| Budget Amount *help |
¥6,500,000 (Direct Cost: ¥6,500,000)
Fiscal Year 1987: ¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 1986: ¥1,900,000 (Direct Cost: ¥1,900,000)
Fiscal Year 1985: ¥2,800,000 (Direct Cost: ¥2,800,000)
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| Research Abstract |
Expression of acid phosphatase genes in Saccharomyces cerevisiae are regulated by a system consisting of five regulatory genes,i.e.,PHO2,PHO4,PHO80,PHO81,and PHO85.The same regulatory genes,except for PHO2,also regulate expression of a gene encoding alkaline phosphatase.A current model proposes that a refulatory molecule (positive factor)specified by PHO4 is indispensable for the transcription of both the genes for acid and alkaline phosphatases.With repressively high amount of inorganic phosphate (Pi)in the medium,a product of the PHO80 gene (negative factor) aggregates with the positive factor,preventing it from activating the transcription of the enzyme structural genes.When the Pi concectration in the medium is low enough,the PHO81 product (mediator)reacts with the negative factor,thereby releasing the positive factor from the negaive factor and allowing it to activate the transcription of the structural ganes.We investigated the mode of expression of the regulatory genes.It was fo
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und that the PHO4 expression (and also PHO80; Toh-e and Shimauchi,Yeast 2:129-139,1986)is low level constitutive.The PHO2 gene was also found to be expressed low level,while it was repressed by Pi and the active PHO2 product.The PHO85 gene might be involved in a mechanism of the PHO80 expression (A. Toh-e,personal communication).We then examined the effect of incresed copy number of the regulatory genes for the acid phosphatase production.More increased phosphatase activities were observed on the increase of the PHO4 gene than the structural genes for enzyme,while the dosage of PHO2 affect little on the enzyme activity. The dosage effect of the PHO4 gene was cancelled by concurrent dosage of the PHO8 gene.These observation strongly suggest that increased dosage or production of the positive factor is effective for a promoter in the protein production by recombinant DNA technology.That the PHO2 gene also directly concerned with gwnw expression was suggested by nuclear localization of the PHO2 proetein and its binding with the5' upstream region of the structural gene.We also investigated the position effect of the positive factor-recognition sites at the5' upstream region of a structural gene with the HIS5 gene encoding histidinol-phosphate aminotransferase as a model.The results showed that one of the recognition sites the most closely located to the open reading frame is essential for the gene regulation and two recognition sites should have an appropriate distance between them. Less
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