Expression of murine DNA methyltransferases Dnmt1 and Dnmt3a in the yeast Saccharomyces cerevisiae.
Murine DNA methyltransferases Dnmt1 and Dnmt3a were expressed in the yeast Saccharomyces cerevisiae. Adjustment to yeast preferences of the nucleotide sequences upstream and downstream of the translation initiation sites of both cDNAs was needed to obtain significant levels of the methyltransferases. Both proteins were correctly localized to the nucleus and their presence had no measurable influence on the functioning of yeast cells. Both Dnmt1 and Dnmt3a expressed in yeast cells were enzymatically active in vitro, and in vivo in the genomic DNA of the transgenic S. cerevisiae ca. 0.06% and 0.4%, respectively, of cytosines became methylated. This level of DNA methylation is about 100- to 10-fold less than that observed in mammalian cells. The constructed system may be used to investigate the in vivo specificity of individual mammalian DNA methyltransferases and to search for additional factors needed to allow more efficient in vivo methylation of chromatin-contained DNA and to study their mechanism of action.
Bulkowska
Urszula
U
Institute of Biochemistry, Faculty of Biology, University of Warsaw, Poland.
Ishikawa
Takao
T
Kurlandzka
Anna
A
Trzcińska-Danielewicz
Joanna
J
Derlacz
Rafał
R
Fronk
Jan
J
D000818:Animals; D001483:Base Sequence; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000235:genetics* / Q000378:metabolism; D019175:DNA Methylation; D000090205:DNA Methyltransferase 3A; D051379:Mice; D008969:Molecular Sequence Data; D012441:Saccharomyces cerevisiae / Q000235:genetics*
C000715535:Dnmt3a protein, mouse; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; D000090205:DNA Methyltransferase 3A; C000619381:Dnmt1 protein, mouse