Effect of methylation on expression of microinjected genes.

Abstract

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...

5-Methylcytosine depletion during tumour development: An extension of the miscoding concept

“…Analysis of the methylation pattern of the viral thymidine kinase (TK) gene in Herpes simplex virus (HSV)-transformed mouse cells showed that when the gene was being actively transcribed it was unmethylated, when inactive it was methylated, and when induced to activity by 5-AzaCyt it was again unmethylated. This finding was extended by Waechter & Baserga (1982) who found that when the cloned gene for HSV-TK was methylated with Eco RI methylase and microinjected into the nucleus of TK"-) cells, methylation at particular sites markedly reduced or abolished the expression of the gene. These authors pointed out the possibility that different genes might respond differently to methylation, since similar Eco RI methylation of the gene coding for Simian virus 40 T antigen had no effect upon its expression after microinjection.…”

Changes in structure and methylation pattern in a cluster of thymidine kinase genes.

“…Studies with inhibitors of DNA methylation suggest that the inhibition of DNA methylation increases the frequency of expression of methylated, inactive genes in vivo (2,4,9,20), and it has been demonstrated that methylated DNA is expressed more poorly than unmethylated DNA after transfer into mammalian cells (18,25,33, 36).…”

Phenotypic variation associated with molecular alterations at a cluster of thymidine kinase genes.

“…When methyladenine was placed at the EcoRI sites with EcoRI methylase and the gene was then microinjected into L cells, TK expression was reduced (35). The authors (35) pointed out that one of the EcoRI sites, the same one we observed to be methylated de novo, was the CCAAT box for the TK gene. This sequence is thought to be an element of the promoter (2,7).…”

“…When the gene was methylated at CCGG by HpaII methylase and then was transferred into L cells, the frequency of TK+ transformants was reduced, but not to zero (25,39). When methyladenine was placed at the EcoRI sites with EcoRI methylase and the gene was then microinjected into L cells, TK expression was reduced (35). The authors (35) pointed out that one of the EcoRI sites, the same one we observed to be methylated de novo, was the CCAAT box for the TK gene.…”