RESUMEN
The Gene Ontology (GO) project (http://www. geneontology.org/) provides structured, controlled vocabularies and classifications that cover several domains of molecular and cellular biology and are freely available for community use in the annotation of genes, gene products and sequences. Many model organism databases and genome annotation groups use the GO and contribute their annotation sets to the GO resource. The GO database integrates the vocabularies and contributed annotations and provides full access to this information in several formats. Members of the GO Consortium continually work collectively, involving outside experts as needed, to expand and update the GO vocabularies. The GO Web resource also provides access to extensive documentation about the GO project and links to applications that use GO data for functional analyses.
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Bases de Datos Genéticas , Genes , Terminología como Asunto , Animales , Bibliografías como Asunto , Correo Electrónico , Genómica , Humanos , Almacenamiento y Recuperación de la Información , Internet , Biología Molecular , Proteínas/clasificación , Proteínas/genética , Programas InformáticosRESUMEN
The BioKnowledge Library is a relational database and web site (http://www.proteome.com) composed of protein-specific information collected from the scientific literature. Each Protein Report on the web site summarizes and displays published information about a single protein, including its biochemical function, role in the cell and in the whole organism, localization, mutant phenotype and genetic interactions, regulation, domains and motifs, interactions with other proteins and other relevant data. This report describes four species-specific volumes of the BioKnowledge Library, concerned with the model organisms Saccharomyces cerevisiae (YPD), Schizosaccharomyces pombe (PombePD) and Caenorhabditis elegans (WormPD), and with the fungal pathogen Candida albicans (CalPD). Protein Reports of each species are unified in format, easily searchable and extensively cross-referenced between species. The relevance of these comprehensively curated resources to analysis of proteins in other species is discussed, and is illustrated by a survey of model organism proteins that have similarity to human proteins involved in disease.
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Bases de Datos Factuales , Proteoma , Animales , Caenorhabditis elegans/genética , Candida albicans/genética , Biología Computacional , Genómica , Servicios de Información , Internet , Saccharomyces cerevisiae/genética , Schizosaccharomyces/genéticaRESUMEN
Translation of mitochondrially coded mRNAs in Saccharomyces cerevisiae depends on membrane-bound mRNA-specific activator proteins, whose targets lie in the mRNA 5'-untranslated leaders (5'-UTLs). In at least some cases, the activators function to localize translation of hydrophobic proteins on the inner membrane and are rate limiting for gene expression. We searched unsuccessfully in divergent budding yeasts for orthologs of the COX2- and COX3-specific translational activator genes, PET111, PET54, PET122, and PET494, by direct complementation. However, by screening for complementation of mutations in genes adjacent to the PET genes in S. cerevisiae, we obtained chromosomal segments containing highly diverged homologs of PET111 and PET122 from Saccharomyces kluyveri and of PET111 from Kluyveromyces lactis. All three of these genes failed to function in S. cerevisiae. We also found that the 5'-UTLs of the COX2 and COX3 mRNAs of S. kluyveri and K. lactis have little similarity to each other or to those of S. cerevisiae. To determine whether the PET111 and PET122 homologs carry out orthologous functions, we deleted them from the S. kluyveri genome and deleted PET111 from the K. lactis genome. The pet111 mutations in both species prevented COX2 translation, and the S. kluyveri pet122 mutation prevented COX3 translation. Thus, while the sequences of these translational activator proteins and their 5'-UTL targets are highly diverged, their mRNA-specific functions are orthologous.
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Regiones no Traducidas 5' , Complejo IV de Transporte de Electrones/genética , Proteínas Fúngicas/genética , Isoenzimas/genética , Proteínas de la Membrana/genética , Proteínas Nucleares/fisiología , Prostaglandina-Endoperóxido Sintasas/genética , Biosíntesis de Proteínas , ARN de Hongos , ARN , Proteínas de Saccharomyces cerevisiae , Secuencia de Aminoácidos , Animales , Ciclooxigenasa 2 , Genes Fúngicos , Prueba de Complementación Genética , Kluyveromyces/clasificación , Kluyveromyces/genética , Proteínas Mitocondriales , Datos de Secuencia Molecular , Mutagénesis , Proteínas Nucleares/genética , Factores de Iniciación de Péptidos , Fenotipo , ARN Mitocondrial , Saccharomyces/clasificación , Saccharomyces/genética , Saccharomyces cerevisiae/clasificación , Saccharomyces cerevisiae/genética , Homología de Secuencia de Ácido NucleicoRESUMEN
The Yeast Proteome Database (YPDtrade mark) has been for several years a resource for organized and accessible information about the proteins of Saccharomyces cerevisiae. We have now extended the YPD format to create a database containing complete proteome information about the model organism Caenorhabditis elegans (WormPDtrade mark). YPD and WormPD are designed for use not only by their respective research communities but also by the broader scientific community. In both databases, information gleaned from the literature is presented in a consistent, user-friendly Protein Report format: a single Web page presenting all available knowledge about a particular protein. Each Protein Report begins with a Title Line, a concise description of the function of that protein that is continually updated as curators review new literature. Properties and functions of the protein are presented in tabular form in the upper part of the Report, and free-text annotations organized by topic are presented in the lower part. Each Protein Report ends with a comprehensive reference list whose entries are linked to their MEDLINE s. YPD and WormPD are seamlessly integrated, with extensive links between the species. They are freely accessible to academic users on the WWW at http://www. proteome.com/databases/index.html, and are available by subscription to corporate users.
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Caenorhabditis elegans/genética , Bases de Datos Factuales , Genoma Fúngico , Proteoma/genética , AnimalesRESUMEN
Translation of mitochondrial mRNAs in Saccharomyces cerevisiae depends on mRNA-specific translational activators that recognize the 5' untranslated leaders (5'-UTLs) of their target mRNAs. We have identified mutations in two new nuclear genes that suppress translation defects due to certain alterations in the 5'-UTLs of both the COX2 and COX3 mRNAs, indicating a general function in translational activation. One gene, MRP21, encodes a protein with a domain related to the bacterial ribosomal protein S21 and to unidentified proteins of several animals. The other gene, MRP51, encodes a novel protein whose only known homolog is encoded by an unidentified gene in S. kluyveri. Deletion of either MRP21 or MRP51 completely blocked mitochondrial gene expression. Submitochondrial fractionation showed that both Mrp21p and Mrp51p cosediment with the mitochondrial ribosomal small subunit. The suppressor mutations are missense substitutions, and those affecting Mrp21p alter the region homologous to E. coli S21, which is known to interact with mRNAs. Interactions of the suppressor mutations with leaky mitochondrial initiation codon mutations strongly suggest that the suppressors do not generally increase translational efficiency, since some alleles that strongly suppress 5'-UTL mutations fail to suppress initiation codon mutations. We propose that mitochondrial ribosomes themselves recognize a common feature of mRNA 5'-UTLs which, in conjunction with mRNA-specific translational activation, is required for organellar translation initiation.
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Mitocondrias/genética , ARN Mensajero/genética , Proteínas Ribosómicas/genética , Ribosomas/genética , Saccharomyces cerevisiae/genética , Supresión Genética , Alelos , Secuencia de Aminoácidos , Clonación Molecular , Ciclooxigenasa 2 , Complejo IV de Transporte de Electrones/genética , Complejo IV de Transporte de Electrones/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Mutación Puntual , Prostaglandina-Endoperóxido Sintasas/genética , Prostaglandina-Endoperóxido Sintasas/metabolismo , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , ARN Mitocondrial , Proteínas Ribosómicas/química , Proteínas Ribosómicas/metabolismo , Ribosomas/metabolismo , Proteínas de Saccharomyces cerevisiae , Alineación de SecuenciaRESUMEN
We determined the seroprevalence of a Sindbis-related virus isolated for the first time in 1975 from ticks in south-east Sicily and typed by Gresikova et al. in 1978. An indirect enzyme immunoassay based on viral membrane antigen for coating microtiter strips was used for the detection of immunoglobulin G to the Sindbis-related virus. The method appeared more sensitive than a similar enzyme immunoassay based on crude lysate antigen. Comparison of the results obtained from sera tested both by membrane antigen enzyme immunoassay and microneutralization test showed 92% agreement, while the agreement between microneutralization test and crude antigen enzyme immunoassay was 76%. An overall elevated seroprevalence (63.66%) was found in a population group living in and around the area of first isolation and seroprevalence in different age groups was also studied.
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Infecciones por Alphavirus/diagnóstico , Anticuerpos Antivirales/sangre , Técnicas para Inmunoenzimas , Virus Sindbis/inmunología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Infecciones por Alphavirus/epidemiología , Antígenos Virales/inmunología , Niño , Preescolar , Humanos , Inmunoglobulina G/sangre , Lactante , Persona de Mediana Edad , Pruebas de Neutralización , Tiras Reactivas , Sicilia/epidemiología , Proteínas de la Matriz Viral/inmunologíaRESUMEN
We used transformation of yeast mitochondria and homologous gene replacement to study features of the 613-base COX3 mRNA 5' untranslated leader (5'-UTL) required for translational activation by the protein products of the nuclear genes PET54, PET122, and PET494 in vivo. Elimination of the single AUG triplet in the 5'-UTL had no detectable effect on expression, indicating that activator proteins do not work by allowing ribosomes to bypass that AUG. Deletion of the entire 5'-UTL completely prevented translation, suggesting that the activator proteins do not function by antagonizing any other negative element in the 5'-UTL. Removal of the 15 terminal bases from the 5' end of the 5'-UTL did not block activator-dependent translation. The largest internal deletion that did not interfere with translation removed 125 bases from the upstream portion of the leader. However, two large deletions that blocked translation could be reverted to activator-dependent expression by secondary changes in the remaining 5'-UTL sequences, indicating that the original deletions had not removed the translational activator target but only deformed it. Taken together, the deletion mutations and revertants define a region of 151 bases (between positions -480 and -330 relative to the start codon) containing sequences that are sufficient for translational activation when modified slightly. Suppression of the respiratory phenotypes of two 5'-UTL mutations by overexpression of PET54, PET122, and PET494 indicated functional interactions between the leader and the three activator proteins. The mature COX3 mRNA is cleaved from a precursor immediately downstream of the preceding tRNAVal in a fashion resembling mRNA processing in vertebrate mitochondria. Our results indicate that the site of this cleavage in Saccharomyces cerevisiae is determined solely by the position of the tRNA.
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Complejo IV de Transporte de Electrones , Proteínas Fúngicas/genética , Proteínas de la Membrana/genética , Procesamiento Postranscripcional del ARN , ARN/metabolismo , Secuencia de Bases , Proteínas Fúngicas/metabolismo , Eliminación de Gen , Proteínas de la Membrana/metabolismo , Mitocondrias/metabolismo , Datos de Secuencia Molecular , ARN Mensajero/genética , ARN Mitocondrial , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiaeRESUMEN
The 613-base 5'-untranslated leader (5'-UTL) of the Saccharomyces cerevisiae mitochondrial COX3 mRNA contains the target of an mRNA-specific translational activator complex composed of at least three nuclearly encoded proteins. We have genetically mapped a collection of cox3 point mutations, using a set of defined COX3 deletions, and found one to be located in the region coding the 5'-UTL. The strain carrying this allele was specifically defective in translation of the COX3 mRNA. Nucleotide-sequence analysis showed that the allele was in fact a double mutation comprised of a single-base insertion in the 5'-UTL (T inserted between bases -428 and -427 with respect to the start of translation) and a G to A substitution at +3 that changed the ATG initiation codon to ATA. Both mutations were required to block translation completely. The effects of the ATG to ATA mutation alone (cox3-1) had previously been analyzed in this laboratory: it reduces, but does not eliminate, translation, causing a slow respiratory growth phenotype. The T insertion in the 5'-UTL had no detectable respiratory growth phenotype as a single mutation. However, the 5'-UTL insertion mutation enhanced the respiratory defective phenotype of missense mutations in pet54, one of the COX3-specific translational-activator genes. This phenotypic enhancement suggests that the -400 region of the 5'-UTL, where the mutation is located, is important for Pet54p-COX3 mRNA interaction.
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Complejo IV de Transporte de Electrones/genética , Mutación Puntual , Biosíntesis de Proteínas , ARN Mensajero/genética , ARN/genética , Saccharomyces cerevisiae/genética , Alelos , Fenotipo , ARN Mitocondrial , Saccharomyces cerevisiae/enzimologíaRESUMEN
The PET54, PET122, and PET494 proteins, which are associated with the yeast inner mitochondrial membrane, specifically activate translation of the mitochondrially encoded COX3 mRNA. We used the two-hybrid system to test whether pairs of these proteins, when fused to either the GAL4 DNA-binding or transcriptional activating domain, can physically associate as measured by the expression of the GAL4-dependent reporter, lacZ. PET54 and PET122 interacted in this system, and an amino-terminally truncated PET494 fragment showed an interaction with PET54. We also detected functional interactions between PET54 and PET122 genetically: a pet54 missense substitution (Phe to Gly at position 244) that caused a severe respiratory defect was suppressed both by a missense substitution affecting PET122 (Gly to Val at position 211) and by overproduction of wild-type PET122. Both Gly and Ala, substituted at PET54 position 244, disrupted the two-hybrid interactions with PET122 and PET494. While Ala at PET54 position 244 caused only a modest respiratory phenotype alone, it caused a severe respiratory defect when combined with a cold-sensitive mitochondrial mutation affecting the COX3 mRNA 5' leader. This synthetic defect was suppressed by a missense substitution in PET122 and by overproduction of wild-type PET122, indicating functional interactions among PET54, PET122, and the mRNA. Taken together with previous work, these data suggest that a complex containing PET54, PET122, and PET494 mediates the interaction of the COX3 mRNA with mitochondrial ribosomes at the surface of the inner membrane.
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Complejo IV de Transporte de Electrones/biosíntesis , Proteínas Fúngicas/metabolismo , Regulación Enzimológica de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Proteínas de la Membrana/metabolismo , Mitocondrias/metabolismo , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , Saccharomyces cerevisiae/enzimología , Alelos , Secuencia de Aminoácidos , Secuencia de Bases , Cartilla de ADN , Proteínas Fúngicas/biosíntesis , Genes Fúngicos , Genes Supresores , Membranas Intracelulares/metabolismo , Proteínas de la Membrana/biosíntesis , Mitocondrias/enzimología , Modelos Biológicos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación Puntual , Reacción en Cadena de la Polimerasa , Ribosomas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
Translation of the Saccharomyces cerevisiae mitochondrial COX3 mRNA, encoding subunit III of cytochrome c oxidase, specifically requires the action of the nuclear gene products PET54, PET122, and PET494 at a site encoded in the 612-base 5' untranslated leader. To identify more precisely the site of action of the translational activators, we constructed two large deletions of the COX3 mRNA 5' untranslated leader. Both deletions blocked translation without affecting mRNA stability. However, one of the large deletions was able to revert to partial function by a small secondary deletion within the remaining 5' leader sequences. Translation of the resulting mutant (cox3-15) mRNA was still dependent on the nuclear-encoded specific activators but was cold sensitive. We selected revertants of this mitochondrial mutant at low temperature to identify genes encoding proteins that might interact with the COX3 mRNA 5' leader. One such revertant carried a missense mutation in the PET122 gene that was a strong and dominant suppressor of the cold-sensitive defect in the mRNA, indicating that the PET122 protein interacts functionally (possibly directly) with the COX3 mRNA 5' leader. The cox3-15 mutation was not suppressed by overproduction of the wild-type PET122 protein but was very weakly suppressed by overproduction of PET494 and slightly better suppressed by co-overproduction of PET494 and PET122.
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Complejo IV de Transporte de Electrones/genética , Regulación Fúngica de la Expresión Génica , Mitocondrias/metabolismo , Biosíntesis de Proteínas , ARN Mensajero/genética , Saccharomyces cerevisiae/genética , Secuencia de Bases , Análisis Mutacional de ADN , Proteínas Fúngicas/metabolismo , Genes Supresores , Datos de Secuencia Molecular , Oligonucleótidos/química , ARN de Hongos/genética , Ribosomas/metabolismo , Eliminación de SecuenciaRESUMEN
The seroprevalence to Toxoplasma gondii (41.1%), rubella virus (88.2%), cytomegalovirus (86.0%), and herpes simplex virus (80.0%) has been evaluated in fertile women living in Catania (Sicily). The population group studied was divided into four age groups to quantify the risk of primary infection in each age group.
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Anticuerpos Antiprotozoarios/sangre , Anticuerpos Antivirales/sangre , Infecciones por Citomegalovirus/epidemiología , Citomegalovirus/inmunología , Fertilidad , Herpes Simple/epidemiología , Vigilancia de la Población , Virus de la Rubéola/inmunología , Rubéola (Sarampión Alemán)/epidemiología , Simplexvirus/inmunología , Toxoplasma/inmunología , Toxoplasmosis/epidemiología , Adolescente , Adulto , Factores de Edad , Animales , Infecciones por Citomegalovirus/sangre , Femenino , Herpes Simple/sangre , Humanos , Prevalencia , Distribución Aleatoria , Factores de Riesgo , Rubéola (Sarampión Alemán)/sangre , Estudios Seroepidemiológicos , Sicilia/epidemiología , Toxoplasmosis/sangreAsunto(s)
Regulación Fúngica de la Expresión Génica , Mitocondrias/metabolismo , Saccharomyces cerevisiae/genética , ADN de Hongos/genética , ADN Mitocondrial/genética , Genes Fúngicos , Procesamiento Postranscripcional del ARN , ARN de Hongos/genética , ARN de Hongos/metabolismo , Transcripción Genética , Transformación GenéticaRESUMEN
The product of the nuclear Saccharomyces cerevisiae gene PET54 is specifically required, along with at least two other nuclear gene products, for translation of the mitochondrial mRNA encoding subunit III of cytochrome c oxidase (coxIII). We have genetically mapped PET54 (to the right arm of chromosome VII, 4.8 cM centromere-distal to SUF15), and have biochemically characterized the gene and its product. We determined the nucleotide sequence of a 1.6-kb DNA fragment carrying PET54 and identified the PET54 reading frame by determining the sequence of an ochre mutant allele as well as frameshift and frameshift-revertant alleles of the gene. The wild-type PET54 gene encodes a slightly basic 293-amino acid protein. PET54 is expressed from two mRNAs, both with unusual features: a major transcript with an extremely short 5'-untranslated leader, and a minor transcript with a relatively long 5'-leader carrying three short open reading frames. Antiserum raised against a trpE-PET54 fusion protein was used to probe subcellular fractions. These experiments showed that the PET54 protein is specifically associated with mitochondria, suggesting that it is likely to act directly in coxIII translation.
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Proteínas Fúngicas/genética , Genes Fúngicos , Genes Reguladores , Biosíntesis de Proteínas , Saccharomyces cerevisiae/genética , Alelos , Secuencia de Aminoácidos , Secuencia de Bases , ADN de Hongos/genética , ADN Mitocondrial/genética , Complejo IV de Transporte de Electrones/biosíntesis , Complejo IV de Transporte de Electrones/genética , Proteínas Fúngicas/metabolismo , Mitocondrias/análisis , Datos de Secuencia Molecular , ARN de Hongos/metabolismo , ARN Mensajero/metabolismo , Proteínas Recombinantes de Fusión/análisisRESUMEN
We have found that agitation of Saccharomyces cerevisiae with glass beads and plasmid DNA using a vortex mixer results in genetic transformation of the yeast cells. This method is less efficient, but considerably more convenient, than other yeast transformation procedures. The fact that the minimal requirements for transformation are simply physical damage and the presence of DNA in an osmotically supportive environment suggests that this process may occur in nature.
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Vidrio , Saccharomyces cerevisiae/genética , Transformación Genética , Ciclo Celular , Permeabilidad de la Membrana Celular , ADN/genética , Técnicas Genéticas , Plásmidos , Saccharomyces cerevisiae/crecimiento & desarrolloRESUMEN
We describe several yeast nuclear mutations that specifically block expression of the mitochondrial genes encoding cytochrome c oxidase subunits II (COXII) and III (COXIII). These recessive mutations define positive regulators of mitochondrial gene expression that act at the level of translation. Mutations in the nuclear gene PET111 completely block accumulation of COXII, but the COXII mRNA is present in mutant cells at a level approximately one-third of that of the wild type. Mitochondrial suppressors of pet111 mutations correspond to deletions in mtDNA that result in fusions between the coxII structural gene and other mitochondrial genes. The chimeric mRNAs encoded by these fusions are translated in pet111 mutants; this translation leads to accumulation of functional COXII. The PET111 protein probably acts directly on coxII translation, because it is located in mitochondria. Translation of the mitochondrially coded mRNA for COXIII requires the action of at least three nuclear genes, PET494, PET54 and a newly discovered gene, provisionally termed PET55. Both the PET494 and PET54 proteins are located in mitochondria and therefore probably act directly on the mitochondrial translation system. Mutations in all three genes are suppressed in strains that contain chimeric coxIII mRNAs with the 5'-untranslated leaders of other mitochondrial transcripts fused to the coxIII coding sequence. The products of all three nuclear genes may form a complex and carry out a single function.(ABSTRACT TRUNCATED AT 250 WORDS)
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Núcleo Celular/metabolismo , ADN Mitocondrial/genética , Complejo IV de Transporte de Electrones/genética , Regulación de la Expresión Génica , Genes Fúngicos , Genes , Biosíntesis de Proteínas , Saccharomyces cerevisiae/genética , Sustancias Macromoleculares , Mitocondrias/enzimología , Mutación , ARN Mensajero/genética , Saccharomyces cerevisiae/enzimología , Transcripción GenéticaRESUMEN
Translation of the yeast mitochondrial mRNA encoding cytochrome c oxidase subunit III (coxIII) is specifically activated by the products of at least three nuclear genes, PET494, PET54, and PET122. To investigate whether the target site for translational activation is within the 5' untranslated leader of the coxIII mRNA, we asked whether translation of another mitochondrial protein, apo-cytochrome b, from a chimeric mRNA bearing the coxIII mRNA leader required PET494, PET54, or PET122. Mutations in any of these three genes abolished translation of cytochrome b from an mRNA bearing the 5' two-thirds of the coxIII mRNA 5' untranslated leader, showing that all three gene products are required for translation of the chimeric mRNA and must act within the 5' two-thirds of the coxIII mRNA leader. Our data suggest that in wild-type cells, the specific activation of coxIII translation by PET494, PET54, and PET122 occurs by the action of these three gene products at a site or sites in a region of the 5' untranslated leader at least 172 nucleotides upstream of the initiation codon.
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Grupo Citocromo b/genética , Complejo IV de Transporte de Electrones/genética , Proteínas Fúngicas/fisiología , Mitocondrias/fisiología , Biosíntesis de Proteínas , ARN Mensajero/genética , Saccharomyces cerevisiae/genética , ADN Recombinante , Regulación de la Expresión Génica , Genes , Mutación , Supresión GenéticaRESUMEN
Mitochondrial translation of the oxi2 mRNA, encoding yeast cytochrome c oxidase subunit III (coxIII), has previously been shown to specifically require the mitochondrially located protein product of the nuclear gene PET494. We show here that this specific translational activation involves at least one other newly identified gene termed PET54. Mutations in PET54 cause an absence of the coxIII protein despite the presence of normal levels of its mRNA. pet494 mutations are known to be suppressible by mitochondrial gene rearrangements that replace the normal 5'-untranslated leader of the oxi2 mRNA with the leaders of other mitochondrial mRNAs. In this study we show that pet54, pet494 double mutants are suppressed by the same mitochondrial gene rearrangements, showing that the PET54 product is specifically required, in addition to the PET494 protein, for translation of the oxi2 mRNA. Since, as we show here, PET54 is not an activator of PET494 gene expression, our results suggest that the products of both of these genes may act together to stimulate coxIII translation.
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Núcleo Celular/metabolismo , ADN Mitocondrial/genética , Complejo IV de Transporte de Electrones/genética , Genes Fúngicos , Genes , Biosíntesis de Proteínas , ARN Mensajero/genética , Saccharomyces cerevisiae/genética , Sustancias Macromoleculares , Mitocondrias/enzimología , Saccharomyces cerevisiae/enzimología , beta-Galactosidasa/genéticaRESUMEN
The product of Saccharomyces cerevisiae nuclear gene PET494 is known to be required for a posttranscriptional step in the accumulation of one mitochondrial gene product, subunit III of cytochrome c oxidase (coxIII). Here we show that the PET494 protein probably acts in mitochondria by demonstrating that both a PET494-beta-galactosidase fusion protein and unmodified PET494 are specifically associated with mitochondria. To define the PET494 site of action, we isolated mutations that suppress a pet494 deletion. These mutations were rearrangements of the mitochondrial gene oxi2 that encodes coxIII. The suppressor oxi2 genes had acquired the 5'-flanking sequences of other mitochondrial genes and gave rise to oxi2 transcripts carrying the 5'-untranslated leaders of their mRNAs. These results demonstrate that in wild-type cells PET494 specifically promotes coxIII translation, probably by interacting with the 5'-untranslated leader of the oxi2 mRNA.
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Núcleo Celular/metabolismo , Genes Fúngicos , Biosíntesis de Proteínas , Procesamiento Postranscripcional del ARN , ARN Mensajero/genética , Saccharomyces cerevisiae/genética , Secuencia de Bases , Genes , Mutación , Plásmidos , ARN Mitocondrial , beta-Galactosidasa/genéticaRESUMEN
The product of the yeast nuclear gene PET494 is required specifically for the translation of the mitochondrially encoded subunit III of cytochrome c oxidase. We have determined the DNA sequence of a 1.9 kb fragment carrying PET494. The sequence contains a single long open reading frame of 489 codons. This open reading frame encodes the PET494 protein since the DNA sequence of the corresponding fragment derived from a strain with a known pet494 amber mutation contained an in frame UAG codon. The results of S1 nuclease protection experiments demonstrated that this region is transcribed and that the 5' ends of the major transcripts lie 30 to 40 base-pairs upstream of the first AUG codon in the PET494 reading frame. The predicted PET494 protein has a highly basic amino-terminal domain of 66 amino acids followed by a stretch of 32 uncharged residues, half of which are hydrophobic. The remainder of the protein is not unusual in amino acid composition or distribution except that the carboxyterminal region is notably basic. The phenotype of mutations generated in vitro around codon 119 by exonuclease digestion and linker insertion indicated that this region is dispensable for function. A mutation caused by deletion of 101 bp of coding sequence behaved like a simple frameshift when inserted into the chromosome: it was partially suppressed by the recessive non-group specific frameshift suppressor suf13 and reverted to Pet+ phenotype by mutations linked to PET494.