Sequence analysis and functional study of thymidylate synthase from zebrafish, Danio rerio

doi:10.1093/jb/mvj100

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	Sequence analysis and functional study of thymidylate synthase from zebrafish, Danio rerio
	Du, CQ ; Niu, RL ; Chu, E ; Zhang, PJ ; Lin, XK
	2006-05-01
发表期刊	JOURNAL OF BIOCHEMISTRY
ISSN	0021-924X
卷号	139 期号:5 页码:913-920
文章类型	Article
摘要	The thymidylate synthase (TS), an important target for many anticancer drugs, has been cloned from different species. But the cDNA property and function of TS in zebrafish are not well documented. In order to use zebrafish as an animal model for screening novel anticancer agents, we isolated TS cDNA from zebrafish and compared its sequence with those from other species. The open reading frame (ORF) of zebrafish TS cDNA sequence was 954 nucleotides, encoding a 318-amino acid protein with a calculated molecular mass of 36.15 kDa. The deduced amino acid sequence of zebrafish TS was similar to those from other organisms, including rat, mouse and humans. The zebrafish TS protein was expressed in Escherichia coli and purified to homogeneity. The purified zebrafish TS showed maximal activity at 28 degrees C with similar K-m value to human TS. Western immunoblot assay confirmed that TS was expressed in all the developmental stages of zebrafish with a high level of expression at the 1-4 cell stages. To study the function of TS in zebrafish embryo development, a short hairpin RNA (shRNA) expression vector, pSilencer 4.1-CMV/TS, was constructed which targeted the protein-coding region of zebrafish TS mRNA. Significant change in the development of tail and epiboly was found in zebrafish embryos microinjected pSilencer4.1-CMV/TS siRNA expression vector.; The thymidylate synthase (TS), an important target for many anticancer drugs, has been cloned from different species. But the cDNA property and function of TS in zebrafish are not well documented. In order to use zebrafish as an animal model for screening novel anticancer agents, we isolated TS cDNA from zebrafish and compared its sequence with those from other species. The open reading frame (ORF) of zebrafish TS cDNA sequence was 954 nucleotides, encoding a 318-amino acid protein with a calculated molecular mass of 36.15 kDa. The deduced amino acid sequence of zebrafish TS was similar to those from other organisms, including rat, mouse and humans. The zebrafish TS protein was expressed in Escherichia coli and purified to homogeneity. The purified zebrafish TS showed maximal activity at 28 degrees C with similar K-m value to human TS. Western immunoblot assay confirmed that TS was expressed in all the developmental stages of zebrafish with a high level of expression at the 1-4 cell stages. To study the function of TS in zebrafish embryo development, a short hairpin RNA (shRNA) expression vector, pSilencer 4.1-CMV/TS, was constructed which targeted the protein-coding region of zebrafish TS mRNA. Significant change in the development of tail and epiboly was found in zebrafish embryos microinjected pSilencer4.1-CMV/TS siRNA expression vector.
关键词	Cloning And Expression Development Of Zebrafish Embryos Enzymatic Activity Thymidylate Synthase Zebrafish
学科领域	Biochemistry & Molecular Biology
DOI	10.1093/jb/mvj100
URL	查看原文
收录类别	SCI
语种	英语
WOS记录号	WOS:000238357000012
引用统计	被引频次：18[WOS] [WOS记录] [WOS相关记录]
文献类型	期刊论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/5822
专题	实验海洋生物学重点实验室
作者单位	1.Chinese Acad Sci, Inst Oceanol, Qingdao 266071, Peoples R China 2.Chinese Acad Sci, Grad Sch, Beijing 100039, Peoples R China 3.Yale Univ, Sch Med, Ctr Canc, New Haven, CT 06520 USA 4.Yale Univ, Sch Med, VACT Healthcare Syst, New Haven, CT 06520 USA
推荐引用方式 GB/T 7714	Du, CQ,Niu, RL,Chu, E,et al. Sequence analysis and functional study of thymidylate synthase from zebrafish, Danio rerio[J]. JOURNAL OF BIOCHEMISTRY,2006,139(5):913-920.
APA	Du, CQ,Niu, RL,Chu, E,Zhang, PJ,&Lin, XK.(2006).Sequence analysis and functional study of thymidylate synthase from zebrafish, Danio rerio.JOURNAL OF BIOCHEMISTRY,139(5),913-920.
MLA	Du, CQ,et al."Sequence analysis and functional study of thymidylate synthase from zebrafish, Danio rerio".JOURNAL OF BIOCHEMISTRY 139.5(2006):913-920.

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