| Project/Area Number |
63580156
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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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 | Nagasaki University |
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Principal Investigator |
KOIKE Masahiko Nagasaki University, School of Medicine, Professor, 医学部, 教授 (10039521)
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| Co-Investigator(Kenkyū-buntansha) |
URATA Yoshishige Nagasaki University, School of Medicine, Instructor, 医学部, 助手 (30185087)
KOIKE Kichiko Nagasaki University, School of Medicine, Associate Professor, 医学部, 助教授 (80039619)
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| Project Period (FY) |
1988 – 1989
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| Project Status |
Completed (Fiscal Year 1989)
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| Budget Amount *help |
¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 1989: ¥400,000 (Direct Cost: ¥400,000)
Fiscal Year 1988: ¥1,300,000 (Direct Cost: ¥1,300,000)
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| Keywords | pyruvate dehydrogenase (PDH) / alpha_2beta_2 subunit structure / PDHalpha, beta cDNAs / PDHalpha, beta mRNAs / biosynthesis / PDHalpha gene / PDHbeta gene / nucleotide sequences |
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| Research Abstract |
1. The cDNAs encoding human pyruvate dehydrogenase (PDH)alpha and beta subunits were isolated from a HeLa cell and human foreskin fibroblast cDNA libraries. They spans 1,363 base pairs (alpha) and 1,557 base pairs (beta), respectively, and encoded alpha precursor of 392 amino acid residues (Mr 43,414) and beta precursor of 359 residues (Mr 39,046), respectively. The amino acid sequences deduced from two cDNAs were highly homologous with those of mature porcine PDHalpha and beta. Two cDNA inserts subcloned into expression vector pTZ18R synthesized mRNAs identical with HeLa cell PDHalpha and betamRNAs in in vitro transcription system. Large scale synthesis of each subunit protein have been attempted by using two cDNAs constructed into prokaryotic expression vectors.
2. Genomic clones encompassing the entire gene for human PDHalpha has been isolated from a leukocyte genomic library. The PDHalpha gene spans 17,082 base pairs and is composed of 11 exons and 10 introns. A total of seven Alu repeats were found in five introns. The entire nucleotide sequence of the gene has been determined and typical consensus promoter sequences in the 5'-flanking region were found. Primer extension analysis indicated that transcription start site is a thymine residue 124 bases upstream from ATG codon in exon 1.
3. Genomic clones encompassing the entire gene for human PDHbeta has been isolated from a leukocyte genomic library constructed into lambdaEMBL3. The clone was characterized by restriction enzyme analysis, extensive DNA sequencing and primer extension analysis. The PDHbeta gene spans 18 kilobase pairs and is composed of 10 exons and 9 introns. Two Alu repeats were found in two introns. The 5'-flanking region of the gene contains CAAT box and 3 Spl binding sequences. The transcription was initiated with a adenine residue 132 bases upstream from the translation initiation codon in exon 1.
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