| Project/Area Number |
63540582
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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 |
動物発生・生理学
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| Research Institution | Tokyo Metropolitan University |
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Principal Investigator |
TOMINO Shiro Tokyo Metropol. University, Dept. of Biology, Professor, 理学部, 教授 (30101075)
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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 |
¥1,900,000 (Direct Cost: ¥1,900,000)
Fiscal Year 1989: ¥400,000 (Direct Cost: ¥400,000)
Fiscal Year 1988: ¥1,500,000 (Direct Cost: ¥1,500,000)
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| Keywords | Insect metamorphosis / Cuticle protein / Gene cloning / mRNA / Nucleotide sequence / Amino acid sequence / Hormonal regulation / Chitin-binding protein / クチクラタンパク質 / cDNAクロ-ニング / タンパク質の一次構造 / キチン結合性 / 遺伝子クローニング |
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| Research Abstract |
Biosynthesis of cuticle proteins was studied during the larval-pupal metamorphosis of the silkworm, Bombyx mori.
1. The urea-soluble proteins extracted from the larval and pupal integuments of B. mori exhibited distinct difference in electrophoretic properties. Major protein component was isolated from each extract and designated as Larval Cutiole Protein (LCP) and Pupal Cuticle Protein. (PCP), respectively, and an antibody was raised against each protein.
2. Immunoblot analysis by use of each antibody demonstrated that LCP first appears 10 hours after hatching and is present in integument throughout the larval stages, while PCP occurs only in the pupal integument.
3. The cDNA libraries in expression vector were prepared from the epidermal mRNA of the fifth instar larvae and of the pupae immediately after larval-pupal ecdysis. The LCP- and PCP-cDNA clones were isolated by screening the libraries with antibody probes.
4. Northern blot analysis of the epidermal RNA provided evidence that the
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biosynthesis of cuticle proteins is regulated in a stage dependent manner at the mRNA level in the epidermal cells. Experiment employing the isolated abdomens of the last instar larvae indicated that the biosynthesis of PCP-mRNA is induced by administration of ecdysteroid.
5. The genomic clone for PCP was isolated from a B. mori genomic library and its nucleotide sequence was determined. The PCP gene comprises a short first exon and 1 kb second exon, which contains majority of protein-coding frame. Two exons are interspersed by a single 4.8 kb intron. The 5' flanking region of the gene contained putative regulatory elements for gene transcription that include a TATA-box and CCAAT-box like structures.
6. The deduced primary structure of PCP revealed the occurrence of unique reiterated structures, termed as Motif-A and Motif-B, respectively. Motif-A, which comprises 13-15 residues rich in hydrophobic amino acids, occurs in three separate locations in the molecule. The eight-residue Motif-B contains a high proportion of alanine and proline. Four consecutive Motives-B are present in the carboxyl-proximal region of PCP. Chromatographic study of the beta-galacto- sidase/PCP fusion protein indicated that these repeating structures are essential for binding PCP to chitin matrix. Less
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