1.Translational regulation of heat shock cognate 70 synthesis in developing zebrafish (Danio rerio) 2.Dectection of UV-dependent DNA incision activity in extracts of zebrafish (Danio rerio) early embryos

• Main Authors: You-Hsin Chang, 張友信
• Other Authors: Todd Hsu
• Format: Others
• Language: zh-TW
• Published: 2008
• Online Access: http://ndltd.ncl.edu.tw/handle/11673741908634603723

碩士 === 國立臺灣海洋大學 === 生物科技研究所 === 96 === PART ONE In our previous studies, we detected a high expression of heat shock cognte 70 (HSC 70) in developing zebrafish and the spontaneous HSC 70 expression was found to be controlled by translational regulation. The purpose of this study was to explore the mechanisms of the translational regulation that produce the spontaneous HSC 70 synthesis. RT-PCR-PAT assay indicated that the poly(A) tail of hsc70 mRNA isolated from 12 to 120 hpf zebrafish carried the same length in developing zebrafish. So translational regulation of HSC 70 synthesis was not conctrolled by changing poly(A) tail length. RNA mobility shift assay showd the sequence of both sense and anti-sense from Hsc70 mRNA 5’UTR and 3’-UTR was shift by specific RNA binding protein. The embryo extracts generated very strong non-specific binding to both sense and anti-sense hsc 70 mRNA, so the mechanism of translational regulation by the binding of specific RNA binding proteins to hsc70 5’ and 3’ -UTR could not be established. PART TWO A strong UV-damaged-DNA binding activity had been detected in the extracts of zebrafish embryos at 12 hrs post-fertilization by gel shift assay. We attempted to study the components of this binding activity and their importance in DNA damage recognition. In incision assay using a biotin-labeled oligonucleotide, we found 12 hpf zebrafish extracts expressed strong UV-independent exonuceloase activities that could be enhanced by ATP, but inhibited by the addition of poly(dI．dC) and poly(lys). When exonuclease activities were blocked, no UV-specific incision fragments were produced along with increasing protein concentration. In incision assay using a supercoiled plasmid in EMSA buffer, a UV-dependent increase in the level of open form plasmid was detected, indicating the association between UV-specific binding and DNA incision. Addition of ATP to 12 hpf zebrafish extracts caused a very intense damage-independent incision. Inclusion of an anti-zebrafish Vg1 antibody in incision mixtures did not inhibit the production of open form plasmid. Taken together, a UV-induced DNA incision activity independent of ATP is expressed in zebrafish early embryos.