RESUMO
The BGL2 gene from Saccharomyces cerevisiae encodes a beta-glucanase which is localized to the yeast cell wall. The ability of a 23-amino acid (aa) signal peptide derived from the BGL2 gene to direct a heterologous protein to the secretory pathway of yeast has been compared to that of the MF alpha 1-encoded signal peptide in a series of gene fusions. As a model protein, the leech anticoagulant, recombinant hirudin variant 2-Lys47 (HIR) has been studied. From a multicopy plasmid chimaeric proteins were produced which carry the BGL2 signal peptide (or the artificial BGL2 pre-Val7 variant) (i) in front of the MF alpha 1 pro sequence (or modified versions of MF alpha 1 pro), i.e., a prepro signal, or (ii) joined directly to the heterologous protein. Accumulation of active HIR in yeast culture supernatants was observed when the BGL2 (or the BGL2 pre-Val7) signal peptide were used in combination with either of three versions of the MF alpha 1 pro peptide: the authentic MF alpha 1 pro, a partially deleted MF alpha 1 pro-delta 22-61, or a pro bearing an aa change (MF alpha 1 pro-Gly22). In each case the BGL2 signal peptide (or its variant) has proven equally productive to the corresponding MF alpha 1 peptide. Four times more active HIR was detected in the culture supernatant when either signal peptide was fused directly to the recombinant protein, as compared to a prepro protein version. Correct signal peptide cleavage was obtained when HIR was produced as a BGL2 pre-Val7::fusion protein.
Assuntos
Variação Genética , Hirudinas/genética , Sinais Direcionadores de Proteínas/genética , Proteínas Recombinantes de Fusão/biossíntese , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Sequência de Bases , Deleção Cromossômica , Genes Fúngicos , Vetores Genéticos , Hirudinas/biossíntese , Dados de Sequência Molecular , Plasmídeos , Processamento de Proteína Pós-Traducional , Sinais Direcionadores de Proteínas/química , Proteínas Recombinantes de Fusão/genética , Saccharomyces cerevisiae/química , Valina/genéticaRESUMO
The URA4 gene of Saccharomyces cerevisiae, coding for the third enzyme of the pyrimidine pathway, has been cloned through phenotypic complementation of a ura4 mutant of S. cerevisiae. Subcloning of an original 9 kb DNA fragment, carrying the yeast URA4 gene, allowed us to localize the gene on a 2 kb ClaI--BamHI fragment. The sequence of the URA4 structural gene and surrounding DNA was determined by the dideoxynucleotide chain termination method. The URA4 gene encodes a dihydroorotase subunit of calculated molecular weight 40,600. S1 nuclease mapping indicated that transcription of URA4 is initiated at four major start sites located at positions -41, -30, -22 and -18. A set of potentially significant sequences was identified in the 5' OH non-coding region of the gene. The deduced amino acid sequence of dihydroorotase was examined and compared with homologous amino acid sequences of Salmonella typhimurium, Escherichia coli and Drosophila melanogaster. S. cerevisiae dihydroorotase shows 40% homology with the S. typhimurium and E. coli enzymes and 23% homology with the D. melanogaster enzyme. A potential active site has been predicted for dihydroorotase from these comparisons.
Assuntos
Amidoidrolases/genética , Di-Hidro-Orotase/genética , Genes Fúngicos , Genes , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Sequência de Bases , Enzimas de Restrição do DNA , Dados de Sequência Molecular , Mapeamento de Nucleotídeos , Plasmídeos , Saccharomyces cerevisiae/enzimologia , Transcrição GênicaRESUMO
Two yeast DNA fragments carrying the URA 1 gene have been isolated. URA 1 mRNA levels were measured in wild-type yeast and ppr 1 (constitutive for DHOdehase synthesis) strains. Results favour the hypothesis that ppr 1 increases the transcription of URA 1. The corresponding E. coli PYR D gene has also been cloned.
