Langner K D, Vardimon L, Renz D, Doerfler W
Proc Natl Acad Sci U S A. 1984 May;81(10):2950-4. doi: 10.1073/pnas.81.10.2950.
The E2a gene of human adenovirus type 2 (Ad2) encodes the 72-kilodalton DNA-binding protein. We previously described perfect inverse correlations between the methylation of all 5' C-C-G-G 3' sequences in the Ad2 E2a gene in virus-transformed hamster cells containing viral DNA sequences in an integrated state and the extent to which this gene is expressed. We subsequently showed that in vitro methylation of all 14 5' C-C-G-G 3' sequences in the cloned E2a gene by prokaryotic Hpa II DNA methyltransferase leads to transcriptional inactivation after microinjection into Xenopus laevis oocytes. The unmethylated cloned E2a gene is expressed in these cells. We report here the construction of partly methylated clones of the E2a gene. In the promoter (5')-methylated construct, three 5' C-C-G-G 3' sequences at the 5' end of the subclone were methylated. One of these sites is located 215 base pairs (bp) upstream (bp 26,169 of Ad2 DNA), and two sites are located 5 and 23 bp downstream from the cap site (bp 25,931 and 25,949 of Ad2 DNA) of the E2a gene. This construct was transcriptionally inactive upon microinjection into nuclei of X. laevis oocytes. In the gene (3')-methylated construct, 11 5' C-C-G-G 3' sequences in the main part of the transcribed gene region were methylated in vitro. This construct was transcribed in X. laevis oocytes, and at least some of the Ad2-specific RNA synthesized was initiated at the same sites as in Ad2-infected human KB cells. Both mock-methylated constructs were transcribed into Ad2-specific RNA in X. laevis oocytes. These results demonstrate that DNA methylations at or close to the promoter and 5' end of the E2a gene cause transcriptional inactivation. Perhaps only one methyl group would be adequate for inactivation; in vivo methylation of more than one cytosine may be a form of safeguard or redundancy.
人腺病毒2型（Ad2）的E2a基因编码72千道尔顿的DNA结合蛋白。我们之前描述过，在整合状态下含有病毒DNA序列的病毒转化仓鼠细胞中，Ad2 E2a基因中所有5' C-C-G-G 3'序列的甲基化与该基因的表达程度之间存在完美的负相关。我们随后表明，原核Hpa II DNA甲基转移酶对克隆的E2a基因中所有14个5' C-C-G-G 3'序列进行体外甲基化后，显微注射到非洲爪蟾卵母细胞中会导致转录失活。未甲基化的克隆E2a基因在这些细胞中表达。我们在此报告E2a基因部分甲基化克隆的构建。在启动子（5'）甲基化构建体中，亚克隆5'端的三个5' C-C-G-G 3'序列被甲基化。其中一个位点位于上游215个碱基对（Ad2 DNA的第26,169位碱基对），两个位点位于E2a基因帽位点（Ad2 DNA的第25,931和25,949位碱基对）下游5和23个碱基对处。将该构建体显微注射到非洲爪蟾卵母细胞核中后转录无活性。在基因（3'）甲基化构建体中,转录基因区域主要部分的11个5' C-C-G-G 3'序列在体外被甲基化。该构建体在非洲爪蟾卵母细胞中被转录，并且合成的至少一些Ad2特异性RNA与Ad2感染的人KB细胞中的起始位点相同。两个模拟甲基化构建体在非洲爪蟾卵母细胞中都被转录为Ad2特异性RNA。这些结果表明，E2a基因启动子及5'端或其附近的DNA甲基化会导致转录失活。也许只需一个甲基就足以导致失活；体内多个胞嘧啶的甲基化可能是一种保障或冗余形式。
