Detecting differences in 5-methylcytosine using restriction enzyme isoschizomers: an endogenous control for complete digestion.

Growing research effort in recent years has focused on the role of 5-methylcytosine (5-MeC) in gene expression. 5-MeC has been implicated in such phenomena as genomic imprinting, maintenance of heterochromatin, and the direct control of gene expression through proteins which bind methylated DNA (1). As interest in 5-MeC has intensified, several new techniques have been developed to find and study genes under the influence of 5-MeC {2-4). Despite these new techniques, Southern blot analysis of genomic DNA cut with methylation-sensitive isoschizomers like MspUHpaU has remained the simplest technique for detecting 5-MeC differences between cell populations. Incomplete digestion by the methylation sensitive isoschizomer, however, can cause both false positives and false negatives; thus a simple control is desirable to ensure that the enzymatic digestions are complete. We have determined that the mitochondrial genome, which is devoid of 5-MeC in mammals, can be used as an endogenous control for complete digestion by HpaU or any other methylationsensitive restriction enzyme (5). Previous efforts to control for complete enzymatic digestion have involved the addition of foreign unmethylated DNA (such as a plasmid) to the digestion reaction or the addition of a digestion reaction aliquot to the foreign DNA (6,7). The mitochondrial genome makes a more powerful control because it is co-isolated with genomic DNA, thus controlling for every step of the DNA isolation and Southern analysis. In addition, using the endogenous mitochondrial genome eliminates the additional steps involved with previous controls. To demonstrate the use of mitochondrial DNA in detecting incomplete digestion and the false negatives that can result a blot was prepared with DNA digested with Mspl, HpaU or HpaU partially inactivated by heat (2.5 or 5 min at 75 °C) or 1% sodium dodecyl sulfate (SDS) in the reaction. Genomic DNA isolation and blot preparation followed standard procedures (8). The blot was first probed with a portion of thymidine kinase (TK), a gene that displays methylation differences between the human multiple myeloma cell lines RPMI 8226 (8226/S) and 8226/V (9). Mspl, which is insensitive to the methylation state of the internal cytosine in its recognition sequence 5'-CCGG-3', produces three gene fragments which were recognized by the TK probe (Fig. 1, left, lanes 1 and 2). The Mspl fragments demonstrate the limit digest of TK in 8226/S and 8226/V and confirm that TK has remained intact in the drug selected 8226/V cell line. In contrast, digestion of 8226/S and 8226/V DNA with HpaU, which does not cut if the internal s v s v s v s v s v