| Project/Area Number |
03453133
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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 |
応用生物化学・栄養化学
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| Research Institution | TOHOKU UNIVERSITY |
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Principal Investigator |
MIZUNO Shigeki Tohoku Univ., Dept. Applied Biol. Chem. Professor, 農学部, 教授 (90112903)
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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 |
¥6,900,000 (Direct Cost: ¥6,900,000)
Fiscal Year 1992: ¥2,400,000 (Direct Cost: ¥2,400,000)
Fiscal Year 1991: ¥4,500,000 (Direct Cost: ¥4,500,000)
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| Keywords | Silkworm / Chromatin / Fibroin gene / Fibroin H-chain / Fibroin L-chain / Disulfide bond / Secretion mutant / Elongation factor 1gamma / 後部絹糸腺 / 延長因子EF1γ / 転写因子 / in vitro転写系 / 遺伝子組換え / cDNA発現ライブラリ- |
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| Research Abstract |
1. Transcriptional regulation of fibroin H- and L-chain genes: Chromatin structures of fibroin H-chain gene in different transcriptional states were compared. The most notable observation was that distributions of DNase I-hypersensitive sites in the 5' upstream region were similar between transcriptionally active (5th instar posterior silk gland) and temporarily inactive (4th molting posterior silk gland) states but those patterns were remarkably different from that of the transcriptionally inactive (5th instar middle silk gland) state. A cDNA clone was isolated from the posterior silk gland cDNA expression library, which produced a DNA-binding protein showing affinity to the L2 sequence, i.e. one of common sequences in the 5' upstream regions of H- and L-chain genes. The nucleotide sequence of this clone exhibited high-level similarity to that of the elongation factor(EF)1gamma. 2. Binding site between fibroin H- and L-chains: Position of the disulfide bond between H- and L-chains was investigated using anti-peptide antibodies raised against synthetic peptides containing putative cysteine residues responsible for the binding in both H- and L-chains. One chymotryptic peptide fraction reacting with both antibodies was identified among fractions eleuted from a reversed phase FPLC column. 3. Analysis of fibroin secretion mutants of the silkworm: Both Nd-s and Nd mutants are unable to secrete fibroin. In Nd-s, a recombination had taken place in the 3rd intron of the L-chain gene and produced a chimeric L-chain molecule. In Nd, degradation products of H-chain which lacked its C terminal region were accumulated in the cytoplasm. 4. Relation between fibroin L-chain and P25: These two proteins produced in the posterior silk gland were shown to be immunologically unrelated.
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