Cloning and determination of a putative promoter region of a mouse ribosomal deoxyribonucleic acid fragment.

An endonuclease EcoRI digest of mouse DNA was subjected to molecular cloning, after partial purification with respect to the ribosomal RNA sequence, using lambda gtWES x lambda B with an in vitro packaging technique. Twelve positive clones were obtained from approximately 2 X 10(4) plaques. One of the clones transferred to the plasmid pBR322 (PMrEL-1) was about 14.9 kb long, hybridizing only with 18S rRNA but not with 28S rRNA. Hybridization of restriction fragments and electron microscopic studies of the R-loop confirmed that this fragment carried about half of the 18S rRNA sequences at one end, suggesting that it contained the initiation site for the 45S preribosomal RNA (pre-rRNA). S1-nuclease protection mapping with hybrids between restriction fragments of the cloned DNA and the 45S pre-rRNA indicated that at least major transcription of the 45S RNA started at a site approximately 4.0 kb upstream from the 5' end of the 18S rRNA. This was confirmed by electron microscopic observations of these hybrids.