Tantravahi U, Guntaka R V, Erlanger B F, Miller O J
Proc Natl Acad Sci U S A. 1981 Jan;78(1):489-93. doi: 10.1073/pnas.78.1.489.
In a rat hepatoma cell line, H4-IIE-C3, a 10-fold excess of 18S and 28S rRNA genes has been found in amplified chromosome regions. Antibodies to 5-methylcytidine bound extensively to the DNA of these regions, indicating a high level of DNA methylation. Most of the amplified rRNA genes were transcriptionally inactive, as shown by their failure to stain with silver. DNAs from the tumor cells and control rat hepatocytes grown with L-[methyl-14C]methionine were digested with restriction endonuclease EcoRI; the DNA fragments were separated by agarose gel electrophoresis, denatured, transferred to nitrocellulose filters, and hybridized to 32P-labeled rRNA or cDNA. Fragments containing the 18S or 28S rRNA coding sequences occurred in three major size classes; all three were rich in 5-methylcytosine. Analysis of EcoRI fragments of DNA from the tumor and control cells after digestion with Hpa II or Msp I endonuclease indicated that the 5'-C-C-G-G-3' sequences in most of the amplified rRNA genes were methylated. Analysis of the fragments produced by digestion with Hha I endonuclease indicated a high degree of methylation within its recognition sequence in the amplified rRNA genes as well. The association of hypermethylation with restricted transcriptional activity suggests that DNA methylation may regulate the activity of the rRNA genes.
在大鼠肝癌细胞系H4-IIE-C3中，已在扩增的染色体区域发现18S和28S rRNA基因过量10倍。与5-甲基胞嘧啶的抗体广泛结合这些区域的DNA，表明DNA甲基化水平很高。如银染显示，大多数扩增的rRNA基因转录无活性。用L-[甲基-14C]甲硫氨酸培养的肿瘤细胞和对照大鼠肝细胞的DNA用限制性内切酶EcoRI消化；DNA片段通过琼脂糖凝胶电泳分离，变性，转移到硝酸纤维素滤膜上，并与32P标记的rRNA或cDNA杂交。含有18S或28S rRNA编码序列的片段出现在三个主要大小类别中；所有这三个类别都富含5-甲基胞嘧啶。用Hpa II或Msp I内切酶消化后对肿瘤细胞和对照细胞DNA的EcoRI片段分析表明，大多数扩增的rRNA基因中的5'-C-C-G-G-3'序列被甲基化。用Hha I内切酶消化产生的片段分析表明，扩增的rRNA基因中其识别序列内也存在高度甲基化。高甲基化与受限转录活性的关联表明DNA甲基化可能调节rRNA基因的活性。
