| Project/Area Number |
02556013
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| Research Category |
Grant-in-Aid for Developmental 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 | Osaka University |
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Principal Investigator |
TANIZAWA Katsuyuki Osaka Univ., ISIR Associate Professor, 産業科学研究所, 助教授 (20133134)
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| Co-Investigator(Kenkyū-buntansha) |
SHIBANO Yuji Suntory Ltd., Inst. for Fund. Res. Research Manager, 基礎研究所, 副参事研究員
TAGAYA Mitsuo Osaka Univ., ISIR Research Associate, 産業科学研究所, 助手 (30179569)
FUKUI Toshio Osaka Univ., ISIR Professor, 産業科学研究所, 教授 (90029843)
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| Project Period (FY) |
1990 – 1992
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| Project Status |
Completed (Fiscal Year 1992)
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| Budget Amount *help |
¥5,300,000 (Direct Cost: ¥5,300,000)
Fiscal Year 1992: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1991: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 1990: ¥2,800,000 (Direct Cost: ¥2,800,000)
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| Keywords | polymerase chain reaction (PCR) / genetic engineering / site-directed mutagenesis / cDNA / random primer / restriction enzyme sites / ホスホリラーゼ / 基質特異性 / 制限酵素 / DNAポリメラ-ゼ反復反応(PCR) / アデニル酸キナ-ゼ / UDP-グルコ-スピロホスホリラ-ゼ / ランダムプライマ- / アスパラギン酸トランスアミナ-ゼ |
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| Research Abstract |
Polymerase chain reaction (PCR) with the extremely thermostable DNA polymerase is useful for specific amplification in not less than 10^5-fold of a very small amount of any genes. The present research utilizing PCR aimed at establishing novel methods of molecular cloning without constructing genomic or cDNA libraries, genetic manipulation by introducing unique restriction sites, and site-directed mutagenesis changing randomly or rapidly into all kinds of amino acid residues. The following results were obtained in this research performed from 1990 to 1992.
1) By using PCR, a new method of site-directed mutagenesis has been developed for replacing the active-site lysyl residue of microbial aspartate transaminase by 20 amino acid residues in a single round of cloning procedure.
2) A system for the efficient expression in Escherichia coli of higher plant UDP-glucose pyrophosphorylase cDNA has been constructed by introducing new restriction enzyme sites with the use of PCR.
3) PCR-based random mutation has been applied to the substrate-binding site of chicken muscle adenylate kinase, and a new method of rapid screening of mutant genes has been devised.
4) Several DNA fragments from cDNAs coding for two higher plant alpha-glucan phosphorylase isozymes have been amplified by PCR with synthetic oligonucleotides having various restriction enzyme sites as primers, and chimeric enzymes composed of the two phosphorylase isozymes and the rabbit muscle enzyme have been constructed. One of the chimeric enzyme has been found to show extraordinarily high affinities for various glucan substrates.
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