Waechter D E, Baserga R
Proc Natl Acad Sci U S A. 1982 Feb;79(4):1106-10. doi: 10.1073/pnas.79.4.1106.
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.
将猿猴病毒40大T抗原基因和单纯疱疹病毒（HSV）胸苷激酶（TK）基因用EcoRI甲基化酶进行甲基化处理。将这些基因显微注射到TK缺陷（tk-）细胞的细胞核中，通过对猿猴病毒40大T抗原进行免疫荧光染色以及对HSV TK进行[3H]胸苷掺入后放射自显影来确定基因的表达情况。我们发现，在EcoRI或EcoRI*条件下，猿猴病毒40基因的甲基化导致基因内部位点及周围序列发生甲基化，当该基因被手动显微注射到哺乳动物细胞核中时，对大T抗原的表达没有影响。然而，HSV tk基因在两个EcoRI位点的甲基化显著降低或消除了该基因的表达。HSV tk的一个EcoRI位点位于基因3'端下游约1.1千碱基处，据信在tk基因的表达中没有调节功能。另一个EcoRI位点位于基因5'端上游79个碱基对处，与[贝诺伊斯特C.、奥黑尔K.、布雷思纳克R.和尚邦P.（1980年）《核酸研究》8，127 - 142]提出的调节序列有相当高的同源性。我们的结果直接证明甲基化可以改变基因表达，而且这种影响严格取决于甲基化的位点。
