteínas Luminescentes/genéticaRESUMO
The advent of systems biology necessitates the cloning of nearly entire sets of protein-encoding open reading frames (ORFs), or ORFeomes, to allow functional studies of the corresponding proteomes. Here, we describe the generation of a first version of the human ORFeome using a newly improved Gateway recombinational cloning approach. Using the Mammalian Gene Collection (MGC) resource as a starting point, we report the successful cloning of 8076 human ORFs, representing at least 7263 human genes, as mini-pools of PCR-amplified products. These were assembled into the human ORFeome version 1.1 (hORFeome v1.1) collection. After assessing the overall quality of this version, we describe the use of hORFeome v1.1 for heterologous protein expression in two different expression systems at proteome scale. The hORFeome v1.1 represents a central resource for the cloning of large sets of human ORFs in various settings for functional proteomics of many types, and will serve as the foundation for subsequent improved versions of the human ORFeome.
Assuntos
Clonagem Molecular , Genômica/métodos , Fases de Leitura Aberta/genética , Fases de Leitura Aberta/fisiologia , Proteômica , Expressão Gênica , Vetores Genéticos , Humanos , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
To initiate studies on how protein-protein interaction (or "interactome") networks relate to multicellular functions, we have mapped a large fraction of the Caenorhabditis elegans interactome network. Starting with a subset of metazoan-specific proteins, more than 4000 interactions were identified from high-throughput, yeast two-hybrid (HT=Y2H) screens. Independent coaffinity purification assays experimentally validated the overall quality of this Y2H data set. Together with already described Y2H interactions and interologs predicted in silico, the current version of the Worm Interactome (WI5) map contains approximately 5500 interactions. Topological and biological features of this interactome network, as well as its integration with phenome and transcriptome data sets, lead to numerous biological hypotheses.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Proteoma/metabolismo , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Biologia Computacional , Evolução Molecular , Genes de Helmintos , Genômica , Fases de Leitura Aberta , Fenótipo , Ligação Proteica , Transcrição Gênica , Técnicas do Sistema de Duplo-HíbridoRESUMO
The National Institutes of Health Mammalian Gene Collection (MGC) Program is a multiinstitutional effort to identify and sequence a cDNA clone containing a complete ORF for each human and mouse gene. ESTs were generated from libraries enriched for full-length cDNAs and analyzed to identify candidate full-ORF clones, which then were sequenced to high accuracy. The MGC has currently sequenced and verified the full ORF for a nonredundant set of >9,000 human and >6,000 mouse genes. Candidate full-ORF clones for an additional 7,800 human and 3,500 mouse genes also have been identified. All MGC sequences and clones are available without restriction through public databases and clone distribution networks (see http:mgc.nci.nih.gov).