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MOLECULAR CLONING OF cDNA ENCODING HUMAN LANOSTEROL SYNTHASE
Sankawa, Ushio,Sung, Chung Ki,Shibuya, Masaaki,Ebizuka, Yutaka 전남대학교 약품개발연구소 1996 약품개발연구지 Vol.4 No.1
A cDNA encoding human lanosterol synthase, the enzyme responsible for the backbone formation step in sterol biosynthesis, was cloned by extensive application of PCRs. Five degenerate oligonucleotide primers(139S, 440S, 528A, 575A and 712A) corresponding to the homologous amino acid sequences among the known 2,3-oxidosqualene cyclase(OSC) were designed. PCR with one pair(440S and 528A) of five primers yielded a 285-by fragment. PCRs with the primers based on the obtained fragment and the degenerate primers (139S and 712A) gave longer fragments. Finally, full nucleotide sequence of cDNA was obtained by a $quot;rapid amplification of cDNA ends$quot; (RACE) method.
A convinient method to evaluate promoter activity with transformed hairy root culture
Jong Soo Lee,Osamu Nakajima,Masaaki Shibuya,Yutaka Ebizuka,Ushio Sankawa 전남대학교 약품개발연구소 1995 약품개발연구지 Vol.3 No.1
Transformed hairy roots of tobacco were used to measure activity of some promoters, horseradish peroxidase promoter (POP), 500 bp and 300 bp region of 5' upstream from Pueraria lobata chalcone synthase gene (CHSpro500, CHSpro300) and cauliflower mosaic virus 35S (CaMV 35S) promoter, which were fused to β-glucuronidase (GUS) gene, respectively. Hairy roots induced by Agrobacterium rhizogenes exhibited GUS activity in strength order of POP, CaMV 35S, CHSpro500 and CHSpro300, and it was suggested that for introduction and expression of foreign gene into the plant genome, POP may be applied as the potent promoter of plant origin in place of CaMV 35S promoter. Moreover, elicitor-response of Pueraria lobata CHS promoter was investigated with this assay system and it was verified that CHS promoter responds well to exogenous elicitor, yeast extract and CaCl₂. Although protoplasts or transgenic plants have been used widely as the materials for that purpose for the last decade, our results suggested that our alternative method was the simple and reasonable one to evaluate promoter activity.