Hypomethylation of HeLa cell DNA and the absence of 5-methylcytosine in SV40 and adenovirus (type 2) DNA: analysis by HPLC

Methylation of the purified virion DNA of both SV40 and adenovirus (type 2) was measured by high performance liquid chromatography (HPLC) and compared to their hosts, African green monkey kidney cells and Hela cells, respectively. Essentially, no 5-methylcytosine was detected in either viral DNA. Implications for viral gene regulation by methylation are discussed. In comparison with normal human cell DNA methylation levels, HeLa cell DNA methylation is reduced significantly.     

Expression of adenovirus type 12 E1A gene in monkey cells, using a simian virus 40 vector.

Simian virus 40 (SV40) recombinants carrying the adenovirus type 12 E1A gene were constructed. The SV40 expression vector was constructed by removing most of the VP1 gene and an internal part of the intervening sequence for late 16S RNA and by joining the 5' and 3' splice sites into a small segment. The adenovirus type 12 E1A gene with or without its own promoter was inserted downstream from the SV40 late promoter and the splicing junctions. The recombinant DNA was propagated and packaged in monkey cells by cotransfection with an early temperature-sensitive mutant (tsA58) DNA as helper. Immunofluorescent staining of the monkey cells infected with the resulting virus stocks showed that up to 20% of the cells overproduced the E1A gene products in the nuclei. Two-dimensional gel electrophoresis of the products indicated that the products were very similar or identical to the authentic polypeptides synthesized in adenovirus type 12-infected human embryo kidney cells. The E1A mRNA was initiated at the SV40 late promoter irrespective of the presence of the E1A promoter and terminated at either the E1A or the SV40 polyadenylation signal. These hybrid mRNAs were correctly spliced in the E1A coding region.