RESUMEN
Sequence analysis of chromosome IX of Saccharomyces cerevisiae revealed an open reading frame of 166 residues, designated TPM2, having 64.5% sequence identity to TPM1, that encodes the major form of tropomyosin in yeast. Purification and characterization of Tpm2p revealed a protein with the characteristics of a bona fide tropomyosin; it is present in vivo at about one sixth the abundance of Tpm1p. Biochemical and sequence analysis indicates that Tpm2p spans four actin monomers along a filament, whereas Tpmlp spans five. Despite its shorter length, Tpm2p can compete with Tpm1p for binding to F-actin. Over-expression of Tpm2p in vivo alters the axial budding of haploids to a bipolar pattern, and this can be partially suppressed by co-over-expression of Tpm1p. This suggests distinct functions for the two tropomyosins, and indicates that the ratio between them is important for correct morphogenesis. Loss of Tpm2p has no detectable phenotype in otherwise wild type cells, but is lethal in combination with tpm1 delta. Over-expression of Tpm2p does not suppress the growth or cell surface targeting defects associated with tpm1 delta, so the two tropomyosins must perform an essential function, yet are not functionally interchangeable. S. cerevisiae therefore provides a simple system for the study of two tropomyosins having distinct yet overlapping functions.
Asunto(s)
Saccharomyces cerevisiae/genética , Tropomiosina/genética , Actinas/metabolismo , Secuencia de Aminoácidos , Haploidia , Datos de Secuencia Molecular , Mutación , Unión Proteica , Saccharomyces cerevisiae/metabolismo , Homología de Secuencia de Aminoácido , Tropomiosina/aislamiento & purificación , Tropomiosina/metabolismoRESUMEN
The genome of the yeast Saccharomyces cerevisiae has been completely sequenced through a worldwide collaboration. The sequence of 12,068 kilobases defines 5885 potential protein-encoding genes, approximately 140 genes specifying ribosomal RNA, 40 genes for small nuclear RNA molecules, and 275 transfer RNA genes. In addition, the complete sequence provides information about the higher order organization of yeast's 16 chromosomes and allows some insight into their evolutionary history. The genome shows a considerable amount of apparent genetic redundancy, and one of the major problems to be tackled during the next stage of the yeast genome project is to elucidate the biological functions of all of these genes.
Asunto(s)
Mapeo Cromosómico , Genes Fúngicos , Genoma Fúngico , Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Secuencia de Bases , Cromosomas Fúngicos/genética , Redes de Comunicación de Computadores , ADN de Hongos/genética , Evolución Molecular , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/fisiología , Biblioteca de Genes , Cooperación Internacional , Familia de Multigenes , Sistemas de Lectura Abierta , ARN de Hongos/genética , Análisis de Secuencia de ADNRESUMEN
Corynebacterium diphtheriae is a Gram-positive, non-spore forming, non-motile, pleomorphic rod belonging to the genus Corynebacterium and the actinomycete group of organisms. The organism produces a potent bacteriophage-encoded protein exotoxin, diphtheria toxin (DT), which causes the symptoms of diphtheria. This potentially fatal infectious disease is controlled in many developed countries by an effective immunisation programme. However, the disease has made a dramatic return in recent years, in particular within the Eastern European region. The largest, and still on-going, outbreak since the advent of mass immunisation started within Russia and the newly independent states of the former Soviet Union in the 1990s. We have sequenced the genome of a UK clinical isolate (biotype gravis strain NCTC13129), representative of the clone responsible for this outbreak. The genome consists of a single circular chromosome of 2 488 635 bp, with no plasmids. It provides evidence that recent acquisition of pathogenicity factors goes beyond the toxin itself, and includes iron-uptake systems, adhesins and fimbrial proteins. This is in contrast to Corynebacterium's nearest sequenced pathogenic relative, Mycobacterium tuberculosis, where there is little evidence of recent horizontal DNA acquisition. The genome itself shows an unusually extreme large-scale compositional bias, being noticeably higher in G+C near the origin than at the terminus.
Asunto(s)
Corynebacterium diphtheriae/genética , Genoma Bacteriano , Anciano , Composición de Base , Cromosomas Bacterianos/genética , Corynebacterium diphtheriae/metabolismo , Corynebacterium diphtheriae/patogenicidad , ADN Bacteriano/química , ADN Bacteriano/genética , Toxina Diftérica/metabolismo , Femenino , Fimbrias Bacterianas/genética , Humanos , Glicoproteínas de Membrana/genética , Datos de Secuencia Molecular , Análisis de Secuencia de ADN , Virulencia/genéticaRESUMEN
We have determined the DNA sequence (46 kilobases) of the short unique region, the short repeat, and part of the long repeat of human cytomegalovirus strain AD169. Analysis of the sequence has revealed at least 38 possible regions that may code for protein. Many of these open reading frames show homology to each other, and five groups of homologous reading frames are identified. Half of the predicted translation products appear to be membrane proteins, and fall into two distinct classes; those that have potential signal and anchor sequences, and those that have seven potential membrane-spanning regions and appear to be integral membrane proteins. A number of the former class contain sites for N-linked glycosylation and may therefore be glycoproteins. None of the 38 open reading frames shows homology to other known herpesvirus proteins.
Asunto(s)
Citomegalovirus/genética , ADN Viral/genética , Secuencias Repetitivas de Ácidos Nucleicos , Secuencia de Aminoácidos , Composición de Base , Secuencia de Bases , Humanos , Biosíntesis de Proteínas , Programas Informáticos , Proteínas ViralesRESUMEN
The powerful combination of genomics and bioinformatics is providing a wealth of information about Mycobacterium tuberculosis, the aetiological agent of human tuberculosis, that will facilitate the conception and development of new therapies. The starting point for genome sequencing was the integrated map of the 4.4 Mb circular chromosome of the widely used, virulent reference strain, M. tuberculosis H37Rv. Cosmids and bacterial artificial chromosomes were selected from ordered libraries and subjected to systematic shotgun sequence analysis. This approach simplified sequence assembly as the genome is rich in repetitive DNA. In common with most bacteria, > 90% of the potential coding capacity is used, and probable or tentative functions could be attributed to > 70% of the genes. The potential biological roles of two of the principal driving forces in genome dynamics, insertion sequence elements and polymorphic multigene families are discussed.
Asunto(s)
Genoma Bacteriano , Mycobacterium tuberculosis/genética , Elementos Transponibles de ADN , Familia de Multigenes , Polimorfismo GenéticoRESUMEN
Everything that we need to know about Mycobacterium leprae, a close relative of the tubercle bacillus, is encrypted in its genome. Inspection of the 3.27 Mb genome sequence of an armadillo-derived Indian isolate of the leprosy bacillus identified 1,605 genes encoding proteins and 50 genes for stable RNA species. Comparison with the genome sequence of Mycobacterium tuberculosis revealed an extreme case of reductive evolution, since less than half of the genome contains functional genes while inactivated or pseudogenes are highly abundant. The level of gene duplication was approximately 34% and, on classification of the proteins into families, the largest functional groups were found to be involved in the metabolism and modification of fatty acids and polyketides, transport of metabolites, cell envelope synthesis and gene regulation. Reductive evolution, gene decay and genome downsizing have eliminated entire metabolic pathways, together with their regulatory circuits and accessory functions, particularly those involved in catabolism. This may explain the unusually long generation time and account for our inability to culture the leprosy bacillus.
Asunto(s)
Genes Bacterianos/genética , Genoma Bacteriano , Lepra/microbiología , Mycobacterium leprae/genética , Evolución Molecular , HumanosRESUMEN
The rate limiting step in a large-scale sequencing project is the generation of single-stranded DNA templates. We describe a fast, semiautomated procedure, using 96-well microtitre plates, in which 192 templates can be readily prepared in 1 day. The technique can be carried out manually or can be semiautomated using a robot pipetting device. We also provide evidence for the reliability and applicability of this method to a large-scale sequencing project.
Asunto(s)
Secuencia de Bases , ADN Viral/aislamiento & purificación , Moldes Genéticos , Bacteriófagos/genética , Escherichia coli/genética , Técnicas Genéticas , RobóticaAsunto(s)
Colifagos/metabolismo , ARN de Transferencia/biosíntesis , 4-Nitrofenilfosfatasa , Secuencia de Bases , Sitios de Unión , Escherichia coli/metabolismo , Mutación , Conformación de Ácido Nucleico , Oligorribonucleótidos/análisis , Hidrolasas Diéster Fosfóricas , Prolina , Ribonucleasas , SerinaAsunto(s)
Colifagos , ARN de Transferencia , ARN Viral , Serina , Supresión Genética , Alanina , Autorradiografía , Isótopos de Carbono , Electroforesis en Gel de Poliacrilamida , Escherichia coli , Genes , Código Genético , Genética Microbiana , Glicina , Mutación , Fenotipo , Fenilalanina , Isótopos de Fósforo , Isótopos de Azufre , Valina , Proteínas Virales/análisisAsunto(s)
Colifagos/análisis , Colifagos/metabolismo , ADN Viral , Genes , Secuencia de Bases , Cromatografía en Capa Delgada , Virus ADN/metabolismo , ADN Viral/análisis , Desoxirribonucleasas , Escherichia coli/metabolismo , Exonucleasas , Nucleasa Microcócica , Conformación de Ácido Nucleico , Receptores de Droga , Ribosomas/metabolismo , Venenos de Serpiente , Streptococcus/enzimologíaAsunto(s)
Nucleótidos/análisis , ARN de Transferencia/análisis , ARN Viral/análisis , Nucleótidos de Adenina/análisis , Autorradiografía , Secuencia de Bases , Cromatografía en Capa Delgada , Colifagos/metabolismo , Nucleótidos de Citosina/análisis , Electroforesis en Gel de Poliacrilamida , Glicina , Nucleótidos de Guanina/análisis , Hidrólisis , Radioisótopos de Fósforo , ARN de Transferencia/biosíntesis , ARN Viral/biosíntesis , Ribonucleasas , Nucleótidos de Uracilo/análisisRESUMEN
We have cloned and sequenced a gene encoding a yeast homologue of the U1 snRNP 70K protein. The gene, SNP1, encodes a protein which has 30% amino acid identity with the human 70K protein and has a predicted molecular weight of 34 kDa. The yeast and human sequences are more closely related to each other than to other (non-U1) RNA-binding proteins, but diverge considerably in their C-terminal portions. In particular, SNP1 lacks the charged carboxy terminus of the human 70K protein. A yeast strain, a alpha 115, was constructed in which one allele of the SNP1 gene contained a 554 bp deletion. Tetrad analysis of a alpha 115 showed that the SNP1 gene is essential for the viability of yeast cells. The complete human 70K gene did not complement snp1, but the lethal snp1 mutation was rescued by plasmids bearing a chimera in which over half the yeast gene was replaced with the homologous region of the human 70K gene, including the RNA-binding domain. These results suggest that SNP1 encodes a functional homologue of the U1 snRNP 70K protein.
Asunto(s)
ARN de Hongos/metabolismo , ARN/metabolismo , Ribonucleoproteínas/genética , Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Secuencia de Bases , Southern Blotting , Clonación Molecular , Sondas de ADN , ADN de Hongos/genética , Genes Fúngicos , Genes Letales , Humanos , Datos de Secuencia Molecular , Mutación , Reacción en Cadena de la Polimerasa , Mapeo Restrictivo , Ribonucleoproteínas Nucleares Pequeñas , Alineación de SecuenciaRESUMEN
The DNA sequence of the HindIII T fragment of human cytomegalovirus strain AD169 has been determined. This 6225 bp sequence has been analysed for transcription signals and probable open reading frames. Similarities with herpes simplex virus, varicella-zoster virus and Epstein-Barr virus genes were observed for three of the predicted open reading frames; a virion protein and a unique DNA helicase are believed to be the functional products of two of these open reading frames. Two other open reading frames are novel in that no homologues could be found, either in the known herpesvirus sequences or in the Protein Identification Resource database. Both of these open reading frames also lie in the genomic coding region of a 5.0 kb RNA which is transcribed throughout the infectious cycle.