Differential activity of DNA methyltransferase in the life cycle of Chlamydomonas reinhardi.

Abstract

Two molecular weight. forms of DNA (cytosine-5-)-methyltransferase [S-adenosyl-L-methionine:DNA (cytosine-5-)-methyltransferase, EC 2.1.1.37], both active in assays in vitro, were isolated from the green alga Chlamydomonas reinhardi at various stages of the life cycle. The enzyme with Mr 60,000 was found in vegetative cells and gametes of both male (mtF) and female (mtt) mating types. The enzyme with Mr 200,000 was specific to gametic cells and zygotes, which are the only stages at which methylation of chloroplast DNA occurs in vivo. Chloroplast DNA from gametes was shown to be methylated on both strands at most if not all methylation sites.and the Mr 200,000 enzyme was shown to methylate both unmethylated. and hemimethylated sites, the latter at an elevated rate. Micrococcus luteus DNA showed the same nearest-neighbor frequencies of methylation after methylation by each molecular weight component. The data suggest strongly that the Mr 20o,ooo enzyme is the active multimeric form of the Mr 60,000 enzyme and that it acts as both initiation and maintenance methylase. It is proposed that methylation of chloroplast DNA in female. gametes and zygotes is regulated by assembly ofthe multimeric Mr 2oo,0o0 active enzyme, which in turn determines the maternal inheritance of-chloroplast DNA.Eukaryotic DNAs contain 5-methylcytosine as a minor component, at concentrations of 27% of total cytosine, depending upon the species (1, 2). The introduction ofmethyl residues into the 5 position ofcytosine occurs after replication and is catalyzed by a DNA methyltransferase (EC 2.1.1.37), which transfers the methyl group from S-adenosylmethionine to cytosine in doublestranded DNA (3). This class of enzymes, found in both plant and animal tissues (3), methylates only a small fraction of cytosines, mainly at C-G doublets (4). Very little evidence of site specificity of a eukaryotic methyltransferase has yet been reported, other than in our preliminary study (5).The physiological roles of methylation in eukaryotic cells are almost entirely unknown (6). Evidence of tissue-specific methylation has been described (7-9), although the significance of the observed methylation patterns is unclear. A function in the regulation of gene expression is suggested especially by the effect of hypomethylation on globin synthesis in Friend cells (10) and the effect of azacytidine on differentiation (11,12). A role for methylation and restriction in the inactivation or destruction of particular sequences of DNA or even whole chromosomes has been proposed (13). In a series ofstudies, we have demonstrated that a methylation and restriction system regulates the inheritance of chloroplast DNA in the sexual alga Chlamydomonas (14). In this organism, as well as in higher plants, chloroplast DNA from the female parent is preferentially transmitted to progeny, leading to the genetic phenomenon of maternal inheritance.In this paper, we describe the isolation and characterization of a DNA methyltransferase from Chlamydomonas. Methylating activity has been ...

A chloroplast-resident DNA methyltransferase is responsible for hypermethylation of chloroplast genes in Chlamydomonas maternal gametes

“…For example, selective methylation of chloroplast DNA has been consistently observed in mt ϩ gametes with buoyant density centrifugation (13), restriction enzyme cleavage patterns (6), and by southwestern hybridization with anti-m 5 C antibodies (3). A gamete-and zygote-specific DNA methyltransferase with a high molecular mass also was identified (20). However, the evidence was indirect, and critical experiments showing a direct association of methylation with persistence of mt ϩ chloroplast DNA during mating have hitherto not been performed to our knowledge.…”

“…In our previous study, we identified a mt ϩ gamete-and zygote-specific DNA methyltransferase of Ϸ200 kDa, which methylates both unmethylated and hemimethylated chloroplast DNA (20). Its identity may in fact be CrMET1, given the molecular mass of Ϸ150 kDa, and the mt ϩ gamete specificity.…”

DNA Methylation--A Regulatory Signal in Eukaryotic Gene Expression

“…Secondly, DNA methylation controls the transcriptional activity of certain algal genes. Site-specific DNA methyltransferases in Chlamydomonas have been isolated and characterized (Sano & Sager, 1980;Sano et al, 1981). In Chlamydomonas, chloroplast genes are maternally inherited.…”

Dynamic changes in the transcriptome and methylome of Chlamydomonas reinhardtii throughout its life cycle

“…Several methyltransferase enzymes that modify cpDNA have been investigated biochemically (Sano et al, 1981), and one candidate chloroplast methyltransfersase gene has been cloned (Nishiyama et al, 2002(Nishiyama et al, , 2004. Since that time, the genome sequence of C. reinhardtii has become available (Merchant et al, 2007) and extensively annotated so that a comprehensive identification of genes encoding DNA methyltransferases can be undertaken.…”