The cloned genes for the simian virus 40 large tumor antigen and for herpes simplex virus (HSV) thymidine kinase (TK) were methylated with EcoRI methylase. The genes were microinjected into the nuclei of TK-deficient (tk-) cells, and expression of the genes was determined by immunofluorescence staining for the simian virus 40 large tumor antigen and by [3H]thymidine incorporation followed by autoradiography for HSV TK. We found that methylation of the simian virus 40 gene, under EcoRI Recently, a number of observations have suggested that methylation may be involved in gene expression (for review, see refs. 1-3). Although the results have not always been devoid of contradictions, evidence has been accumulating that nonexpressed genes are more heavily methylated than genes that are being expressed (4)(5)(6)(7)(8)(9)(10). A direct approach to the problem is to methylate cloned genes and microinject them manually into the nuclei ofmammalian cells in which their expression can be studied (11-13). We report here that methylation of the simian virus 40 (SV40) A gene at up to 24 different sites has no effect whatsoever on its expression, as measured by immunofluorescence, when it is microinjected into mammalian cells. On Dulbecco's minimal essential medium/10% fetal calf serum. Cells used for microinjection were plated on small cover glasses marked with small circles. Microinjection was carried out as described by Graessmann and Graessmann (11) under direct visual control on a fixed stage of an inverted phase-contrast microscope (Leitz Diavert 400) with a micromanipulator.Recombinant Plasmids. pSV2G, a plasmid constructed in our laboratory and containing the early region of SV40, extending from map unit 1 counterclockwise to map unit 0.14, has been described and characterized (12). This plasmid has been shown to induce positive immunofluorescence for the large tumor (T) antigen and cellular DNA synthesis when microinjected into ts13, and 3T3 cells (12).pTK, aplasmid containing the BamHI fragment ofthe tk gene of HSV cloned in pBR322, was generously given by Carlo Croce, The Wistar Institute, and it has been described in a paper from his laboratory (15). The third plasmid, pC6, which contains the full SV40 DNA and the HSV tk gene in pBR322, was also obtained from Carlo Croce and has been described in the same paper (15).Plasmids were grown in HB101 cells. After amplification with chloramphenicol, plasmid DNA was isolated and purified on a CsCl gradient (16). Enzymes and Reagents. Restriction enzymes and EcoRI methylase were purchased from New England BioLabs or from Bethesda Research Laboratories. DNA Modification. Restriction or methylation of plasmid DNA at the canonical EcoRI sites (G-A-A-T-T-C), was done as suggested by the suppliers of the enzymes.Methylation of plasmid DNA at the noncanonical EcoRI* sites (N-A-A-T-T-N') was done as described by Woodbury et al. (17). Plasmid DNA (15-20 ,ug) 1106The publication costs ofthis article were defrayed in part by page charge payment. This article must ther...

Different domains of the SV40 A gene have different functions, such as viral DNA replication, cell DNA replication, and stimulation of cellular RNA synthesis. The sequences in the SV40 A gene that are critical for the induction of cell DNA synthesis lie on the map between nucleotide 4360 and nucleotide 4001, a stretch of 360 nucleotides coding for 120 of the 708 amino acids of the large T antigen. The sequences critical for stimulation of rRNA synthesis lie on the map further downstream, between nucleotides 3827 and 3506, thus indicating that the signals for growth in size and for cell DNA replication can be dissociated. Methylation of the SV40 A gene at multiple ECoRI* sites has no effect on its expression. However, methylation of the HSV-TK gene at one single ECoRI site 70 base pairs upstream from the cap site inhibits its expression. The results indicate that methylation of genes affects their expression, but only when methylation occurs at specific sites.

tsAF8 cells are a temperature-sensitive (ts) mutant of BHK cells that arrest in the G1 phase of the cell cycle at the non-permissive temperature of 40.6 degrees C. Previous reports had suggested that the temperature-sensitivity of these cells was based on a defect in either the synthesis, assembly or turnover of RNA polymerase II. We now show that the direct microinjection of purified RNA polymerase II into nuclei of tsAF8 cells corrects the ts defect and allows these cells to enter the S phase of the cell cycle.

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