METHYLATION OF SINGLE SITES WITHIN THE HERPES-SIMPLEX VIRUS TK CODINGREGION AND THE SIMIAN-VIRUS-40 T-ANTIGEN INTRON CAUSES GENE INACTIVATION

In order to determine whether partial methylation of the herpes simple x virus (HSV) tk gene prevents tk gene expression, the HSV fk gene was cloned as single-stranded DNA. By in vitro second-strand DNA synthesi s, specific HSV tk gene segments were methylated, and the hemimethylat ed DNA molecules were microinjected into thymidine kinase-negative rat 2 cells. Conversion of the hemimethylated DNA into symmetrical methyla ted DNA and integration into the host genome occurred early after gene transfer, before the cells entered into the S phase. HSV tk gene expr ession was inhibited either by promoter methylation or by methylation of the coding region. Using the HindIII-SphI HSV tk DNA fragment as a primer for in vitro DNA synthesis, all cytosine residues within the co ding region, from +499 to +1309, mere selectively methylated. This spe cific methylation pattern caused inactivation of the HSV tk gene, whil e methylation of the cytosine residues within the nucleotide sequence from +811 to +1309 had no effect on HSV tk gene activity. We also meth ylated single HpaII: sites within the HSV tk gene using a specific met hylated primer for in vitro DNA synthesis. We found that of the 16 HSV tk HpaII sites, methylation of 6 single sites caused HSV tk inactivat ion. All six of these ''methylation-sensitive'' sites are within the c oding region, including the HpaII-6 site, which is 571 bp downstream f rom the transcription start site. The sites HpaII-7 to HpaII-16 were a ll methylation insensitive. We further inserted separately the methyla tion-sensitive HSV fk HpaII-6 site and the methylation-insensitive Hpa II-13 site as DNA segments (32-mer) into the intron region of the simi an virus 40 T antigen (TaqI site). Methylation of these HpaII sites ca used inhibition of simian virus 40 T-antigen synthesis.