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The origin and function of the large amount of 5-methylcytosine in plant DNA is not well understood. As a tool for in vitro studies of methylcytosine formation in plants we have isolated and characterized the DNA methyltransferase present in germinating wheat embryo. An enzyme fraction enriched 300-fold over the tissue homogenate was obtained by salt extraction of nuclei, chromatography on DEAE-cellulose, Sephadex (3-75, blue Sepharose and on DNA immobilized on cellulose. It catalyzes the methylation of cytosine residues in doublestranded DNAs isolated from wheat, maize, calf thymus or bacteria using S-adenosylmethionine as methyl donor. The efficient methylation of both an unmethylated plasmid DNA and its hemimethylated derivative indicate that the wheat DNA methylase can function de now and in maintenance methylation. A relative molecular mass of 50000 -55 000 was estimated by gel permeation chromatography and sucrose density gradient centrifugation. Polyacrylamide gel electrophoresis showed the presence of a protein of M , = 50000 and one other componentThe preference for endogenous, double-stranded DNA as substrate and the lower molecular mass distinguish wheat DNA methyltransferase from the DNA methylases obtained from mammalian sources. The properties of the wheat enzyme resemble, however, those of the DNA methylase isolated from the alga Chlamydomonas reinhardii, suggesting that plant cells possess their own type of DNA methyltransferase for the biosynthesis of their high methylcytosine content in DNA. ( M , = 35000).Plant DNA is distinguished from the DNA of all other organisms by its substantial content of the modified base, 5-methylcytosine, which can constitute up to 8 mo1/100 mol of the total base composition, or one-third of all cytosine residues [l, 21. The origin and functions of this high level of DNA methylation in plants are unknown. Vertebrates have much lower proportions of their cytosine residues in DNA methylated (approximately 0.9 mo1/100 mol). Here, changes in methylation are involved in the control of gene expression during development and cell differentiation, with a general correlation between undermethylation in active gene sequences and full methylation in non-transcribed regions [3, 41. Whether a similar signal function occurs in plant tissues is not well established; the above correlation seems to hold for zein genes, but not for the alcohol dehydrogenase gene of maize [5, 61. In any event it is difficult to conceive that the entire amount of msCyt in plants takes part in control mechanisms.In spite of the abundance of 5-methylcytosine in plants, its biosynthesis has not received much attention. A DNA methyltransferase was characterized in the green algae, Chlumydomonus reinhardii [7, 81 but because the unicellular Correspondence to H . Follmann,

Synchronous cells of the green alga, Scenedesmus obliquus, cultured in a 14‐h/10‐h light/dark regime, contain a peak of ribonucleoside‐diphosphate reductase activity and maximum deoxyribonucleoside 5′‐triphosphate concentrations at the 12th hour of the cell cycle, coinciding with DNA synthesis and preceding the formation of eight daughter cells. The intracellular dTTP pool reaches 4.5 pmol and the other pools 2–3 pmol/106 cells. Algal reductase activity is sensitive to cycloheximide, but not to lincomycin. These correlations demonstrate the functioning of the NDP → dNDP → dNTP pathway of DNA precursor biosynthesis in plant cells. In the presence of 20 μg 5‐fluorodeoxyuridine/ml, an inhibitor of thymidylate synthesis, the dTTP pool is rapidly depleted and DNA synthesis ceases. 5‐Fluorouracil and methotrexate produce similar effects. At the same time the ribonucleotide reductase activity and also the dATP pool are greatly increased, especially when fluorodeoxyuridine treatment is combined with continued illumination of the algae. In contrast, arabinosylcytosine, an inhibitor of DNA replication, has no effect on ribonucleotide reduction. The control of de novo enzyme synthesis in the eucaryotic algae therefore appears to depend on the presence of dTTP (or a related nucleotide), but not directly coupled to DNA synthesis. This interdependence resembles the situation observed in HeLa cells, while it may differ in detail from control mechanisms of ribonucleotide reductase studied in bacteria.

not received Cell cycleAnaerobiosis Deoxyribonucleotide pool Ribonucleotide reduction

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