RESUMO
Two EcoRI restriction fragments carrying Saccharomyces cerevisiae repressible acid phosphatase genes were analyzed. Transcripts were mapped by restriction endonuclease cleavage of glyoxal-stabilized R-loops and by gel blot hybridizations to cDNA. Homology between the two fragments was examined by gel blots and heteroduplex analysis. Each fragment carried a region of about 1.5 kilobases that coded for a repressible acid phosphatase, and these regions showed homology to one another. In addition, one fragment carried a second region of somewhat lower homology that probably codes for the so-called constitutive acid phosphatase.
Assuntos
Fosfatase Ácida/genética , Saccharomyces cerevisiae/genética , Mapeamento Cromossômico , DNA Fúngico/genética , Indução Enzimática , Regulação da Expressão Gênica , Genes , Peso Molecular , Hibridização de Ácido Nucleico , RNA Mensageiro/genética , Transcrição GênicaRESUMO
The nucleotide sequence of 5'-noncoding and N-terminal coding regions of two coordinately regulated, repressible acid phosphatase genes from Saccharomyces cerevisiae were determined. These unlinked genes encode different, but structurally related polypeptides of molecular weights 60,000 and 56,000. The DNA sequences of their 5'-flanking regions show stretches of extensive homology upstream of, and surrounding, a "TATA" sequence and in a region in which heterogeneous 5' ends of the p60 mRNA were mapped. The predicted amino acid sequences encoded by the N-terminal regions of both genes were confirmed by determination of the amino acid sequence of the native exocellular acid phosphatase and the partial sequence of the presecretory polypeptide synthesized in a cell-free protein synthesizing system. The N-terminal region of the p60 polypeptide was shown to be characterized by a hydrophobic 17-amino acid signal polypeptide which is absent in the native exocellular protein and thought to be necessary for acid phosphatase secretion.
Assuntos
Fosfatase Ácida/genética , Regulação da Expressão Gênica , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Sequência de Bases , DNA Fúngico/genética , Genes , Genes Reguladores , Peptídeos/fisiologia , Sinais Direcionadores de Proteínas , RNA Mensageiro/genéticaRESUMO
In Pichia pastoris, alcohol oxidase (AOX) is the first enzyme in the methanol utilization pathway and is encoded by two genes, AOX1 and AOX2. The DNA and predicted amino acid sequences of the protein-coding portions of the genes are closely homologous, whereas flanking sequences share no homology. The functional roles of AOX1 and AOX2 in the metabolism of methanol were examined. Studies of strains with disrupted AOX genes revealed that AOX1 was the major source of methanol-oxidizing activity in methanol-grown P. pastoris. The results of two types of experiments each suggested that the difference in AOX activity contributed by the two genes was a consequence of sequences located 5' of the protein-coding portions of the genes. First, the coding portion of AOX2 was able to functionally substitute for that of AOX1 when placed under the control of AOX1 regulatory sequences. Second, when labeled oligonucleotide probes specific for the 5' nontranslated region of each gene were used, it was apparent that the steady-state level of AOX1 mRNA was much higher than that of AOX2. Except for the difference in the amount of mRNA present, the two genes appeared to be regulated in the same manner. A physiological reason for the existence of AOX2 was sought but was not apparent.
Assuntos
Oxirredutases do Álcool/genética , Genes Fúngicos , Família Multigênica , Pichia/genética , Saccharomycetales/genética , Alelos , Clonagem Molecular , Mutação , Pichia/metabolismo , Plasmídeos , RNA Fúngico/genética , RNA Fúngico/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Mapeamento por RestriçãoRESUMO
We characterized the organization and expression of PHO5 and PHO3, the tightly linked repressible and constitutive acid phosphatase genes of Saccharomyces cerevisiae. The "constitutive" gene, PHO3, is expressed only when PHO5 is not. Altering PHO5 expression, either through promoter deletions or through mutations in trans-acting regulatory genes, showed that PHO5 expression is sufficient to block transcription of PHO3. An active genomic copy of PHO5 was able to block expression of PHO3 from a high-copy-number plasmid, showing that some trans-acting product of PHO5 is involved. This is probably a translation product, since the presence of a nontranslatable PHO5 RNA did not inhibit transcription of PHO3.
Assuntos
Fosfatase Ácida/genética , Saccharomyces cerevisiae/genética , Mapeamento Cromossômico , Clonagem Molecular , Regulação da Expressão Gênica , Genes , Família Multigênica , Mutação , Fosfatos/fisiologia , Regiões Promotoras Genéticas , RNA Mensageiro/genética , Homologia de Sequência do Ácido NucleicoRESUMO
Segments of yeast genomic DNA containing two enolase structural genes have been isolated by subculture cloning procedures using a cDNA hybridization probe synthesized from purified yeast enolase mRNA. Based on restriction endonuclease and transcriptional maps of these two segments of yeast DNA, each hybrid plasmid contains a region of extensive nucleotide sequence homology which forms hybrids with the cDNA probe. The DNA sequences which flank this homologous region in the two hybrid plasmids are nonhomologous indicating that these sequences are nontandemly repeated in the yeast genome. The complete nucleotide sequence of the coding as well as the flanking noncoding regions of these genes has been determined. The amino acid sequence predicted from one reading frame of both structural genes is extremely similar to that determined for yeast enolase (Chin, C. C. Q., Brewer, J. M., Eckard, E., and Wold, F. (1981) J. Biol. Chem. 256, 1370-1376), confirming that these isolated structural genes encode yeast enolase. The nucleotide sequences of the coding regions of the genes are approximately 95% homologous, and neither gene contains an intervening sequence. Codon utilization in the enolase genes follows the same biased pattern previously described for two yeast glyceraldehyde-3-phosphate dehydrogenase structural genes (Holland, J. P., and Holland, M. J. (1980) J. Biol. Chem. 255, 2596-2605). DNA blotting analysis confirmed that the isolated segments of yeast DNA are colinear with yeast genomic DNA and that there are two nontandemly repeated enolase genes per haploid yeast genome. The noncoding portions of the two enolase genes adjacent to the initiation and termination codons are approximately 70% homologous and contain sequences thought to be involved in the synthesis and processing messenger RNA. Finally there are regions of extensive homology between the two enolase structural genes and two yeast glyceraldehyde-3-phosphate dehydrogenase structural genes within the 5- noncoding portions of these glycolytic genes.
Assuntos
DNA Fúngico , Genes , Glicerolfosfato Desidrogenase/genética , Fosfopiruvato Hidratase/genética , Saccharomyces cerevisiae/enzimologia , Sequência de Bases , Enzimas de Restrição do DNA , Código Genético , Peso Molecular , Hibridização de Ácido Nucleico , Saccharomyces cerevisiae/genéticaRESUMO
An analog of growth hormone releasing factor (GRF), [Leu27]GRF(1-40)-OH, has been expressed and secreted in Saccharomyces cerevisiae under the control of the alpha-factor gene promoter and prepro sequence. A single pair of consecutive basic residues served as a processing site between the alpha-factor sequences and the GRF sequences. [Leu27]GRF(1-40)-OH from fermentor broth containing 20-30 mg/L of immunoreactive peptides was shown to be correctly processed and to possess biological activity as measured in vitro and in vivo. Additional peptides purified from broth appear to result from proteolytic degradation of the original translation product. Analysis of the amino acid compositions and sequences of these peptides suggests that processing enzymes may be responsible for some of the degradation.
Assuntos
Expressão Gênica , Hormônio Liberador de Hormônio do Crescimento/análogos & derivados , Fragmentos de Peptídeos/genética , Adeno-Hipófise/fisiologia , Engenharia de Proteínas/métodos , Sequência de Aminoácidos , Aminoácidos/análise , Animais , Bioensaio , Células Cultivadas , Vetores Genéticos , Hormônio Liberador de Hormônio do Crescimento/genética , Fator de Acasalamento , Dados de Sequência Molecular , Fragmentos de Peptídeos/metabolismo , Peptídeos/genética , Adeno-Hipófise/citologia , Regiões Promotoras Genéticas/genética , Sinais Direcionadores de Proteínas/genética , Ratos , Saccharomyces cerevisiae/genética , Transfecção/genéticaRESUMO
A synthetic gene encoding aprotinin (bovine pancreatic trypsin inhibitor) was fused to the Saccharomyces cerevisiae prepro alpha mating factor leader sequence at the dibasic amino acid processing site. Pichia pastoris strains were developed to express one or multiple copies of a methanol-inducible expression cassette containing the gene fusion. P. pastoris containing a single copy of the vector secreted approximately 150 mg/l of immunoreactive protein. A construct bearing five copies of the expression cassette secreted 930 mg/l of aprotinin. The purified aprotinin molecule was equipotent with the native molecule in a trypsin inhibition assay. Protein sequence analysis showed that the alpha factor-aprotinin fusion was not processed at the basic amino acid residues Lys-Arg. Instead, recombinant aprotinin had additional N-terminal amino acids derived from prepro alpha factor. The N-terminal extension was variably 11 or 4 amino acids. Inclusion of the spacer DNA sequence encoding Glu and Ala between aprotinin and the Lys-Arg processing site led to the secretion of a biologically active aprotinin containing only a Glu-Ala N-terminal extension.