1991 Fiscal Year Final Research Report Summary
Cloning and expression of genes responsible for secondary metabolism in medicinal plants.
| Project/Area Number |
02807199
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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 |
Biological pharmacy
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| Research Institution | Chiba University |
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Principal Investigator |
SAITO Kazuki CHiba Univ., Fac. Pharm. Sci. Lecturer, 薬学部, 講師 (00146705)
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| Co-Investigator(Kenkyū-buntansha) |
MURAKOSHI Isamu Chiba Univ., Fac. Phorm. Sci. Professor, 薬学部, 教授 (30009162)
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| Project Period (FY) |
1990 – 1991
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| Keywords | Secondary metabolism / Molecular cloning / Transgenic Plant / Cysteine synthase |
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| Research Abstract |
Cysteine synthase(CSase)(O-acetylserine(thiol)-lyase, EC 4.2.99.8)catalyzes the formation of L-cysteine, the key step in sulfur assimilation in plants, from O-acetyl-L-serine and hydrogen sulfide. We report here isolation and characterization of cDNA clones encoding cysteine synthase from spinach(Spinacia oleracea L.). Internal peptide sequences were obtained from V8 protease-digested fragments of purified CSase. A lambdagt10 cDNA library was constructed from poly(A)^+ RNA of young green leaves of spinach. Screening with two synthetic mixed nucleotides encoding the partial peptide sequences gave 19 positively hybridized clones from 2x10^5 clones. Nucleotide sequence analysis of two independent cDNA clones revealed a continuous open reading frame encoding a polypeptide of 325 amino acids with a calculated molecular mass of 34, 185 Da. Sequence comparison of deduced amino acids showed 53% identity with CSases of Escherichia coli and Salmonella typhimurium. Sequence homology was also observed with other metabolic enzymes of amino acids in bacteria and yeast, and rat hemoprotein H-450. A bacterial expression vector was constructed and this plasmid was able to genetically complement an E. coli auxotroph deficient CSases. The accumulation of functionally active spinach CSase in E. coli was also demonstrated by Western blotting and the assay of enzymatic activity.
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Research Products
(8 results)
