Open AccessEffect of methylation on expression of microinjected genes.
Author(s) -
Dieter E. Waechter,
Renato Baserga
Publication year - 1982
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.79.4.1106
Subject(s) - ecori , microbiology and biotechnology , biology , methylation , gene , gene expression , thymidine kinase , dna methylation , cpg site , virus , herpes simplex virus , genetics , restriction enzyme
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 or EcoRI* conditions, resulting in methylation at sites within the gene and in the surrounding sequences, has no effect on expression of the large tumor antigen when the gene is manually microinjected into mammalian nuclei. However, methylation of the HSV tk gene at the two EcoRI sites markedly reduces or abolishes the expression of this gene. One of the EcoRI sites of HSV tk is approximately 1.1 kilobases downstream from the 3' end of the gene and is believed to have no regulatory function in the expression of the tk gene. The other EcoRI site is 79 base pairs upstream from the 5' end of the gene and has considerable homology to the regulatory sequence proposed by [Benoist C., O'Hare, K., Breathnach, R., & Chambon, P. (1980) Nucleic Acids Res. 8, 127-142]. Our results are direct proof that methylation can alter gene expression and also that the effect depends strictly on the sites that are methylated.