MOLECULAR CLONING AND EXPRESSION ANALYSIS OF Dnmt1 IN GOLDFISH, CARASSIUS AURATUS

Fish is the lowest vertebrate. Its external fertilization and development, large reproductive potential, and optically clear embryos give fish a number of distinct advantages over other vertebrates as an exploitable experimental system for the study of developmental processes as well as the mechanism and function of epigenetic modification in vertebrates. Studying the mechanism of epigenetic modification by DNA methylation and the function of epigenetic modification in regulating gene expression in fish will provide insights into the biological function of DNA methylation, the evolution of epigenetic modification mechanism in vertebrates and the role of epigenetic modification in the evolution of vertebrates. In vertebrates, DNA methyltransferase (Dnmt1) Ⅰ plays a pivotal role in maintaining the correct genome DNA methylation pattern. In low vertebrate including fish, however, Dnmt1 has still been poorly investigated. Here, the full length cDNA of goldfish Dnmt1, Carassius auratus, was cloned by reverse transcription polymerase chain reaction(RT-PCR)method. The temporal and spatial expression patterns of goldfish Dnmt1 were examined by real-time PCR and whole mount in situ hybridization. The entire Dnmt1 cDNA sequence was 4912bp long and the open read frame was 4512bp long, which encoded a protein with 1503 amino acids. Amino acid sequence alignment of Dnmt1s (Carcasses auratus, Danio rerio, Xenopus laevis, Gallus gallus and Homo sapiens) revealed that the structure of Dnmt1 protein also consisted of an N-terminal regulatory domain and a C-terminal catalytic domain. The conservation of N-terminal domain among Carcasses auratus, Danio rerio, Xenopus laevis, Gallus gallus and Homo sapiens was very low, but C-terminal domain was highly conserved. Phylogenetic tree analysis showed that the homology of Dnmt1 protein between Carassius auratus and Danio rerio was the highest, Xenopus laevis was the second and Gallus gallus and Homo sapiens took the last. The high conservation of C-terminal domain implied that the catalytic function might be crucial for the survival of the species. The variation of N-terminal domain may reflect adaptive synergistic evolutions between the Dnmt1 structure and chromatin in different species.Whole mount in situ hybridization and real-time PCR results reveled that Dnmt1 mRNA was of abundantly maternal origin in Carassius auratus. During cleavage and blastula stages, the level of Dnmt1 mRNA was gradually reduced. As a result, it hit the lowest during gastrula stage. But in 2-day old embryos, the transcription level of Dnmt1 was gradually increased. No regional difference was detected in the pre-neurula stage embryos by in situ hybridization, but it was observed in the embryos after period of organogenesis. Dnmt1 mRNA expression was significantly higher in eyes, brain and matured somites than in other tissues. RT-PCR and real-time PCR results demonstrated that Dnmt1 expressed in all the examined adult tissues such as liver, heart, spleen, kidney, brain, muscle and retina. However, the transcription level of Dnmt1 mRNA was higher in those tissues with vigorous cell proliferation. The difference is apparently in association with the copying and maintenance of cell lineage specific methylation patterns in the genome of the newly forming cells.