PUBLICATION
Sequence Analysis and Functional Study of Thymidylate Synthase from Zebrafish, Danio rerio
- Authors
- Du, C., Niu, R., Chu, E., Zhang, P., and Lin, X.
- ID
- ZDB-PUB-060616-27
- Date
- 2006
- Source
- J. Biochem. (Tokyo) 139(5): 913-920 (Journal)
- Registered Authors
- Keywords
- none
- MeSH Terms
-
- Amino Acid Sequence/genetics
- Animals
- Base Sequence/genetics
- Cloning, Molecular
- DNA, Complementary/isolation & purification*
- DNA, Complementary/metabolism
- Escherichia coli
- Gene Silencing
- Humans
- Mice
- Phylogeny
- Rats
- Recombinant Proteins/isolation & purification
- Recombinant Proteins/metabolism
- Sequence Alignment
- Sequence Analysis
- Thymidylate Synthase/chemistry*
- Thymidylate Synthase/genetics*
- Thymidylate Synthase/metabolism
- Zebrafish/genetics*
- Zebrafish/metabolism
- Zebrafish Proteins/genetics
- Zebrafish Proteins/metabolism
- PubMed
- 16751599 Full text @ J. Biochem. (Tokyo)
Citation
Du, C., Niu, R., Chu, E., Zhang, P., and Lin, X. (2006) Sequence Analysis and Functional Study of Thymidylate Synthase from Zebrafish, Danio rerio. J. Biochem. (Tokyo). 139(5):913-920.
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 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.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping