Bovine papillomavirus (BPV) was methylated in vitro at either the 29 HpaII sites, the 27 HhaI sites, or both. Methylation of the HpaII sites reduced transformation by the virus two-to sixfold, while methylation at HhaI sites increased transformation two-to fourfold. DNA methylated at both HpaII and HhaI sites did not differ detectably from unmethylated DNA in its efficiency of transformation. These results indicate that specific methylation sites, rather than the absolute level of methylated cytosine residues, are important in determining the effects on transformation and that the negative effects of methylation at some sites can be compensated for by methylation at other sites. BPV molecules in cells transformed by methylated BPV DNA contained little or no methylation, indicating that the pattern of methylation was not faithfully retained in these extrachromosomally replicating molecules. Methylation at the HpaII sites (but not the HhaI sites) in the cloned BPV plasmid or in pBR322 also inhibited transformation of the plasmids into Escherichia coli HB101 cells.

Cell line 101 is a thymidine kinase (TK)-positive derivative of Ltk-which contains ca. 20 copies of the herpes simplex virus TK gene organized in a tandem array. DNA methylation at three sites within the gene and flanking sequences was inversely correlated with expression: the sites were unmethylated in line 101, methylated in each of 4 TK-negative derivatives of 101, and unmethylated in each of 21 TK-positive derivatives derived from them. The same three sites were affected in most of the 20 copies of the TK gene, whereas other sites between them were not affected. Although the entire gene cluster was never lost, indicating that integration into the genome was stable, internal rearrangements occurred at a high frequency. The rearrangements had no obvious correlation with the state of methylation or with the expression of the genes.There is a well-established inverse correlation between the level of DNA methylation and the expression of many genes, including globin (31), integrated viral genomes (11,27,28,32), ovalbumin (16), and the genes coding for adenine phosphoribosyltransferase and dihydrofolate reductase (26). Studies with inhibitors of DNA methylation suggest that the inhibition of DNA methylation increases the frequency of expression of methylated, inactive genes in vivo (2,4,9,20), and it has been demonstrated that methylated DNA is expressed more poorly than unmethylated DNA after transfer into mammalian cells (18,25,33, 36).Conversely, other studies have failed to detect the involvement of methylation in the expression of some genes (8, 19), indicating either that methylation does not play a role in the expression of those genes or that the sites of methylation which were important for the regulation were not easily detected by restriction endonuclease digestion. Data from a number of studies indicate that methylation of one or a few sites in specific regions (often in the 5' noncoding region) is sufficient to abolish the expression of some genes (1,22,26), so that if the particular sites of importance do not fall within a sequence recognized by one of the methylation-sensitive restriction endonucleases, they are difficult to identify and might be missed.Several laboratories have studied the modulation of expression of thymidine kinase (TK) genes which had been transferred into cultured cells (3-5, 12, 21, 24, 29), and several mechanisms by which expression of the TK gene is regulated have been identified. Robins et al. (24) demonstrated that of 40 lines which expressed the TK genes, half retained the genes after selection against TK expression, indicating that mechanisms in addition to gene loss were involved in modulating the expression of the transferred genes. Davies et al. (5) described cell lines in which changes in expression were correlated with changes in the nuclease sensitivity of the TK-containing chromatin but in which no changes in DNA methylation could be detected. Ostrander * Corresponding author. t Contribution 1239 from the Department of Biology. et al. (21) and a previous publicati...