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DNA methylation is found almost ubiquitously in nature and the methyltransferases show evidence of a common evolutionary origin. It will be a fascinating study in protein evolution to follow the ways in which the structures of the various enzymes have developed. Although methylation may have a direct effect on DNA structure the evidence for the importance of this in vivo is accumulating only slowly. In contrast, there is now abundant evidence that methylation of DNA affects DNA-protein interactions and so may have a function in all processes in which such interactions occur. The binding of nucleases is affected in the processes of mismatch repair, DNA restriction and possibly demethylation during differentiation in vertebrates. The binding of transcription factors is affected by DNA methylation and the association of DNA with packaging and segregation proteins may play a part in the control of transcription and replication. The interplay of these effects makes DNA methylation a complex but rewarding area for study. Perhaps we should no longer refer to methylcytosine and methyladenine as minor bases, but rather as key bases which help regulate the functions of DNA.

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The detailed mechanisms of inhibition of transcription by DNA methylation are still unknown, but it has become obvious that the formation of chromatin plays an important role in this process. Using an approach enabling us to methylate, in vitro, chosen regions in a plasmid, we now show that specific methylation of nonpromoter sequences results in transcriptional inhibition of a reporter gene construct and that this inhibition is independent of the position of the methylated region within the plasmid. In plasmid minichromosomes containing a short region of methylated DNA, both methylated and unmethylated sequences are protected from MATERIALS AND METHODS Plasmids. Plasmid pVHCk was constructed by cloning a 2-kb KpnI-EcoRI fragment containing the chloramphenicol acetyltransferase (CAT) reporter gene under the control of the SV40 promoter/enhancer and the SV40 terminator region from pVHC1 (7) into pBluescript KSII-. A map of the plasmid is shown in Fig. 1. The promoterless plasmid pPLk was generated by deleting the SV40 promoter/enhancer from * Corresponding author.pVHCk by digestion with K1nI-HindIII, generation of blunt ends with S1 nuclease, and religation. Two constructs containing the SV40 promoter/enhancer at both sides of the CAT gene/terminator region were generated by cloning the BamHI-linkered promoter fragment in both orientations into the BamHI site of pVHCk immediately 3' of the terminator region. They were named pPPk (promoters in same orientation) and pPRk (second promoter in reverse orientation), respectively.Generation of patch-methylated constructs. Phagemid single-stranded DNA (ssDNA) was isolated by the method of Blondel and Thillet (4). Restriction fragments were gel purified and annealed to ssDNA at a molar ratio of 3:1 (fragment DNA:ssDNA) in 20 mM Tris-HCl (pH 7.5)-10 mM MgCl2-50 mM NaCl-1 mM dithiothreitol by heating for 2 min at 95°C and allowing to cool down from 70 to 30°C in 1 h. To prevent binding to and methylation of the singlestranded region by methylase SssI (M.SssI), T4 gene 32 protein (Boehringer Mannheim) was added (10 ,ug/,g of ssDNA), and the reaction mixture was incubated at 37°C for 15 min. The double-stranded DNA patch was methylated for 16 h, using M.SssI (New England Biolabs) under conditions recommended by the manufacturer. After proteinase K treatment and phenol extraction, the unmethylated gap was filled in and ligated by standard procedures (35). For every patch-methylated construct, a mock-methylated control was generated in the same way but omitting M.SssI. To confirm the location of the methylated region, constructs were isolated immediately or 2 days after transfection, and the methylated region was released with the appropriate restriction enzymes and subjected to HpaII digestion. The DNA was then separated on a 1.5% agarose gel, transferred to a nylon membrane (Hybond N+; Amersham), and hybridized to the appropriate labeled fragment. Membranes were reprobed after stripping with boiling sodium dodecyl sulfate (SDS) solution (0.5%).Transfections and analysis of CA...