A new promoter-probe vector for Saccharomyces cerevisiae using fungal glucoamylase cDNA as the reporter gene.
A system is described for the selection of DNA sequences showing promoter activity in the yeast Saccharomyces cerevisiae using a heterologous reporter enzyme which is efficiently secreted by the yeast host. A multicopy shuttle plasmid of the YEp-type was constructed so as to carry multiple unique cloning sites at the 5' end of the Aspergillus awamori glucoamylase cDNA. Glucoamylase can only be expressed upon insertion at one of these unique cloning sites of a DNA fragment from any source, provided it is endowed with promoter function in S. cerevisiae. As the glucoamylase signal-peptide is functional in S. cerevisiae, the enzyme is efficiently secreted by the yeast transformants. This phenotype can be very easily detected on plate assays and accurately quantified by spectrophotometric analysis of the culture supernatant. Since S. cerevisiae naturally lacks amylolytic activity, any wild-type strain can be used as a host in this system. To evaluate the system, a DNA pool of random fusions was created by ligating sau 3A digested S. cerevisiae genomic DNA to the BglII-linearized vector. The resulting hybrid plasmids were transformed into S. cerevisiae and several transformants secreting glucoamylase to varying degrees were obtained.
Scorpione
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RC
Departamento de Biologia Celular, Universidade de Brasília, DF.
De Camargo
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Schenberg
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Astolfi-Filho
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D001230:Aspergillus / Q000201:enzymology; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000235:genetics; D005800:Genes, Fungal / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics*; D005087:Glucan 1,4-alpha-Glucosidase / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics*; D021382:Protein Sorting Signals / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*
D004271:DNA, Fungal; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D005087:Glucan 1,4-alpha-Glucosidase