Analysis of DNA (cytosine 5) methyltransferase mRNA sequence and expression in bovine preimplantation embryos, fetal and adult tissues.
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abstract
Mammalian preimplantation development is a critical stage for establishment of the genomic methylation pattern and proper function of the enzymes responsible for this appear essential for normal development. To date, the vast majority of work concerning the developmental expression of the DNA cytosine 5-methyltansferases (Dnmts) has been conducted in mice. Here we report the sequence and expression of the Dnmt family during bovine preimplantation and fetal development. Bovine Dnmt mRNAs display strong sequence homology to those of human and mouse and similar to other species, exist as multiple isoforms. Two of these splice variants, which have been termed Dnmt2gamma and Dnmt3a4, represent previously unreported sequence combinations. Work presented here demonstrates early bovine embryos express mRNA coding for the somatic form of Dnmt1 and that this transcript fractionates with the ribosome. Unlike the murine model, mRNA encoding the de novo methyltransferases, Dnmt3a and 3b are present during preimplantation development and can also be found in the ribosomal subcellular fraction. Further, results of Real Time PCR analysis indicate significant differences in Dnmt mRNA expression levels exist among different tissue types as well as between fetal and adult stages. Recently, it has been postulated that the cause of abnormal methylation observed in cloned embryos may be due in part to misexpression of the Dnmt1o isoform during preimplantation development. Work presented here raises new and significant hypotheses that must be considered both regarding the cadre of DNA methyltranferases that direct epigenetic programming during normal development and regarding the implication of abnormal DNMT expression in cloned embryos.
