| Project/Area Number |
60570109
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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 |
General medical chemistry
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| Research Institution | Toyama Medical and Pharmaceutical University |
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Principal Investigator |
OGAWA Hirofumi Associate Professor of Biochemistry at Toyama Med.& Pharmaceu. Univ., 医学部, 助教授 (30111743)
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| Co-Investigator(Kenkyū-buntansha) |
FUJIOKA Motoji Professor of Biochemistry at Toyama Med. & Pharmaceu. Univ., 医・生化学, 教授 (30030000)
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| Project Period (FY) |
1985 – 1986
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| Project Status |
Completed (Fiscal Year 1986)
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| Budget Amount *help |
¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 1986: ¥400,000 (Direct Cost: ¥400,000)
Fiscal Year 1985: ¥1,100,000 (Direct Cost: ¥1,100,000)
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| Keywords | cDNA cloning / rat glycine methyltransferase / amino acid sequence / enhancer core element / genomic structure / acetylvaline |
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| Research Abstract |
1. The N-terminal of rat liver glycine methyltransferase (EC2.1.1.20) is blocked. The peptide containing the N-terminal residue was obtained from various protease-digests through HPLC, and its sequence was determined as Acetylval-Asp-Ser-Val-Tyr-Arg by means of Edman degradation, HPLC and mass spectrometric analysis.
2. A lot of cDNA clones for this enzyme were screened from a lambda gtll library containing liver cDNA inserts by plaque hybridization, using the previously obtained cDNA clone as a probe. Sequence analysis of the largest insert DNA showed that the cDNA consisted of 4 bp of 5' noncoding region, an open reading frame of 882 bp, and 102 bp of 3' noncoding region. The cDNA-deduced amino acid sequence contained both the amino and carboxyl terminal sequences.
3. The genomic DNA clone for glycine methyltransferase was obtained from a lambda Charon 4A library by in situ plaque hybridization. Five clones were found to have an insert of 6500 bp long. Sequence determination indicated that the gene consisted of 6 exons split by 5 introns as compared to the cDNA sequence. The sequences of exon/intron splice junctions clearly obeyed the "GT...AG" rule. Sl nuclease mapping and primer extension analysis allowed us to propose that the A residue located 19 bp upstream from the translation initiation codon is the site of transcription initiation. "TATA", "CAT" and "GC" boxes,and the complementary sequence to the enhancer core element were located at the expected positions upstream of the transcription initiation site.
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