We found a match
Your institution may have rights to this item. Sign in to continue.
- Title
Sequence Analysis and Functional Study of Thymidylate Synthase from Zebrafish, Danio rerio.
- Authors
Changqing Du; Rongli Niu; Chu, Edward; Peijun Zhang; Xiukun Lin
- Abstract
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°C with similar Km 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.
- Subjects
THYMIDYLATE synthase; ANTINEOPLASTIC agents; ZEBRA danio; NUCLEOTIDES; IMMUNOBLOTTING
- Publication
Journal of Biochemistry, 2006, Vol 139, Issue 5, p913
- ISSN
0021-924X
- Publication type
Article
- DOI
10.1093/jb/mvj100