title: Molecular cloning, expression, and characterization of starfish DNA (cytosine-5)-methyltransferases.
abstract: To determine whether and if so how a DNA methylation-dependent epigenetic mechanism for transcriptional gene silencing functions in Echinoderms, we cloned and sequenced dnmt1 and dnmt3 cDNAs of the starfish Asterina pectinifera. Since the Strongylocentrotus purpuratus genome has only two loci of DNA (cytosine-5)-methyltransferase genes encoding Dnmt1 and Dnmt3, they might constitute a sufficient set of dnmt genes in Echinoderms. The starfish Dnmt3 whose cDNA we cloned showed highest homology to a mammalian Dnmt3a2 splicing variant. Essentially all the characteristic motifs and sequences of the mammalian counterparts were found in the starfish Dnmts as well, except that a typical PCNA binding domain motif was lacking in the starfish Dnmt1. RT-PCR analysis indicated that the dnmt1 mRNA exists in both ovary and oocytes, but its levels in other tissues were very low or almost negligible. In contrast, the dnmt3 mRNA was detected only in the ovary, and not at all in the oocytes. The size of a dnmt1 transcript was about 6.5 kb on Northern blot analysis. On heterologous expression, the starfish Dnmt1 protein was expressed in insect cells in catalytically active form.
authors.affiliation: Department of Bioscience and Biotechnology, Graduate School of Natural Science and Technology, Okayama University, Tsushima-naka, Okayama 700-8530, Japan.
authors.forename: Yoshihito
authors.initials: Y
authors.lastname: Fujihara
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Miyasako
authors.forename: Kumiko
authors.initials: K
authors.lastname: Kato
authors.forename: Tadahiro
authors.initials: T
authors.lastname: Hayashi
authors.forename: Tetsuo
authors.initials: T
authors.lastname: Toraya
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D047288:Asterina / Q000201:enzymology / Q000235:genetics*; D003001:Cloning, Molecular; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D015723:Gene Library; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009865:Oocytes / Q000201:enzymology*; D009928:Organ Specificity; D010053:Ovary / Q000201:enzymology*; D010802:Phylogeny; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D061987:Sf9 Cells / Q000378:metabolism; D047270:Strongylocentrotus purpuratus / Q000201:enzymology / Q000235:genetics
chemical_list: D007527:Isoenzymes; D011993:Recombinant Fusion Proteins; D004248:DNA (Cytosine-5-)-Methyltransferases