Amplified ribosomal RNA genes in a rat hepatoma cell line are enriched in 5-methylcytosine.

Abstract

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-'4C]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.Genes for mammalian 18S and 28S rRNA (rDNA) occur in multiple copies that are clustered at one or more sites per haploid genome. These sites can be visualized by in situ hybridization (1, 2) or a silver staining technique (3,4 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.489

5‐Methylcytosine in genes with methylation‐dependent regulation

“…The second observable fact was a striking intermittence of dcm-enriched and dcm-poor regions along all genes. This also conformed with the demonstratioI1 that methylation preferentially occurs on regulatory sequences that do not code for mRNAs, while the coding sequences are SmC-poor [1,6]. Fig.…”

Changes in DNA methylation during development in the B chromosome NOR of the grasshopper Eyprepocnemis plorans

“…From the first demonstration that DNA methylation may regulate the activity of the rRNA genes in a rat hepatoma cell line (Tantravahi et a!., 1981), the phenomenon has been extended to other organisms such as, for instance, peas (Watson et a!., 1987), wheat (Flavell et a!., 1988), frogs (Ruiz & Brison, 1989) and mice (Suzuki et a!., 1992). Interestingly, DNA methylation also seems to be involved in the inactivation of extra rRNA genes contained in a supernumerary chromosome segment that has arisen from amplification of a nucleolus organiser region (NOR) in the grasshopper Pyrgomorpha conica (Suja eta!., 1993).…”

Transcription of mouse rDNA and associated formation of the nucleolus organizer region after gene transfer and amplification in Chinese hamster cells.

“…The number of rRNA genes and the size of the silver-stained NORs (AgNORs) generally parallel the level of nucleolus organizer activity and, presumably, the rRNA transcriptional activity (24,40). Silver staining is restricted to potentially active NORs; it is absent from the nonexpressed rDNA clusters on chromosomes of one species in-rodent-human hybrid cells (7,27,32) and is also absent from homogeneously staining chromosomal regions containing greatly amplified and hypermethylated rDNA (28,37).…”

“…The number of rRNA genes and the size of the silver-stained NORs (AgNORs) generally parallel the level of nucleolus organizer activity and, presumably, the rRNA transcriptional activity (24,40). Silver staining is restricted to potentially active NORs; it is absent from the nonexpressed rDNA clusters on chromosomes of one species in-rodent-human hybrid cells (7,27,32) and is also absent from homogeneously staining chromosomal regions containing greatly amplified and hypermethylated rDNA (28,37).Transcription of rDNA requires both RNA polymerase I and several protein cofactors; the cofactors from rodent cells do not support in vitro transcription from human rDNA and vice versa (13, 20, 23, 31 predominates (8,26,41,42), and it is unclear whether mouse and hamster rDNAs can use the same transcription cofactors.In this paper, we show that mouse rDNA can be transcribed by using only Chinese hamster components. We describe the transfer of a cloned mouse rRNA gene fragment into dihydrofolate reductase-deficient (dhfr-) hamster cells by DNA-mediated cotransformation with a constructed dhfr gene (9), coamplification of the two genes by stepwise selection of methotrexate-resistant (MTXr) sublines, faithful transcription of some of the rDNA fragments, and silver staining of a fraction of this rDNA chromatin.…”