The gene for the small ribosomal RNA on yeast mitochondrial DNA: Physical map, direction of transcription and absence of an intervening sequence

Abstract

1. We have constructed a physical map of the yeast mtDNA region coding for the small RNA component of the mitochondrial ribosome. The map includes restriction sites for HindII, BamHI, MboI, MboII, HapII, HaeIII and Sau-961. 2. We have cloned Mbol mtDNA fragments containing parts of the 15S ribosomal gene in the BamHI site of the plasmid pBR325, using standard recombinant DNA techniques. With the exception of one border fragment, more than 5,800 base pairs long, all four MboI DNA fragments were cloned. 3. We have digested DNA fragments, containing part of the 15S ribosomal gene at one end only, with the 3'-specific exonuclease III of Escherichia coli and the 5'-specific exonuclease of bacteriophage lambda in opposite directions with respect to the physical map. From the hybridization of (32)P-labelled 15S RNA to the single-stranded ends of these DNA fragments we conclude that transcription of this gene runs clock-wise on the map (i.e. from oxi-2 towards oxi-3). 4. We have studied the position of the 15S ribosomal gene on the physical map with the S1 nuclease mapping procedure of Berk and Sharp (Cell 12 (1977) 721-732). It is asymmetrically localized on a 2,000 base pair HapII fragment and the length of the protected hybrids adds to approximately 1,600 base pairs. 5. We have found no evidence for an intervening sequence in the 15S ribosomal gene, since S1 nucleasetreated hybrids, analysed by electrophoresis in alkaline gels, showed no indication for interruptions in the DNA strand of the hybrid.