Overexpression of Medaka (Oryzias latipes) photolyase gene in Medaka cultured cells and early embryos.
To study the role and the regulation of the photolyase gene in the Medaka (small teleost), we constructed a eukaryotic expression plasmid of the Medaka photolyase gene and introduced it into Medaka cells in vivo and in vivo. The expression plasmid contains a cytomegalovirus enhancer and a thymidine kinase promoter to overexpress the photolyase gene of the Medaka. First, we transfected this construct into cultured Medaka cells and established several lines of transfectant. Every transfectant showed enhanced ability of pyrimidine dimer repair in the presence of fluorescent light. In the transfectant that showed the most enhanced ability of photorepair, the augmented transcription of photolyase gene was observed compared with that of progenitor OL32 cells. In this transfectant, we also observed an enhanced rate of UV survival with 20 min of fluorescent light treatment after irradiation with a 400 J/m2 UV sunlamp. Next, the expression construct was microinjected into the embryos of the Medaka at the one cell stage. Compared with the nontreated counterparts, the overexpression of a photolyase gene was detected in the microinjected embryos, but we failed to detect a significant increase in photo-reactivability of death at the midblastula stage.
Funayama
T
T
Department of Biological Sciences, University of Tokyo, Japan. funayama@uts2.s.utokyo.ac.jp.
Mitani
H
H
Shima
A
A
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002470:Cell Survival / Q000528:radiation effects; D017931:DNA Primers; D004255:Deoxyribodipyrimidine Photo-Lyase / Q000096:biosynthesis*; D004625:Embryo, Nonmammalian / Q000201:enzymology*; D015971:Gene Expression Regulation, Enzymologic / Q000528:radiation effects*; D008969:Molecular Sequence Data; D009990:Oryzias; D016133:Polymerase Chain Reaction; D012867:Skin / Q000166:cytology / Q000201:enzymology*; D014466:Ultraviolet Rays
D017931:DNA Primers; D004255:Deoxyribodipyrimidine Photo-Lyase