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...

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E coli tRNA2Phe was modified at 25 degrees C with 3M sodium bisulphite, pH6.0, for periods of up to 48 hours, Three cytadinine residues, at position 17, 74 and 75 from the 5' end were each deaminated to uridine. The 2-methylthio-N6-isopentenyl adenosine at position 37 formed a 1:1 bi-sulphite addition product which was stable to alkaii. No other residues were permanently modified. The rate of modification of each residue was first order with respect to remaining unmodified nucleotide, the time of half reaction, t1/2, being different for each residue. C17 reaction reacted at twice the rate of cytidine in PolyC, indicating that it occupied a very exposed position in the tRNA.

We have previously shown that DNA methylation acts as a focus for the formation of inactive chromatin in vivo. We have investigated the mechanism further by in vitro transcription of a template containing two tRNA genes and an extensive (G+C)-rich sequence characteristic of a CpG island. The extent of transcription from the unmethylated or fully methylated template was assayed in the presence of varied levels of histone H1. The transcriptional activity of both templates was inhibited by increasing amounts of histone H1, although inhibition with the methylated template occurs at a lower H1:DNA ratio. The H1c variant shows the greatest preferential inhibition of the methylated template. We demonstrated that histone H1 complexed to DNA is one of the factors that inhibits transcription by preventing the formation of initiation complexes, particularly on methylated template, rather than the formation of disordered H1.DNA aggregates.

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A gene for tRNAGlu has been assigned to human chromosome 1p36 by in situ hybridisation using a [3H]-labelled or biotinylated 2.4-kb (human) DNA fragment containing a tRNAGlu gene as a probe. With the biotinylated DNA probe a secondary statistically significant site of hybridisation was observed at 1q21-22 which might represent a pseudogene or related sequence. In fibroblasts from gorilla (Gorilla gorilla) using biotin labelling, a single site of hybridisation occurred at 1qter which provides further support for homology of 1q in the higher apes and human 1p.

A mixture of low molecular weight RNAs, in which only tRNAs were radiolabelled, was used as a hybridisation probe to select for tRNA-like sequences within a bank of human genomic DNA in lambda Charon 4A. A restriction enzyme digest of one of the selected lambda Charon 4A recombinants contained two fragments (2.4 Kb & 1.8 Kb) which hybridised tRNA and which, when subcloned into pAT153, were transcribed in Xenopus oocyte nuclei. Analysis of the subcloned 2.4 Kb fragment, which was of remarkably high transcriptional activity, revealed the presence of a single gene for tRNAGlu in the middle of the fragment. The sequence immediately preceding the gene has the potential for forming a tRNA-like structure.

The reaction of 14C methyl-labelled HeLa cell 28 S ribosomal RNA with sodium bisulphite was studied. Using conditions under which 30% of the total cytidine residues were de-aminated to uridine, the reactivities of individual cytidine residues near particular methylation sites differed widely; some underwent almost quantitative reaction, some showed intermediate reactivity and others were almost inert. The possible value of this method as a conformational probe for ribosomal RNA is discussed.