Isolation and expression of a chicken DNA methyltransferase cDNA

Shoji Tajima, Hideki Tsuda, Nobunao Wakabayashi, Akira Asano, Shigeki Mizuno, Katsuhiko Nishimori

Research output: Contribution to journalArticle

35 Citations (Scopus)

Abstract

A 0.5 kb fragment of chicken DNA methyltransferase cDNA was PCR-amplified using a set of degenerate primers. A clone harboring a 5 kb insert was isolated from a cDNA library by screening with the PCR-amplified cDNA fragment as a probe. The elucidated nucleotide sequence gave a 4,614 nucleotide open reading frame, and the predicted protein was highly homologous to the mouse and human DNA methyltransferases, especially in the amino acid sequence of the catalytic domain in the carboxyl-terminal region. The cysteine-rich region and Lys-Gly repeat first found in the mouse sequence were also conserved in chicken. However, about 250 amino acid residues in the amino-terminal portion of chicken DNA methyltransferase diverged from the amino-terminus of the mouse or human sequence. Northern blot analysis showed that the message of chicken DNA methyltransferase was expressed at high levels in the testis, in the lung and in Marek's virus-transformed chicken T-lymphoma cells. Expression of the chicken DNA methyltransferase in COS1 cells demonstrated that the enzyme is a so-called maintenance-type methylase. When poly(dG-dC)-poly(dG-dC) was used as the methyl acceptor, to provide a measure of de novo methylase activity, the Km value for S-adenosyl L-methionine was about 5 μM, which was 10 times higher than that when poly(dI-dC)-poly(dI-dC) was used. The affinity of DNA methyltransferase for S-adenosyl L-methionine in catalyzing de novo-type methylation activity was lower than that in catalyzing maintenance-type activity, though it was still high enough for the enzyme to work as a de novo-type methylase under physiological conditions.

Original languageEnglish
Pages (from-to)1050-1057
Number of pages8
JournalJournal of biochemistry
Volume117
Issue number5
DOIs
Publication statusPublished - 1995 May

Keywords

  • CDNA cloning
  • Chiken
  • DNA methylation
  • DNA methyltransferase
  • Nucleotide sequence

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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    Tajima, S., Tsuda, H., Wakabayashi, N., Asano, A., Mizuno, S., & Nishimori, K. (1995). Isolation and expression of a chicken DNA methyltransferase cDNA. Journal of biochemistry, 117(5), 1050-1057. https://doi.org/10.1093/oxfordjournals.jbchem.a124805