| Project/Area Number |
03680174
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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 Kichiko Nagasaki University School of Medicine, Associate Professor, 医学部, 助教授 (80039619)
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| Co-Investigator(Kenkyū-buntansha) |
GOTO Sinji Same Institute and Department, Instructor, 医学部, 助手 (50186889)
URATA Yosisige Same Institute and Department, Instructor, 医学部, 助手 (30185087)
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| Project Period (FY) |
1991 – 1992
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| Project Status |
Completed (Fiscal Year 1992)
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| Budget Amount *help |
¥1,900,000 (Direct Cost: ¥1,900,000)
Fiscal Year 1992: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1991: ¥1,000,000 (Direct Cost: ¥1,000,000)
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| Keywords | Pyruvate dehydrogenase / alpha and beta subunits / Gene / Expression / Deficiency / PCR-amplification / Point mutation / ヒトピルビン酸脱水素酵素 / αーサブユニット遺伝子 / βーサブユニット遺伝子 / 変異(欠損症)遺伝子 |
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| Research Abstract |
Human pyruvate dehydrogenase (PDH) is a mitochondrial enzyme encoded by nuclear genes and is a component enzyme of the PDH complex. PDH composed of two nonidentical subunits, alpha (41 kDa, PDHalpha) and beta (36 kDa, PDHbeta) and a tetrameric structure, alpha_2beta_2 (153 kDa). It utilyzes thiamin pyruphosphate (TPP) as coenzyme and the binding site of TPP is located on PDHalpha. Human PDHalpha and beta genes have been cloned using radiolabeled PDHalpha and beta cDNAs. The 5'-flanking regeons of two genes contain typical consensus promotor sequences. Primer extension analysis indicated that the transcriptional start sites of PDHalpha and beta genes are thymine and adenine located at 124 and 132 bases upstream from initiation codon in exon 1, respectively. The regulation and enhancer of expression of two genes have been examined by CAT assay., gel retardation assay and foot print method and are still underway. All exon segments of genomic DNAs of PHDalpha and beta isolated from the patient's liver with fatal PDH deficiency and from control leucocytes were amplified by PCR. Sequences of both PCR-amplified products were compared with normal PDHalpha and beta genes. Two single base-pair substitutions in two exons of the patient's PDHalpha gene, one G to T, in exon 5 and the other, T to c, in exon 6, were detected. These mutations generate a Phe-116 in PDHalpha in place of the normal Cys-116 and a Pro-162 in place of the Leu-162. The absence of both immunoreactive PDHalpha and beta proteins in the liver extract from the patient sugests that the point mutations in the PDHalpha gene lead to a decrease two subunit proteins.
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