| Project/Area Number |
08672503
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Biological pharmacy
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| Research Institution | Tokyo Medical and Dental University |
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Principal Investigator |
ISHIDATE Kozo Tokyo Medical and Dental University, Medical Research Institute, Associate Professor, 難治疾患研究所, 助教授 (40014287)
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| Co-Investigator(Kenkyū-buntansha) |
MATSUO Ritsuko Tokyo Medical and Dental University, Medical Research Institute Research Associa, 難治疾患研究所, 助手 (00126260)
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| Project Period (FY) |
1996 – 1997
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| Project Status |
Completed (Fiscal Year 1997)
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| Budget Amount *help |
¥2,300,000 (Direct Cost: ¥2,300,000)
Fiscal Year 1997: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1996: ¥1,400,000 (Direct Cost: ¥1,400,000)
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| Keywords | choline kinase / ethanolamine kinase / cDNA cloning / cell growth / phosphocholine / phosphoethanolamine / phosphatidylcholine biosynthesis / cDNAクローニング |
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| Research Abstract |
By means of peptide sequence information, several cDNA clones encoding a 42kDa choline/ethanolamine kinase (CK/EK) were isolated from a rat kidney cDNA library. Eight clones were sequenced with all of them resulting in identical overlapping nucleotide sequences. Four of them posessed entire open reading frame which could encode 394 amino acids with a calculated molecular size of 45,100. The predicted amino acid sequence contained all of the internal petide fragment sequences derived from the purified 42kDa enzyme. When the open reading frame was introduced into pGEX-2T vector and transfected into E.coli cells, a significant CK/EK activity did appear in the cell lysate. A homology comparison with the previously reported CK cDNAs from rat liver showed 66-68% in entire nucleotide sequences and 57-59% in amino acid sequences, indicating that the cloned cDNA here must be a novel CK/EK gene product.
Complementary DNAs homologous to a rat 42kDa CK/EK and to a 50kDa CK were isolated from a 17-day post coitum mouse embryo cDNA library and their sequences were compared btween the two murine species, respectively. The nucleotide sequence homology (within the coding sequence) between mouse and rat 50kDa CKs (96.0%) was considerably higher than that between their 42kDa CK/EKs (92.4%). Northern blot and RT-PCR studies on several rat tissues demonstrated that both isozymes are expressed ubiquitously with the highest level in testis.
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