Specific transcription of mouse ribosomal DNA in a cell-free system that mimics control in vivo

Abstract

Cloned ribosomal DNA (rDNA) from mouse, which contains the initiation site of 45S pre-rRNA transcription and 5' flanking sequences, has been used as the template in an in vitro transcription system. In the presence of extracts from rapidly growing Ehrlich ascites cells, RNA polymerase I initiates specifically in that region of purified rDNA where the 5' end of 45S rRNA has been mapped. This is shown by electrophoretic analysis of the length of run-off transcripts synthesized from truncated templates, by S1 nuclease mapping, and by hybridization analysis of the in vitro products. The ability of the crude extracts to promote faithful transcription of mouse rDNA correlates with the proliferation rate of the cells. Only extracts prepared from exponentially growing mouse cells contain the factor(s) required for the faithful transcription of mouse ribosomal genes. Extracts from nongrowing or slowly growing mouse cells show very little activity. Thus, the cell-free system somehow reflects the rRNA synthetic activity of the cell and will prove valuable for the identification and purification of the various factors that are involved in the specific read-out of rDNA and may play a central role in the regulation of transcription of the ribosomal genes.