GENOMIC SEQUENCING REVEALS ABSENCE OF DNA METHYLATION IN THE MAJOR LATE PROMOTER OF ADENOVIRUS TYPE-2 DNA IN THE VIRION AND IN PRODUCTIVELYINFECTED-CELLS

By using methylation-sensitive restriction endonucleases, we have prev iously provided evidence that adenovirus type 2 (Ad2) virion DIVA or f ree intranuclear Ad2 DNA in productively infected hamster or human cel ls is not methylated. We have now chosen a different experimental appr oach and have investigated the major late promoter (MLP) sequence of A d2 DNA for the presence of 5-methyldeoxycytidine (5-mC) residues with the genomic sequencing technique. This study has been prompted by the finding that the MLP of Ad2 DNA can be inactivated by sequence-specifi c methylation in experiments in which a MLP-chloramphenicol acetyltran sferase construct has been transcribed in a cell-free system from HeLa cell nuclear extracts. Virion Ad2 DIVA and Ad2 DNA isolated from prod uctively infected human or hamster cells between 1 and 48 h postinfect ion (p.i.) have now been analyzed, There is no evidence for the presen ce of 5-mC in the cytidine positions in the MLP of any of these Ad2 pr eparations. We conclude that DNA methylation does not seem to play a r ole in the early-late control of this viral promoter. The sensitivity of the genomic sequencing technique does not permit us to exclude the unlikely presence of 5-mC in a few Ad2 DNA molecules.