Nucleic Acids Research: VOLUME 39 ISSUE 6 2011

The pattern of methyiation of mouse mitochondrial DNA (mtDNA) was studied using several techniques. By employing a sensitive analytical procedure it was possible to show that this DNA contains the modified base 5-methylcytosine (mCyt). This residue occurred exclusively at the dinucleotide sequence CpG at a frequency of 3 to 5%. The pattern of methyiation was further investigated by determining the state of methyiation of several MspI(Hpall) sites. Different sites were found to be methylated to a different extent, implying that methyiation of mtDNA is nonrandom. Based on the known base composition and nucleotide sequence of mouse mtDNA, the dinucleotide sequence CpG was found to be underrepresented in this DNA. The features of mtDNA methyiation (CpG methyiation, partial methyiation of specific sites and CpG underrepresentation) are also characteristic of vertebrate nuclear DNA. This resemblance may reflect functional relationship between the mitochondrial and nuclear genomes. INTRODUCTION Modified bases occur, with very few exceptions, in the DNA of all prokaryotes and eukaryotes (1). In Escherichia coli DNA both 6-methyladenine (mAde) and 5-methylcytosine (mCyt) are found (2), in GATC and CC^GG sequences respectively (3). In higher eukaryotes, mCyt is the only methylated base (4) and is present in the dinucleotide sequence CpG (5). In plant DNA where high levels of m Cyt are found (6), many of the methylations are also found in the dinucleotide sequence CpG (7-10). In addition, methylated cytosine is also found in a variety of other cytosine-containing dinucleotides all of which, however, are part of the basic trinucleotide CXG (11) . The biological role of DNA methyiation is now becoming clear. In E. coli, methyiation is probably involved in protecting the DNA from the action of specific nucleases. Such is the case with the restriction-modification system where it was shown that methylated bases are playing a role (12,13). In addition, other findings (14,15) suggest that methyiation may serve as an indirect signal for processes involving single stranded breaks in DNA (e.g. mismatch © IRL Press Limited, Oxford, England. 4811 Nucleic Acids Research repair, genetic recombination). As it is suggested that methylation protects DNA from single stranded breaks (14), a limited number of transiently unmethylated sites over a high methylation background, may serve as a signal for initiating these processes (15). In higher eukaryotes, methylation is correlated with gene expression (16) . Analysis of the state of methylation of different genes revealed that in nany instances hypomethylation of specific sites in a gene coincides with the initiation of its expression (17). Although it is an established fact that mitochondria of all organisms contain DNA (18), whose nucleotide sequence in several species is partly or completely known (19-23), the occurrence of modified bases in this DNA is still in dispute. Thus, m Cyt has not been detected in mitochondrial DNA (mtDNA) from Paramecium aurelia (24), HeLa cells and Xenopus laevis (25) . The internal cytosine residue in the sequence CCGG of mtDNA from different strains of yeast, Neurospora crassa, rat and calf, was also found to be unmethylated (26). On the other hand, varying amounts of m Cyt were observed in mtDNA from mouse, hamster, rat, calf, fish and birds (27-30). In none of these cases, however, has the distribution of this methylated residue among the different cytosine containing dinucleotides been determined. The increasing amount of data pointing to the role of m Cyt in gene expression on one hand, and the unclear situation concerning the existence of this residue in mtDNA on the other hand, prompted us to examine in detail the state of methylation of mouse mtDNA. MATERIALS AND METHODS Strains and culturing conditions. E. coli K12 JC5183 (31) was used for cloning. Ltk~aprt~ mouse cells, a derivative of clone D (32) were used as a source for mitochondrial DNA, and were maintained in Dulbecco modified Eagle's medium supplemented with 10% (vol/vol) new born calf serum. Enzymes and chemicals. Restriction enzymes Bam HI, Taq I, Cla I, Xho I, Msp I and Hpa II, DNA polymerase and T4 DNA ligase were purchased from New England Biolabs. Conditions used for digesting DNA with restriction enzymes were those recomaended by the manufacturer. Excess quantities of enzymes (20U/iJg) were used in Msp I and Hpa II digestions. The restriction pattern was not changed even when higher quantities of Hpa II (60U/)jg) were used, thus excluding the possibility of incomplete digestion. DNase, micrococcal nuclease and spleen phosphodiesterase were from Sigma, calf intestinal alkaline phosphatase was from Boehringer. The four [a?P]labelled deoxyribonucleoside triphosphates(3000 Ci/mmole) were purchased from Amersham.