Molecular Cloning of zMSH2 from Zebrafish ( Danio rerio) and Effects of Heat Shock on MSH Gene Expression

• Main Author: 王詩雅
• Other Authors: 許濤
• Format: Others
• Language: zh-TW
• Published: 2002
• Online Access: http://ndltd.ncl.edu.tw/handle/23862586041157138599

碩士 === 國立海洋大學 === 水產生物技術研究所 === 90 === Mismatched nucleotides in eukaryotic DNA are recognized by heterodimeric complexes of proteins homologus to the bacterial MutS protein. We attempted to study gene expression of MutS homologues in developing vertebrates using zebrafish ( Danio rerio ) as a model system. A novel cDNA encoding a zebrafish MutS homologue, zfMSH2, was obtained by reverse transcription followed by rapid amplification of cDNA ends ( RACE ). The zfMSH2 cDNA has an open reading frame of 2811 nucleotides that can be translated into a polypeptide of 936 amino acids with a molecular mass about 105 kDa. The amino acid sequence of zfMSH2 shares 72 %、69 % and 69 % identity to African clawed frog 、human and mouse MSH2, respectively. The zfMSH2 protein contains four ATP-binding motifs and a mismatch recognition region conserved among MutS homologues. Recombinant zfMSH2 obtained from in vitro transcription/translation showed a preferential binding to a heteroduplex containing a G-T mismatch as shown by affinity adsorption. Northern blot analysis detected apparent levels of zfMSH2 and zfMSH6 mRNA expressions in 12 and 36-hr-old zebrafish embryos, while these expressions are significantly reduced in 60-hr-old late embryos and 84-hr-old larvae, indicating a stage-dependent regulation of MSH2 and MSH6 expression in developing zebrafish. Incubation of G-T and G-G heteroduplex probes with the extracts of 12 to 60-hr-old embryos generated predominantly high-shifting binding complexes with similar band intensities. Although low in zfMSH2 and zfMSH6 productions, mismatch recognition proteins contained in 84-hr-old zebrafish bound apparently stronger to a G-T than to a G-G heteroduplex probe, producing both high and low-shifting retardation complexes. The strong G-T complexes generated by 84-hr-old zebrafish extracts might result from the binding of G-T recognition proteins specifically produced in more mature zebrafish to mismatched DNA. The expressions of zfMSH2 and zfMSH6 mRNA in 36 and 84-hr-old zebrafish were sensitive to heat stress at 37 ℃ for 30 min, as a reduction in the levels of these MSH mRNA species were observed immediately after heat treatment. The expressions of zfMSH2 and zfMSH6 mRNA returned to normal levels about 2hrs after incubation at 28.5 ℃. Heat stress did not affect the expression of β-actin mRNA in developing zebrafish at all. Whether heat shock inhibited the transcription of zfMSH2 and zfMSH6 genes or caused an accelerated mRNA degradation remain to be determined.