RESUMO
The role of cyclic AMP (cAMP) in the regulation of mammalian cell proliferation has been the subject of controversy. Negative control was demonstrated in the 1970s, but evidence of positive control in other cell types has been neglected. Recent evidence which demonstrates such a control in the yeast Saccharomyces cerevisiae has now made this concept acceptable.
Assuntos
Divisão Celular/efeitos dos fármacos , AMP Cíclico/farmacologia , Animais , Saccharomyces cerevisiaeRESUMO
The bloodstream form of Trypanosoma brucei contains transcripts of at least four genes showing partial sequence homology to the genes for eucaryotic adenylate and guanylate cyclases (S. Alexandre, P. Paindavoine, P. Tebabi, A. Pays, S. Halleux, M. Steinert, and E. Pays, Mol. Biochem. Parasitol. 43:279-288, 1990). One of these genes, termed ESAG 4, belongs to the polycistronic transcription unit of the variant surface glycoprotein (VSG) gene. Whereas ESAG 4 is transcribed only in the bloodstream form of the parasite, the three other genes, GRESAG 4.1, 4.2, and 4.3, are also expressed in procyclic (insect) forms. These genes differ primarily in a region presumed to encode a large extracellular domain. We show here that ESAG 4-related glycoproteins of about 150 kDa can be found in the trypanosome membrane, that they are detected, by light and electron gold immunocytochemistry, only at the surface of the flagellum, and that the products of at least two of these genes, ESAG 4 and GRESAG 4.1, can complement a Saccharomyces cerevisiae mutant for adenylate cyclase. The recombinant cyclases are associated with the yeast membrane fraction and differ with respect to their activation by calcium: while the GRESAG 4.1 and yeast cyclases are inhibited by calcium, the ESAG 4 cyclase is stimulated. ESAG 4 thus most probably encodes the calcium-activated cyclase that has been found to be expressed only in the bloodstream form of T. brucei (S. Rolin, S. Halleux, J. Van Sande, J. E. Dumont, E. Pays, and M. Steinert. Exp. Parasitol. 71:350-352, 1990). Our data suggest that the trypanosome cyclases are not properly regulated in yeast cells.
Assuntos
Adenilil Ciclases/genética , Flagelos/enzimologia , Trypanosoma brucei brucei/genética , Glicoproteínas Variantes de Superfície de Trypanosoma/genética , Adenilil Ciclases/metabolismo , Animais , Teste de Complementação Genética , Immunoblotting , Microscopia Eletrônica , Família Multigênica , Mutação , Saccharomyces cerevisiae/genética , Trypanosoma brucei brucei/enzimologia , Trypanosoma brucei brucei/ultraestrutura , Glicoproteínas Variantes de Superfície de Trypanosoma/metabolismoRESUMO
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.
Assuntos
Sequência Conservada , DNA Mitocondrial/genética , Proteínas Fúngicas/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais , Proteínas/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Animais , Anticorpos/imunologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Clonagem Molecular , Replicação do DNA , DNA Mitocondrial/biossíntese , Evolução Molecular , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/imunologia , Deleção de Genes , Genoma , Humanos , Membranas Intracelulares/química , Membranas Intracelulares/metabolismo , Microscopia Eletrônica , Mitocôndrias/química , Mitocôndrias/genética , Mitocôndrias/ultraestrutura , Dados de Sequência Molecular , Fenótipo , Transporte Proteico , Proteínas/química , Proteínas/genética , Proteínas/imunologia , Ratos , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestrutura , Alinhamento de Sequência , SolubilidadeRESUMO
The complete nucleotide (nt) sequence of the PUT4 gene, whose product is required for high-affinity proline active transport in the yeast Saccharomyces cerevisiae, is presented. The sequence contains a single long open reading frame of 1881 nt, encoding a polypeptide with a calculated Mr of 68,795. The predicted protein is strongly hydrophobic and exhibits six potential glycosylation sites. Its hydropathy profile suggests the presence of twelve membrane-spanning regions flanked by hydrophilic N- and C-terminal domains. The N terminus does not resemble signal sequences found in secreted proteins. These features are characteristic of integral membrane proteins catalyzing translocation of ligands across cellular membranes. Protein sequence comparisons indicate strong resemblance to the arginine and histidine permeases of S. cerevisiae, but no marked sequence similarity to the proline permease of Escherichia coli or to other known prokaryotic or eukaryotic transport proteins. The strong similarity between the three yeast amino acid permeases suggests a common ancestor for the three proteins.
Assuntos
Transportadores de Cassetes de Ligação de ATP , Sistemas de Transporte de Aminoácidos Básicos , Sistemas de Transporte de Aminoácidos Neutros , Sistemas de Transporte de Aminoácidos , Proteínas de Bactérias , Proteínas de Membrana Transportadoras/genética , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Sequência de Bases , Dados de Sequência Molecular , Mapeamento por Restrição , Proteínas de Saccharomyces cerevisiae , Homologia de Sequência do Ácido NucleicoRESUMO
In Saccharomyces cerevisiae, mutations at the GAP1 locus selectively abolish the activity of the general amino acid transport system. This permease catalyses active transport of apparently all biological amino acids across the plasma membrane. We have determined the nucleotide sequence of the GAP1 gene. The sequence contains an open reading frame of 601 codons corresponding to a polypeptide of Mr 65578. This polypeptide is strongly hydrophobic; it exhibits three potential glycosylation sites. Hydropathy analysis suggests 12 membrane-spanning regions. The N-terminal domain is charged, it does not resemble hydrophobic signal sequences found in secreted proteins. Hence the GAP1 gene encodes a protein with characteristics typical of integral membrane proteins translocating ligants across cellular membranes. The deduced amino acid sequence of GAP1 protein presents strong similarities to those of the yeast arginine, histidine and proline permeases, suggesting a common evolutionary origin for these amino acid permeases. Nitrogen-source regulation of the GAP1 permease is believed to occur at two distinct levels, i.e. permease synthesis and permease activity [Grenson (1983) Eur. J. Biochem. 133, 135-139]. Northern analysis of GAP1-specific transcripts in wild-type and in mutant strains is in agreement with these views and indicates that nitrogen catabolite repression of GAP1 synthesis occurs at the RNA level.
Assuntos
Regulação Enzimológica da Expressão Gênica , Genes Fúngicos , Proteínas de Membrana Transportadoras/genética , Nitrogênio/metabolismo , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Sistemas de Transporte de Aminoácidos , Sequência de Bases , Northern Blotting , Repressão Enzimática/genética , Proteínas de Membrana Transportadoras/biossíntese , Proteínas de Membrana Transportadoras/isolamento & purificação , Dados de Sequência Molecular , Peptídeos/isolamento & purificação , RNA Fúngico/análise , Mapeamento por Restrição , Saccharomyces cerevisiae/enzimologiaRESUMO
The complete sequence of a 36775 bp DNA segment located on the right arm of chromosome XV of Saccharomyces cerevisiae has been determined and analysed. The sequence encodes 26 open reading frames of at least 100 amino acids. Eight of these correspond to known genes, whereas 18 correspond to new genes.
Assuntos
Cromossomos Fúngicos/genética , Genes Fúngicos/genética , Fases de Leitura Aberta/genética , Saccharomyces cerevisiae/genética , Dados de Sequência Molecular , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
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.
Assuntos
Genes Fúngicos , Fases de Leitura Aberta/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/genética , Cromossomos Fúngicos/genética , Meios de Cultura , Deleção de Genes , Genes Essenciais , Plasmídeos/genética , Reação em Cadeia da Polimerase/métodos , Temperatura , Transformação GenéticaRESUMO
The cargB or CAR2 gene, coding for ornithine aminotransferase, was isolated by functional complementation of a cargB- mutation in Saccharomyces cerevisiae. It was used as a hybridization probe to analyse RNA and chromosomal DNA from four strains bearing cis-dominant regulatory mutations leading to constitutive, mating-type-dependent, ornithine aminotransferase synthesis. The four mutations appear to be insertions. Their size and restriction pattern suggested that they were transposable elements, Ty1. All were inserted in the same orientation with respect to the cargB gene. We cloned the cargB gene with its associated insertion from two constitutive mutants (cargB+ Oh-1 and cargB+ Oh-2). We determined the sequence of the cargB 5' region from the wild-type gene and from the two mutated genes. The DNA sequences of the extremities of the two insertions were very homologous but not identical and were similar to previously reported Ty1 element direct repeats (delta). The same five-base-pair sequence, ATATA, was found at both ends of both Ty1 elements, indicating that both Ty1 were transposed to the same site. This site is located 115 base pairs upstream from the putative cargB coding region. The 5' end of cargB transcript as determined by S1 mapping was the same in the wild-type strain and in the four mutants. The cargB transcript was not detected in the wild-type strain grown under non-induced conditions, while under the same conditions it was present in all four mutants.
Assuntos
Ornitina-Oxo-Ácido Transaminase/genética , Saccharomyces cerevisiae/genética , Transaminases/genética , Sequência de Bases , Clonagem Molecular , Enzimas de Restrição do DNA , Elementos de DNA Transponíveis , DNA Fúngico/análise , Regulação da Expressão Gênica , Mutação , Ornitina-Oxo-Ácido Transaminase/biossíntese , RNA Fúngico/análise , Saccharomyces cerevisiae/enzimologia , Transcrição Gênica , Transformação GenéticaRESUMO
The NPR1 gene codes for a protein, called the nitrogen permease reactivator protein or Npr1, which appears to promote the activity of several permeases for nitrogenous substances under conditions of nitrogen catabolite derepression, but fails to do so in the presence of ammonium ions. This gene has been cloned. Its transcription seems unaffected by growth on ammonia, so any ammonia regulation of Npr1 function most likely occurs at another level. In order to elucidate further the mechanism of permease inactivation, which requires an intact NPI1 gene product (NPI1 for nitrogen permease inactivator gene, formerly termed MUT2) and the role of Npr1 in counteracting this process, we have studied the effects of NPR1 and NPI1 gene dosage on general amino-acid permease activity. On nitrogen-derepressing media, NPR1 gene dose can be increased from 1 copy in a diploid to 16 plasmid-borne copies in a haploid strain without altering general amino-acid permease activity. On minimal ammonia medium, the plasmid-bearing haploid cells exhibit low but increased general amino-acid permease activity with respect to non-transformed cells. The adverse effect of the NPI1 gene product on general amino-acid permease activity is reduced when NPI1 gene dose is decreased to 1 gene copy in a diploid strain, regardless of the nitrogen source. We hypothesize that this product inactivates the permease by stoichiometric binding and that the Npr1 protein or a product of its catalytic action opposes this binding under conditions of nitrogen derepression.
Assuntos
Amônia/farmacologia , Genes Fúngicos , Proteínas de Membrana Transportadoras/genética , RNA Fúngico/análise , Saccharomyces cerevisiae/enzimologia , Sistemas de Transporte de Aminoácidos , Clonagem Molecular , DNA Fúngico/análise , Ativação Enzimática , Regulação da Expressão Gênica , Mutação , Saccharomyces cerevisiae/genéticaRESUMO
Despite the important role of the ammonium ion in metabolism, i.e. as a form of nitrogen that is taken up from the soil by microorganisms and plants, little is known at the molecular level about its transport across biomembranes. Biphasic uptake kinetics have been observed in roots of several plant species. To study such transport processes, a mutant yeast strain that is deficient in two NH4+ uptake systems was used to identify a plant NH4+ transporter. Expression of an Arabidopsis cDNA in the yeast mutant complemented the uptake deficiency. The cDNA AMT1 contains an open reading frame of 501 amino acids and encodes a highly hydrophobic protein with 9-12 putative membrane spanning regions. Direct uptake measurements show that mutant yeast cells expressing the protein are able to take up [14C]methylamine. Methylamine uptake can be efficiently competed by NH4+ but not by K+. The methylamine uptake is optimal at pH 7 with a Km of 65 microM and a Ki for NH4+ of approximately 10 microM, is energy-dependent and can be inhibited by protonophores. The plant protein is highly related to an NH4+ transporter from yeast (Marini et al., accompanying manuscript). Sequence homologies to genes of bacterial and animal origin indicate that this type of transporter is conserved over a broad range of organisms. Taken together, the data provide strong evidence that a gene for the plant high affinity NH4+ uptake has been identified.
Assuntos
Arabidopsis/genética , Proteínas de Transporte/genética , Proteínas de Transporte de Cátions , Proteínas de Plantas/genética , Compostos de Amônio Quaternário/metabolismo , Sequência de Aminoácidos , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Clonagem Molecular , Genes de Plantas/genética , Teste de Complementação Genética , Transporte de Íons , Metilaminas/metabolismo , Modelos Biológicos , Dados de Sequência Molecular , Proteínas de Plantas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
The complete sequence of a 40247 bp DNA segment located on the left arm of chromosome X of Saccharomyces cerevisiae has been determined and analysed. The sequence encodes the 5' coding region of the URA2 gene and 18 open reading frames of at least 100 amino acids. Ten of these correspond to known genes, whereas eight correspond to new genes. In addition, the sequence contains a tRNA-Ala gene, a tRNA-Asp gene, a Ty4 transposable element and three delta elements.
Assuntos
Cromossomos Fúngicos/genética , DNA Fúngico/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Elementos de DNA Transponíveis , Fatores de Transcrição GATA , Genes Fúngicos , Dados de Sequência Molecular , Fases de Leitura Aberta , Análise de Sequência de DNA , Fatores de Transcrição/genéticaRESUMO
The NPR1 gene of Saccharomyces cerevisiae plays a central role in controlling permease activity; its product is required to promote the activity of at least six distinct transport systems for nitrogenous nutrients under conditions of nitrogen catabolite derepression. We report here the nucleotide sequence of the cloned NPR1 gene. The predicted amino acid sequence indicates that NPR1 encodes a protein of 86 kDa which appears to be organized into two distinct structural domains. The amino-terminal domain of NPR1 (residues 1 to 440) contains 26% serine residues and several regions strongly enriched for PEST residues suggesting a short half-life for the NPR1 protein. The carboxy-terminal region of NPR1 contains consensus sequences characteristic of the catalytic domains of protein kinases. Therefore, NPR1-dependent positive control of nitrogen transport systems most likely involves protein phosphorylation. Northern analysis indicates that the absence of general amino acid permease (GAP1) activity in npr1 mutants is not due to reduction in transcription or messenger stability. Hence, the NPR1 protein probably acts at the post-transcriptional level. Proteins that may serve as substrates for phosphorylation are discussed.
Assuntos
Amônia/metabolismo , Proteínas Fúngicas/genética , Genes Fúngicos , Proteínas de Membrana Transportadoras/metabolismo , Nitrogênio/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Sequência de Bases , Transporte Biológico , Northern Blotting , Dados de Sequência Molecular , Mutação , Proteínas Quinases/metabolismo , RNA Fúngico/biossíntese , Mapeamento por Restrição , Saccharomyces cerevisiae/enzimologia , Schizosaccharomyces/enzimologia , Schizosaccharomyces/genética , Homologia de Sequência do Ácido Nucleico , Serina , Transcrição GênicaRESUMO
The regulatory mutations in Saccharomyces cerevisiae designated cargA + Oh, cargB + Oh, and durOh are alterations in the control regions of the respective structural genes. The alteration causing the cargA + Oh mutation has been shown to be an insertion of a Ty1 element in the 5' noncoding region of the CAR1 ( cargA ) locus. All three mutations cause overproduction of their corresponding gene products and belong to the ROAM family of mutations (Regulated Overproducing Allele responding to Mating signals) in yeast. The amount of overproduction in ROAM mutants is determined, at least in part, by signals that control mating functions in yeast. We report the identification of two genetic loci that regulate Oh mutant gene expression but that do not affect mating ability. These loci are defined by the recessive roc mutations ( ROAM mutation control) that reduce the amount of overproduction caused by the cargA + Oh, cargB + Oh, and durOh mutations. RNAs homologous to CAR1 ( cargA ), DUR1 ,2 and Ty1 DNA probes were analyzed by the Northern hybridization technique. In comparison with wild-type strains, cargA + Oh and durOh mutant strains grown on ammonia medium contain increased amounts of CAR1 and DUR1 ,2 RNA. This RNA overproduction is diminished in MATa/MAT alpha diploid strains as well as in haploid strains that also carry the ste7 mutation which prevents mating or that carry either of the roc1 or roc2 mutant alleles. The amount of RNA homologous to Ty1 DNA is also reduced in ste7 , roc1 , and roc2 mutant strains. This reduction is not observed in a strain with the ste5 mutation, which prevents mating but has no effect on overproduction of ROAM mutant gene products.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Elementos de DNA Transponíveis , Genes Fúngicos , Genes Reguladores , Genes , Mutação , Saccharomyces cerevisiae/genética , Transcrição Gênica , Alofanato Hidrolase/genética , Arginase/genética , Ornitina-Oxo-Ácido Transaminase/genética , Saccharomyces cerevisiae/enzimologia , Especificidade da EspécieRESUMO
Subcellular localization of enzymes of arginine metabolism in Saccharomyces cerevisiae was studied by partial fractionation and stepwise homogenization of spheroplast lysates. These enzymes could clearly be divided into two groups. The first group comprised the five enzymes of the acetylated compound cycle, i.e., acetylglutamate synthase, acetylglutamate kinase, acetylglutamyl-phosphate reductase, acetylornithine aminotransferase, and acetylornithine-glutamate acetyltransferase. These enzymes were exclusively particulate. Comparison with citrate synthase and cytochrome oxidase, and results from isopycnic gradient analysis, suggested that these enzymes were associated with the mitochondria. By contrast, enzymatic activities going from ornithine to arginine, i.e., arginine pathway-specific carbamoylphosphate synthetase, ornithine carbamoyltransferase, argininosuccinate synthetase, and argininosuccinate lyase, and the two first catabolic enzymes, arginase and ornithine aminotransferase, were in the "soluble" fraction of the cell.
Assuntos
Saccharomyces cerevisiae/enzimologia , Acetamidas , Acetilcoenzima A , Acetiltransferases/análise , Aldeído Oxirredutases/análise , Arginase/análise , Argininossuccinato Liase/análise , Argininossuccinato Sintase/análise , Carbamoil Fosfato Sintase (Glutamina-Hidrolizante)/análise , Glutamatos , Mitocôndrias/enzimologia , Compostos Organofosforados , Ornitina , Ornitina Carbamoiltransferase/análise , Ornitina-Oxo-Ácido Transaminase/análise , Fosfotransferases/análise , Fosfotransferases (Aceptor do Grupo Carboxila) , Frações Subcelulares/enzimologia , Transaminases/análiseRESUMO
Transport of 4-aminobutyric acid (GABA) in Saccharomyces cerevisiae is mediated by three transport systems: the general amino acid permease (GAP1 gene), the proline permease (PUT4 gene), and a specific GABA permease (UGA4 gene) which is induced in the presence of GABA. The UGA4 gene encoding the inducible GABA-specific transporter was cloned and sequenced and its expression analyzed. The predicted amino acid sequence shows that UGA4 encodes a 62 kDa protein having 9-12 putative membrane-spanning regions. The predicted UGA4 protein shares significant sequence similarity with the yeast choline transporter (CTR gene), exhibiting but limited similarity to the previously reported GABA transporters, i.e. the yeast GAP1 and PUT4 permeases and the rat brain GAT-1 transporter. Induction of UGA4 in the presence of GABA is exerted at the level of UGA4 mRNA accumulation, most probably at the level of transcription itself. This induction is conferred by the 5' flanking region and requires the integrity of two positive regulatory proteins, the inducer-specific factor UGA3 and the pleiotropic factor UGA35/DURL/DAL81. In the absence of the pleiotropic UGA43/DAL80 repressor, UGA4 is constitutively expressed at high level.
Assuntos
Proteínas de Transporte/genética , Proteínas de Membrana/genética , Proteínas de Membrana Transportadoras , Transportadores de Ânions Orgânicos , Saccharomyces cerevisiae/genética , Ácido gama-Aminobutírico/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Proteínas de Transporte/biossíntese , Clonagem Molecular , Escherichia coli/genética , Proteínas de Escherichia coli , Proteínas da Membrana Plasmática de Transporte de GABA , Teste de Complementação Genética , Óperon Lac/genética , Proteínas de Membrana/biossíntese , Dados de Sequência Molecular , Conformação Proteica , RNA Mensageiro/análise , Proteínas Recombinantes de Fusão/biossíntese , Mapeamento por Restrição , Proteínas de Saccharomyces cerevisiae , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Transcrição GênicaRESUMO
We have recently isolated a rat cDNA encoding a novel cellular protein able to interact with the major nonstructural protein NS1 of parvovirus H-1 and have termed this protein SGT, for small glutamine-rich tetratricopeptide repeat (TPR)-containing protein. Here we report the isolation of a cDNA from human placenta encoding the human homologue, human SGT. SGT from rat and human contain 314 and 313 amino acids, respectively, and share 91% sequence identity at the protein level. The highest degree of similarity is present within the central region containing three TPR motifs in tandem array. The similarities, however, also extend beyond this region. Human SGTtranscript was found to be ubiquitously present in all human tissues tested. By fluorescence in situ hybridization analysis we have mapped the human gene to chromosome 19p13. The SGT-coding sequences are evolutionarily conserved, since we could identify genes encoding proteins of similar size and structure in the genomes of Saccharomyces cerevisiae and Caenorhabditis elegans.
Assuntos
Caenorhabditis elegans/genética , Proteínas/química , Proteínas/genética , Saccharomyces cerevisiae/genética , Homologia de Sequência de Aminoácidos , Sequência de Aminoácidos , Animais , Northern Blotting , Western Blotting , Proteínas de Transporte , Mapeamento Cromossômico , Cromossomos Humanos Par 19/genética , Clonagem Molecular , DNA Complementar/análise , DNA Complementar/isolamento & purificação , Humanos , Chaperonas Moleculares , Dados de Sequência Molecular , Especificidade de Órgãos , Proteínas/isolamento & purificação , Ratos , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
The Saccharomyces cerevisiae gene cargA + or CAR1 , encoding arginase has been cloned by recovering function in transformed yeast cells. It was used to analyse RNA and chromosomal DNA from six strains bearing cis-dominant regulatory mutations leading to constitutive arginase synthesis. The DNA from the four cargA + O- strains in which constitutive arginase synthesis was independent of the mating-type functions showed no detectable differences with the wild- typye . The cargA + O- mutations were, therefore, small alterations, possibly single base substitutions. On the other hand, the cargA + Oh-1 and cargA + Oh-2 mutations, leading to a constitutive and mating-type dependent arginase synthesis, were identified as insertions. Their size and restriction pattern strongly suggested that they were induced by the Ty1 yeast transposable element. This was confirmed by cloning and analysis of the cargA + Oh-1 mutant gene. The concentration of arginase RNA was significantly increased in the mutants, indicating that the regulation of arginase synthesis was exerted, at least in part, at the level of RNA synthesis or stability. In the cargA + Oh-2 strain the Ty1 element was located at a distance of approximately 600 base pairs from the insertion present in the cargA + Oh-1 strain. This result suggests either a surprisingly large arginase regulatory region or an indirect influence of the Ty1 element on gene expression over long distances.
Assuntos
Arginase/genética , Clonagem Molecular , Genes Dominantes , Genes Fúngicos , Genes Fúngicos Tipo Acasalamento , Genes Reguladores , Genes , Sequência de Bases , Enzimas de Restrição do DNA , DNA Fúngico/genética , Hibridização de Ácido Nucleico , Plasmídeos , Biossíntese de Proteínas , RNA Fúngico/genéticaRESUMO
The proline permease gene PUT4 has been cloned. Nitrogen-source regulation ('ammonia sensitivity') of this and at least two other amino-acid permeases is believed to occur at two distinct levels, i.e. permease synthesis and permease activity. Therefore, PUT4 transcription/messenger stability was examined in the ammonia- and proline-grown wild type as well as in mutant strains supposedly affected at only one or at both of these levels. We report transcript-level repression of proline permease synthesis in ammonia-grown cells. Repression is lifted at this level in gdhCR, gln1ts and gdhA mutants which exhibit pleiotropically derepressed permease and catabolic enzyme activities. On the other hand, the npi1 and npi2 mutations, formerly called mut2 and mut4, relieve an inactivation process which seems only to affect permeases. These mutations do not affect the detected PUT4 RNA level. The only known positive factor in proline permease regulation, the nitrogen permease reactivator protein Npr1, is believed to counteract the inactivation process on derepressing media. This protein appears to have an additional, indirect effect on PUT4 transcription/messenger stability: it would actually mediate repression via its activating effect on ammonia uptake.
Assuntos
Sistemas de Transporte de Aminoácidos Neutros , Genes Fúngicos , Genes , Proteínas de Membrana Transportadoras/genética , Nitrogênio/metabolismo , RNA Fúngico/análise , Saccharomyces cerevisiae/enzimologia , Clonagem Molecular , Proteínas de Membrana Transportadoras/metabolismo , Mutação , Prolina/metabolismo , Saccharomyces cerevisiae/genética , Transformação GenéticaRESUMO
The nonstructural protein NS1 of autonomous parvoviruses is essential for viral DNA amplification and gene expression and is also the major cytopathic effector of these viruses. NS1 acts as nickase, helicase, and ATPase and upregulates P38-driven transcription of the capsid genes. We report here the identification of a novel cellular protein that interacts with NS1 from parvovirus H-1 and which we termed SGT, for small glutamine-rich tetratricopeptide repeat (TPR)-containing protein. The cDNA encoding full-length SGT was isolated through a two-hybrid screen with, as bait, the truncated NS1dlC69 polypeptide, which lacks the C-terminal transactivation domain of NS1. Full-length NS1 and SGT interacted in the two-hybrid system and in an in vitro interaction assay. Northern blot analysis revealed one major transcript of about 2 kb that was present in all rat tissues investigated. Rat sgt cDNA coded for 314 amino acids, and the protein migrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular mass of 34 kDa. SGT could be detected in both the nucleus and the cytoplasm of rat cells, as determined by indirect immunofluorescence analysis and Western blotting of fractionated cellular extracts with an affinity-purified antiserum raised against recombinant SGT (AC1.1). In H-1 virus-infected rat and human cells, compared to mock-infected controls, differences in the migration of SGT polypeptides were revealed after Western blot analysis of total cellular extracts. Moreover, the transient expression of NS proteins was sufficient to induce SGT modification. These results show that cellular SGT, which we have identified as an NS1-interacting protein, is modified by parvovirus infection as well as NS expression.