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

Abstract

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 .cntdot. .lambda.B with an in vitro packaging technique. Twelve positive clones were obtained from approximately 2 .times. 104 plaques [of Escherichia coli]. One of the clones transferred to the plasmid pBR322 (pMrEL-1) was about 14.9 kb[kilobase]/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 EM observations of these hybrids.