Methylation of the Deoxyribonucleic Acid of Physarum polycephalum at Various Periods during the Mitotic Cycle*

Abstract The methylation of major nuclear DNA-cytosine was found to occur throughout the mitotic cycle of Physarum polycephalum, during the period of DNA synthesis (S), as well as during the remainder of interphase (G2) when essentially no synthesis de novo of the major nuclear DNA takes place. After incubation of the mold with methionine methyl-3H, 5-methylcytosine was the only radioactive methylated minor base detected in major nuclear DNA, although 3H was also found to be incorporated during the S period into DNA-adenine, -guanine, and -thymine, presumably by conversion of the methionine methyl group to 1-carbon intermediates incorporated into DNA nucleotides during synthesis de novo. During the G2 period, methylation of major nuclear DNA occurred at a relatively constant rate which was approximately 50% of that observed during the S period. Incorporation of 3H from methionine methyl-3H into 5-methylcytosine of both mitochondrial DNA and the nuclear heavy satellite DNA was also observed. It was estimated that the three DNA fractions of Physarum contain 1 methylcytosine residue for every 12 to 24 cytosine residues.

during the period of DNA synthesis (S), as well as during the remainder of interphase (62) when essentially no synthesis de novo of the major nuclear DNA takes place. After incubation of the mold with methionine methyl-3H, 5-methylcytosine was the only radioactive methylated minor base detected in major nuclear DNA, although 3H was also found to be incorporated during the S period into DNA-adenine, -guanine, and -thymine, presumably by conversion of the methionine methyl group to l-carbon intermediates incorporated into DNA nucleotides during synthesis de novo. During the 62 period, methylation of major nuclear DNA occurred at a relatively constant rate which was approximately 50% of that observed during the S period. Incorporation of 3H from methionine methyl-3H into 5-methylcytosine of both mitochondrial DNA and the nuclear heavy satellite DNA was also observed. It was estimated that the three DNA fractions of Physarum contain 1 methylcytosine residue for every 12 to 24 cytosine residues.
The enzymatic methylation of pre-formed DNA was first described by Gold, Hurwitz, and Anders (2). Subsequently it has been reported that DNA methylation appears to be associated with DNA synthesis in both prokaryot.ic and eukaryotic cells: Lark (3) and Billen (4) have demonstrated that DNA is normally methylated as it is replicated in Escherichia coli, while inhibitors of DNA synthesis added to cultures of mammalian cells have been found to reduce the level of DNA methylation by 60 to 85% (5,6). In the present work, we have studied the methylation of DNA at various periods during the mitotic cycle of Physarum polycephalum, a myxomycete whose nuclei undergo naturally synchronous division and in which a clearly defined DNA synthetic period (S) occurs immediately following * This work was supported by Contract W-31-109-Eng-78 with the United States Atomic Energy Commission, Report COO-78-230. A preliminary report of this work has been presented (1).

mitosis.
There is no Gl period in Physarum. We have found that methylation of the major nuclear DNA occurs not only during the S period but also during the G2 period, a time when essentially no synthesis de no2ro of the major nuclear DNA takes place. The rate of DNA methylation during the G2 period, which was found to be relatively constant, was about 50% of the rate observed during the S period.

Growth
of Organism-Stock shake cultures were maintained axenically as microplasmodia grown at 23" in semidefined medium in the dark as described by Daniel and Baldwin (7). Synchronously dividing microplasmodia were prepared by fusion of microplasmodia on filter paper surfaces (Carl Schleicher and Schuell Company, Keene, New Hampshire, type 576) supported on a layer of glass beads, as described by Nygaard, Giittes, and Rusch (8). The time of mitosis was ascertained by microscopic examination of alcohol-fixed smears. Interdivision time under our conditions was approximately 7 hours.
Incorporation of Radioactive Precursors-Labeled precursors, generally obtained from New England Nuclear or Schwarz BioResearch, were added to the medium at the desired time at the following levels: thymidine methylJH, 5 $Zi per ml; methionine methyl-3H, 5 to 30 MCi per ml; sodium formate-"C, 2 &i per ml; phosphate-32P, 10 #.Zi per ml. The specific activity of the labeled precursors varied but was found not to affect the level of incorporation, probably because of the content of these precursors in the semidefined medium.
At the end of the incubation period, shake cultures were harvested by a brief centrifugation, after which the pellets were frozen. Macroplasmodia were scraped from the filter paper into a cold solution composed of 0.15 M NaCl and 0.015 M sodium citrate, which was then frozen.
Hot Acid Extraction of Nucleic Acids-In experiments involving the incorporation of thymidine-3H, molds were washed with cold 4% trichloracetic acid in acetone and then with cold 0.6 N HC104. Total nucleic acids were extracted from the acidinsoluble pellet by twice heating at 85" for 15 min in the presence of 0.6 N HC104. The hot acid extract was assayed for radioactivity and for DNA by the Burton modification of the diphenylamine reaction (9). In experiments involving the incorporation of formateJ4C, molds were washed with cold acid as described 6436 Issue of December 10, 1970 H. H. Evans and T. E. Evans 6437 above, and RNA and DNA were then separated by the Schmidt-Thannhauser procedure (10). Both RNA and DNA were then concentrated under reduced pressure and subjected to hydrolysis and base separation as described subsequently.
Extraction of Native DNA-In experiments involving the incorporation of methionine methyl-3H or phosphate-32P, a more rigorous purification of DNA was employed. Total DNA was extracted according to the method described by Evans (11). The heavy satellite DNA and mitochondrial DNA were selectively extracted in some experiments by the method of Braun and Evans (12). In experiments involving the incorporation of 32P, the extracted DNA was subjected to chromatography on a column, 2 x 15 cm, of Sepharose 6-B (Pharmacia) at 40" to separate the DNA from radioactive low molecular weight contaminants.
The DNA samples were added to solutions of Radio-Tracer Grade CsCl (Harshaw Chemical Company, Cleveland, Ohio) which were subsequently adjusted to the appropriate density (1.701 to 1.712 g per ml) and centrifuged in a Beckman-Spinco model L2 centrifuge for 66 hours at 32,000 rpm at 25' in a 40.3 fixed-angle head rotor.
At the end of the run, the tubes were punctured at the bottom, and 11-drop fractions were collected in test tubes. After the addition of 1 ml of HzO, the absorbance at 260 nm and the radioactivity of the fractions were determined.
The contents of tubes containing DNA of the same type (i.e. heavy satellite DNA, major nuclear DNA, and mitochondrial DNA) were combined. When the three types of DNA were not cleanly separated, the CsCl density gradient centrifugation was repeated with each of the initially isolated fractions.
Initial and final CsCl fractionations are illustrated in Fig. 1. The final CsCl fractions, which were then dialyzed exhaustively against 0.01 M ammonium acetate, were concentrated under reduced pressure.
Hydrolysis and Chromatographic Separation of Bases and Nucleotides-Before hydrolysis of the isolated DNA samples, nonradioactive bulk Physarum DNA was added to bring the total amount of DNA to 0.5 to 1.0 mg. Carrier 5-methylcytosine (0.42 pmole) or 5-methylcytosine deoxyribonucleotide (0.15 pmole) was also added to facilitate subsequent chromatographic identification of these compounds. The DNA, or RNA, was hydrolyzed to yield free bases by heating for 1 hour in the presence of 0.04 ml of 70% HClOd at 100". The hydrolysate was centrifuged, and the supernatant solution was subjected to two-dimensional chromatography on Whatman No. 4 chromatography paper, with the use of isopropyl alcohol-HCl-Hz0 (170:41:39) (13) in the first direction and 86% butanol-Hz0 in an NH, atmosphere (14) in the second direction.
When complete separation of the bases was not obtained, chromatography was repeated with the isopropyl alcohol-HCl-Hz0 solvent system. The absorbance at 260 nm and the radioactivity of the eluted bases were then determined.
DNA was hydrolyzed to deoxyribonucleotides by enzymatic digestion with DNase I (EC 3 .1.4.5, Worthington) and snake venom phosphodiesterase (EC 3.1.4.1, Worthington). DNase I (100 pg) and 1 mg of DNA were dissolved in a l-ml solution composed of ammonium acetate (0.01 M, pH 7.0), MgC& (0.025 M), and NaF (0.025 M). The solution, which was incubated for 2 hours at 37" was then heated rapidly to 70" and maintained at this temperature for 5 min. After cooling, the pH was adjusted to 9.0 with NHdOH, phosphodiesterase (0.125 mg) was added, A, first density gradient centrifugation of bulk DNA extracted from a shake flask incubated for 24 hours with 10 &i per ml of methionine methyl-aH.
Fractions 10 to 12 were combined for the heavy satellite DNA, Fractions 14 to 18 for major nuclear DNA, and Fractions 24 to 28 for mitochondrial DNA.
The increase in absorbance in Fractions 34 to 40 is due to a polysaccharide contaminant of the DNA preparation. The initial density of the C&l solution was 1.707 g per ml. B, third and final density gradient centrifugation in the purification of mitochondrial DNA.
Fractions 20 to 25 were combined for base analysis. Nonradioactive bulk Physarum DNA, 100 e, was added as an absorbance marker.
The initial density of the CsCl solution was 1.707 g per ml. C, third and final density gradient centrifugat.ion in the purification of the heavy nuclear satellite DNA.
Fractions 21 to 24 were combined for base andysis. Nonradioactive bulk Physarum DNA, 100 pg, was added as an absorbance marker.
The initial density of the CsCl solution was 1.712 g per ml. and the solution was incubated first at 37" for 90 min and then at 70" for 5   HCl and HzO. Material adsorbed on the charcoal was eluted with 50% ethanol-O.15 N NHJOH, the eluate was concentrated under reduced pressure, and the residue was hydrolyzed by heating with 70% HC104 at 100" for 1 hour. After two-dimensional chromatography as described in the preceding section, adenine was eluted and assayed for ultraviolet absorbance and radioactivity.
Determination of Incorporation of Methionine Methyl-3H into Protein-Total protein was extracted according to the method described by Greenberg and Rothstein (16) from a shake culture previously incubated with methionine methyl-3H for 24 hours.
The extracted protein, 2 mg, was hydrolyzed with 6 N HCl, and the amino acids were separated as described by Moore and Stein (17), with a Beckman ammo acid analyzer with attached Nuclear-Chicago liquid scintillation flow counter. Measurement of Radioactivity-Radioisotopes were assayed by liquid scintillation counting in a solution containing 125 g of naphthalene, 6.75 g of Omnifluor (New England Nuclear), 100 ml of absolute methanol, and 40 g of Cab-O-W (Cabot Corporation, Boston, Massachusetts) per liter of p-dioxane. Counts were corrected for quenching by means of an external standard.

DNA Bases Labeled after Incubation of Physarum with Methionine Methyl-3H-In
an initial experiment, the occurrence of the methylation of DNA in Physarum was determined. A shake culture was incubated with methionine methyl-3H for 24 hours, and the distribution of 3H in the individual bases of major nuclear DNA was determined.
The results are shown in Table  I. Most of the radioactivity was located in adenine, thymine, and 5-methylcytosine.
In this experiment, 6-methylaminopurine, in addition to 5-methylcytosine, was added to the hydrolysate before chromatography.
No 3H was found in the re-isolated 6-methylaminopurine.
Thus, no radioactive methylated minor base other than 5-methylcytosine was detected in major nuclear DNA after incubation of Physarum with methionine methyl-3H.
Conversion of Methionine Methyl Group to I-Carbon Intermediates in Physarum-It is probable that the radioactivity incorporated into DNA-adenine, -guanine, and -thymine arises through conversion of the methyl group of methionine to lcarbon intermediates utilized subsequently in the formation of purine and thymine nucleotides which would be incorporated into DNA during its synthesis de novo. The occurrence of these reactions in Physarum was indicated indirectly by the finding that 3H from methionine methyl-3H was incorporated into serine of total protein and into acid-soluble adenine compounds. Amino acid analysis of the total protein and the distribution of 3H in the amino acids are shown in Table II. The labeled fractions were methionine, methionine sulfoxides, serine, aspartic acid, lysine, histidine, two unknown basic amino acids, and two unknown ninhydrin-negative fractions. The 3H in the lysine and histidine is probably due to the presence in these fractions of methylated lysine and methylated histidine, which have been found to occur in the proteins of a variety of tissues and which would not be separated from lysine and histidine under the Issue of December 10, 1970  Unknowns 1 and 2, which were ninhydrin-positive, were eluted with the basic amino acids from the short column in the order listed.
Unknowns 3 and 4, which were ninhydrinnegative, were eluted from the long column in the order listed.
To investigate the incorporation of 3H from methionine methyLaH into acid-soluble adenine compounds, synchronously dividing plasmodia were incubated with 38 aCi per ml of methionine methyLaH during the S period.
Total acid-soluble adenine was found to have a specific activity of 36,000 cpm per pmole, while the specific activity of DNA-adenine was 2,400 cpm per fimole.
DNA Methylation during Mitotic Cycle-The occurrence of DNA methylation at various periods throughout the mitotic cycle was determined by incubating macroplasmodia with methionine methyL3H for successive periods of 14 hours each following mitosis. Two small portions of each plasmodium were also incubated in media containing thymidine methyl-3H or formate-"% for the same time periods.
The incorporation of 3H from methionine methylL3H is shown in Table III, the  incorporation of thymidine-3H in Table IV, and the incorporation of formate-% in Table V. The methylation of major nuclear DNA-cytosine occurred at a relatively constant rate during G2, a period in which essentially no synthesis de nova of major nuclear DNA takes place (Table III).
The rate of DNA methylation during the G2 period was about 50% of the rate occurring during the S period.
In contrast, the incorporation of 3H into DNA-purines during the G2 period was only 5 to 10% of that occurring during the S period.
Incorporation of thymidine-3H and formate-'4C into bulk DNA during G2 was 10 to 24% of that occurring during S (Tables IV and V). The relatively high amount of radioactivity found in bulk DNA during the G2 period in these experiments is probably due to the omission of the CsCl density gradient centrifugation step in the extraction of DNA. This procedure removes both of the satellite DNA fractions (which are synthesized throughout the cycle (12)), as well as any remaining low molecular weight radioactive contaminants.
RNA synthesis, as indicated by the incorporation of formate-14C into RNA-adenine (Table V), reached a maximum during the period from l+ to 3 hours following mitosis.
The pattern of RNA synthesis during the mitotic cycle has been described by Mittermayer, Braun,and Rusch (20). Methylation of Satellite DNAs-A comparison of the incorporation of 3H from methionine methyl-SK into the three DNA fractions of Physarum during the G2 period (3 to 6 hours following mitosis) is shown in Table VI. Incorporation  into 5methylcytosine was noted in the case of the heavy nuclear satellite DNA and mitochondrial DNA, as well as into major nuclear DNA.
In this as well as in other experiments, a relatively high percentage of the 3H incorporated into heavy nuclear DNA was localized in thymine.
The mechanism involved in this incorporation will require further investigation (see under "Discussion").
We have not investigated the presence of methylated minor bases other than 5-methylcytosine in the case of the satellite DNAs, nor have we measured the methylation of satellite DNAs at various times throughout the mitotic cycle.
Determination of Amount of 5-Methylcytosine in Physarum DNA-To determine the amount of 5-methylcytosine in each  Rechromatography of the dMCMP in the same solvent reduced the amount of 3H to 29ib of the radioactivity originally present in dCMP-3H.
Although the results were highly variable, probably because of variable contamination of dMCMP with dCMP, it may be estimated that these DNA fractions contain 1 residue of methylcytosine for every 12 to 24 residues of cytosine (Table VII).

DISCUSSION
The incorporation of 3H of methionine methyl-3H into Physurum DNA apparently involves two mechanisms: (a) conversion of the methionine methyl group to l-carbon intermediates that are subsequently utilized for the formation of purine and thymine nucleotide precursors of DNA, and (5) methylation of cytosine residues of the pre-formed DNA polymer.
Formation of lcarbon intermediates in this organism is indicated by the experiments demonstrating the incorporation of the label into serine of the total protein and into acid-soluble adenine compounds.
The lo-to 20-fold decrease in the incorporation into major nuclear DNA-purines during the G2 period as compared with the S period also suggests that this incorporation involves synthesis de novo rather than methylation of the pre-formed DNA polymer.
The appearance of 3H in DNA-thymine might be explained either by conversion of the methionine methyl group to l-carbon intermediates incorporated into thymidylic acid and then into DNA during synthesis de novo or by deamination of pre-formed DNA-Bmethylcytosine.
Sneider and Potter (5) have suggested that deamination of pre-formed DNA-5methylcytosine might occur in viva, producing mispairing and possible initiation sites on the DNA template for replication or transcription.
It is also possible that deamination of DNA-5-methylcytosine could occur in vitro during hydrolysis of the DNA to free bases. In Physarum, incorporation of 3H from methionine methyl-3H into DNAthymine appears to be due in part to synthesis de novo of DNA from previously formed 3H-TMP, since inclusion of nonradioactive thymidine, fluorodeoxyuridine, and uridine in the medium reduced the incorporation into DNA-thymine by 90%, while not affecting the incorporation of 3H from methionine methyl-3H into DNA-5-methylcytosine or the incorporation of 32P into bulk DNA. ' The question of whether or not 3H from methionine methyl-3H is incorporated into DNA-thymine in Physarum by an additional mechanism, such as deamination of pre-formed DNA&methylcytosine, must await further investigation. The occurrence of methylation of the major nuclear DNA during the G2 period, in the absence of synthesis de novo of this DNA, is of interest when considering the possible function of DNA methylation.
Methylation of DNA has been shown to be involved in bacterial modification-restriction processes (21), in which infecting phage DNA, methylated by permissive host enzymes, becomes resistant to host endonuclease degradation. If DNA methylation is involved with the control of nuclease action in eukaryotic organisms, the process might serve as a negative control of transcription or replication initiator endonucleases, if such exist.