1990 Fiscal Year Final Research Report Summary
Structure and Function of Genes for Phospholipid Synthesis
| Project/Area Number |
01480527
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
代謝生物化学
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| Research Institution | Gunma University |
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Principal Investigator |
YAMASHITA Satoshi Gunma University, Biochemistry, Professor, 医学部, 教授 (50025623)
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| Co-Investigator(Kenkyū-buntansha) |
KODAKI Tsutomu Gunma University, Biochemistry, Assistant, 医学部, 助手 (70170264)
UCHIDA Tsutomu Gunma University, Biochemistry, Assistant, 医学部, 助手 (00160276)
NIKAWA Jun-ichi Gunma University, Biochemistry, Lecturer, 医学部, 講師 (00134271)
HOSAKA Kohei Gunma University, Biochemisty, Lecturer, 医学部, 講師 (70108992)
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| Project Period (FY) |
1989 – 1990
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| Keywords | Phospholipid synthesis / Gene cloning / Promoter / Phosphatidylethanolamine methylation pathway / Choline kinase |
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| Research Abstract |
Phosphatidylcholine is involved in a variety of cellular functions as an essential membrane component in eukaryotes. This investigation was darried out to elucidate how phosphatidylcholine synthesis is regulated. To this end, we studied the structure, function, and expression of the genes encoding the phosphatidylethanolamine methylation pathway in yeast and the CDP-choline pathway in rat liver.
1. PEM1 and PEM2 genes encoding the yeast Phosphatidylethanolamine methylation pathway. We disrupted the PEM1 and PEM2 loci in the wild-type yeast genome and determined methyltransferase activities in the disruptants. The assay showed that the PEM1-encoding enzyme was specific for the first methylation of the pathway whereas the PEM2-encoding one catalyzes all three methylation steps with a preference for the second and third methylation ones. Since the PEM1 and PEM2 genes were both subject to repression by inositol and choline, we analyzed the 5' upstream regions of these genes to define the up
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stream activation sequence responsible for this transcriptional contol. Deletion analysis and UAS assay identified a arepeat of the Catrtgaa sequence as an essential component of the regulatory regions of the PEM1 gene and a comblination of Catrtgaa and GRE1 site as that of the PEM2 gene. The proteins which specifically bind to these sequences were identified.
2. Rat liver choline Kinase. Choline kinase is known to be regulatory enzyme in the CDP-choline pathway. To elucidate its control, we purified the enzyme from rat brain by 15,000-fold to homogeneity. The enzyme was a homodimer consisting of two identical 44-kd subunits. Antiboty was raised against the enzyme and used for cloning choline kinase cDNA from rat liver lambda 11 cDNA library. The cDNA was ligated into an expression vector and transformed into E. coli cells, where the clone directed the synthesis of an active choline kinase indistinguishable from the rat liver enzyme. Significant homology was noted in the deduced amino acid sequences between rat liver and yeast choline kinases. Less
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