RESUMEN
A novel protein family (p14.5, or YERO57c/YJGFc) highly conserved throughout evolution has recently been identified. The biological role of these proteins is not yet well characterized. Two members of the p14.5 family are present in the yeast Saccharomyces cerevisiae. In this study, we have characterized some of the biological functions of the two yeast proteins. Mmf1p is a mitochondrial matrix factor, and homologous Mmf1p factor (Hmf1p) copurifies with the soluble cytoplasmic fraction. Deltammf1 cells lose mitochondrial DNA (mtDNA) and have a decreased growth rate, while Deltahmf1 cells do not display any visible phenotype. Furthermore, we demonstrate by genetic analysis that Mmf1p does not play a direct role in replication and segregation of the mtDNA. rho(+) Deltammf1 haploid cells can be obtained when tetrads are directly dissected on medium containing a nonfermentable carbon source. Our data also indicate that Mmf1p and Hmf1p have similar biological functions in different subcellular compartments. Hmf1p, when fused with the Mmf1p leader peptide, is transported into mitochondria and is able to functionally replace Mmf1p. Moreover, we show that homologous mammalian proteins are functionally related to Mmf1p. Human p14.5 localizes in yeast mitochondria and rescues the Deltammf1-associated phenotypes. In addition, fractionation of rat liver mitochondria showed that rat p14.5, like Mmf1p, is a soluble protein of the matrix. Our study identifies a biological function for Mmf1p and furthermore indicates that this function is conserved between members of the p14.5 family.
Asunto(s)
Secuencia Conservada , ADN Mitocondrial/genética , Proteínas Fúngicas/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales , Proteínas/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Animales , Anticuerpos/inmunología , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Clonación Molecular , Replicación del ADN , ADN Mitocondrial/biosíntesis , Evolución Molecular , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/inmunología , Eliminación de Gen , Genoma , Humanos , Membranas Intracelulares/química , Membranas Intracelulares/metabolismo , Microscopía Electrónica , Mitocondrias/química , Mitocondrias/genética , Mitocondrias/ultraestructura , Datos de Secuencia Molecular , Fenotipo , Transporte de Proteínas , Proteínas/química , Proteínas/genética , Proteínas/inmunología , Ratas , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestructura , Alineación de Secuencia , SolubilidadRESUMEN
We describe the disruption and basic functional analysis of five novel open reading frames (ORFs) discovered during the sequencing of the Saccharomyces cerevisiae genome: YJL118w, YJL122w, YJL123c, YJL124c, YJL125c, located on chromosome X. Disruptions have been realized using the long-flanking homology-PCR replacement strategy (LFH-PCR; Wach et al., 1996) in the FY1679 diploid strain. Sporulation and tetrad analysis of these heterozygous deletants were performed, as well as a functional analysis on the haploid deleted strains: different growth conditions (complete glucose and glycerol, minimal media) at three temperatures 15, 30 and 37 degrees C were tested. Analysis revealed YJL125c as an essential gene; the four other ORFs were non-essential and showed no particular phenotype. In addition, the five kanMX4 disruption cassettes were cloned in pUG7 vector. Finally, the five ORFs with their promoter and terminator regions were cloned in the centromeric yeast vector pRS416. The vectors containing the disruption cassettes, the cognate wild-type genes, as well as the deletant strains are available at the EU EUROFAN (EUROSCARF, Frankfurt, DE) genetic and stock centre.
Asunto(s)
Genes Fúngicos , Sistemas de Lectura Abierta/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/genética , Cromosomas Fúngicos/genética , Medios de Cultivo , Eliminación de Gen , Genes Esenciales , Plásmidos/genética , Reacción en Cadena de la Polimerasa/métodos , Temperatura , Transformación GenéticaRESUMEN
From lidar signals detected with a shot per shot differential absorption lidar instrument tuned for tropospheric ozone measurements and recording each individual return, we reconstruct histograms of their sampled values for each channel of our digitizers. The analysis of their shape permits the correction of our measurements for experimental biases. In particular, a negative correlation is found between the skew of the histograms and the intensity of the backscattered light. The skew comes from a tail at high sampled values, interpreted as due to a raise of the relative contribution to the signal of a signal-induced noise when this intensity diminishes. By fitting a Gaussian function to the histograms without considering their tails, we calculate average signals unbiased by the corresponding noise. This approach allows us to increase the range of our ozone profiles, up to as much as double it in some cases.
RESUMEN
The organisation of the URA1 gene of Schizosaccharomyces pombe was determined from the entire cDNA cloned by the transformation of an ATCase-deficient strain of Saccharomyces cerevisiae. The URA1 gene encodes the bifunctional protein GLNase/CPSase-ATCase which catalyses the first two steps of the pyrimidine biosynthesis pathway. The complete nucleotide sequence of the URA1 cDNA was elucidated and the deduced amino-acid sequence was used to define four domains in the protein; three functional domains, corresponding to GLNase (glutamine amidotransferase), CPSase (carbamoylphosphate synthetase) and ATCase (aspartate transcarbamoylase) activities, and one cryptic DHOase (dihydroorotase) domain. Genetic investigations confirmed that both GLNase/CPSase and ATCase activities are carried out by the same polypeptide. They are also both feedback-inhibited by UTP (uridine triphosphate). Its organization and regulation indicate that the S. pombe URA1 gene product appears very similar to the S. cerevisiae URA2 gene product.
Asunto(s)
Antranilato Sintasa , Aspartato Carbamoiltransferasa/genética , Carbamoil-Fosfato Sintasa (Glutamina-Hidrolizante)/genética , Dihidroorotasa/metabolismo , Complejos Multienzimáticos/genética , Transferasas de Grupos Nitrogenados , Saccharomyces cerevisiae/enzimología , Schizosaccharomyces/genética , Secuencia de Aminoácidos , Aspartato Carbamoiltransferasa/antagonistas & inhibidores , Aspartato Carbamoiltransferasa/metabolismo , Secuencia de Bases , Carbamoil-Fosfato Sintasa (Glutamina-Hidrolizante)/antagonistas & inhibidores , Carbamoil-Fosfato Sintasa (Glutamina-Hidrolizante)/metabolismo , Clonación Molecular , ADN de Hongos , Datos de Secuencia Molecular , Complejos Multienzimáticos/antagonistas & inhibidores , Complejos Multienzimáticos/metabolismo , Schizosaccharomyces/enzimología , Homología de Secuencia de Aminoácido , Transferasas/metabolismo , Uridina Trifosfato/metabolismoRESUMEN
The positive screening procedure previously described was used in order to select, clone and characterize mutants defective in negative feedback control by UTP of the yeast carbamoylphosphate synthetase-aspartate transcarbamylase protein (CPSase-ATCase). The selection procedure was improved by adding a general mapping method for dominant mutations in order to avoid sequencing the whole URA2 allele (7 kb). All 16 mutants obtained carry missense mutations leading to single amino acid replacements: five of them are located in the CPSase domain while the other 11 are in the ATCase domain. In these 16 mutants, ATCase is no longer inhibited by UTP although CPSase retains full sensitivity to the effector, suggesting that the regulation of the two activities involve distinct mechanisms. Amino acid replacements in the ATCase domain were located on a three-dimensional model structure of the yeast ATCase domain. They are clustered in two regions of this domain which must be directly involved in the feedback process.
Asunto(s)
Aspartato Carbamoiltransferasa/metabolismo , Carbamoil-Fosfato Sintasa (Glutamina-Hidrolizante)/metabolismo , Complejos Multienzimáticos/metabolismo , Saccharomyces cerevisiae/enzimología , Uridina Trifosfato/metabolismo , Regulación Alostérica/efectos de los fármacos , Secuencia de Aminoácidos , Aminoácidos/fisiología , Aspartato Carbamoiltransferasa/antagonistas & inhibidores , Aspartato Carbamoiltransferasa/genética , Carbamoil-Fosfato Sintasa (Glutamina-Hidrolizante)/antagonistas & inhibidores , Carbamoil-Fosfato Sintasa (Glutamina-Hidrolizante)/genética , Análisis Mutacional de ADN , Retroalimentación , Genes Fúngicos , Datos de Secuencia Molecular , Complejos Multienzimáticos/antagonistas & inhibidores , Complejos Multienzimáticos/genética , Mutación Puntual , Conformación Proteica , Saccharomyces cerevisiae/genéticaAsunto(s)
Evolución Biológica , Genes Fúngicos , Saccharomyces cerevisiae/enzimología , Schizosaccharomyces/enzimología , Acetiltransferasas/genética , Aspartato Carbamoiltransferasa/genética , Carbamoil-Fosfato Sintasa (Glutamina-Hidrolizante)/genética , Dihidroorotasa/genética , Complejos Multienzimáticos/genética , Saccharomyces cerevisiae/genética , Schizosaccharomyces/genéticaAsunto(s)
Aspartato Carbamoiltransferasa/metabolismo , Carbamoil-Fosfato Sintasa (Amoniaco)/metabolismo , Carbamoil-Fosfato Sintasa (Glutamina-Hidrolizante)/metabolismo , Mutación , Saccharomyces cerevisiae/enzimología , Alelos , Regulación Alostérica , Aspartato Carbamoiltransferasa/genética , Carbamoil-Fosfato Sintasa (Amoniaco)/genética , Carbamoil-Fosfato Sintasa (Glutamina-Hidrolizante)/genética , Clonación Molecular , Farmacorresistencia Microbiana , Retroalimentación , Fluorouracilo/farmacología , Genes Fúngicos , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genéticaRESUMEN
We have undertaken an in vivo genetic approach to the analysis of negative feedback control by uridine triphosphate (UTP) of the yeast carbamoylphosphate synthetase-aspartate transcarbamoylase multifunctional protein (CPSase-ATCase). Using an analog of uracil, 5-fluorouracil, we have constructed a screening system leading, in one step, to selection and cloning of a functional aspartate transcarbamoylase that is defective in negative feedback control by UTP. Due to the nature of the screen, spontaneous or UV-induced mutants could be recovered. Well-characterized cloned mutants have been sequenced and reveal one or two modifications in single codons leading to single amino acid replacements. These amino acid changes occurred either in the CPSase or ATCase domains, abolishing their sensitivity to regulation but not their catalytic activities. Hence the regulatory and catalytic sites are distinct. With the same screening system, it may also be possible to enlarge the scope of the molecular study of the feedback processes to include equivalent proteins in fungi as well as higher eukaryotes.