Transcription of mouse rDNA and associated formation of the nucleolus organizer region after gene transfer and amplification in Chinese hamster cells.
Mouse rDNA can initiate transcription by using only Chinese hamster cell components, and this is associated with nucleolus organizer activity. To demonstrate this, we transferred a 3.2-kilobase segment of mouse rDNA containing the promoter, the transcription initiation site, and part of the external transcribed spacer to dihydrofolate reductase-deficient Chinese hamster cells by cotransformation with an abbreviated mouse dhfr gene. Stepwise selection for methotrexate resistance produced sublines in which the mouse rDNA was usually coamplified with the donor dhfr DNA and occupied the same site or sites in the hamster genome, as shown by in situ hybridization. Transcription from mouse rDNA was demonstrated in two such lines, and S1 protection mapping indicated faithful initiation of the transcript. In some cells from both lines, the chromosome segments containing amplified mouse rDNA showed multiple silver-staining regions (i.e., active nucleolus organizers). Although the transferred mouse rDNA was able to use the rDNA transcriptional machinery of the Chinese hamster, the level of transcription was much lower than expected from the rDNA copy number, and a large fraction of each amplified region showed no silver staining. Since the absence of silver staining is generally correlated with the absence of transcription, many copies of the amplified mouse rDNA may have been in a chromatin conformation in which they could not be transcribed. This was not associated with the extensive methylation seen in other amplified, inactive rDNA sequences.
D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D004262:DNA Restriction Enzymes; D004275:DNA, Ribosomal / Q000235:genetics*; D004351:Drug Resistance; D005784:Gene Amplification; D005796:Genes; D007621:Karyotyping; D008677:Metaphase; D008727:Methotrexate / Q000494:pharmacology; D051379:Mice; D008808:Mice, Inbred CBA; D009693:Nucleic Acid Hybridization; D009697:Nucleolus Organizer Region / Q000502:physiology / Q000648:ultrastructure*; D010957:Plasmids; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics; D014158:Transcription, Genetic
D004275:DNA, Ribosomal; D013762:Tetrahydrofolate Dehydrogenase; D004262:DNA Restriction Enzymes; D008727:Methotrexate