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Fine structure of the 21S ribosomal RNA region on yeast mitochondria DNA. I. Construction of the physical map and localization of the cistron for the 21S mitochondrial ribosomal RNA.

1. We have used restriction enzyme analysis of petite mtDNAs to construct a detailed physical map of the 21S region on the mtDNA of the Saccharomyces cerevisiae strain JS1-3D. The map covers a segment of about 20,000 bp, on which the recognition sites of the enzymes HapII, HindII, HindIII, Sa1I, XhoI and HhaI have been localized (22 sites in total). This map has been checked in various ways against the independently constructed overall physical map of the mtDNA of strain JS1-3D. In addition, we have constructed a physical map with a resolution of about 200 bp of a HapII fragment of 1850 bp long, which carries the loci omega, RIB-1 and probably RIB-2. 2. The 21S rRNA hybridizes with the five adjacent HindII + III fragments TD9, DT19, TD15, DT14 and TT1, which lie in that order on the physical map of the 21S region. Of these, the two non-adjacent fragments TD9 and DT14 show a much stronger hybridization with 21S rRNA than DT19, TD15, and TT1. 3. The fragment DD5 (= DT19 + TD15) and part of DT14 belong to a sequence of about 1000 bp, which is absent from Saccharomyces carlsbergensis mtDNA. Although DD5 and DT14 show (very weak, respectively stronger) hybridization with 21S rRNA, the 1000 bp insert probably does not code for the 21S rRNA: the 21S rRNA of S. carlsbergensis comigrates with the 21S rRNA of JS1-3D on polyacrylamide gels under denaturing conditions. 4. Fragment DT14 hybridizes with the HindII + III fragment TD9, which shows the strongest hybridization with 21S rRNA. The presence of these sequence homologies has hampered the precise mapping of the 21S rRNA cistron. Our results are compatible, however, with the hypothesis that the sequences, coding for 21S rRNA, are located on HindII + III fragments that are not adjacent on JS1-3D mtDNA, namely TD9, DT14 and TT1.

DNA Restriction Enzymes↗

RNA, RIBOSOMAL/*BIOSYNon of ribosomal 5S RNA genes in Chironomus thummi by in situ hybridization of iodinated 5S RNA.

5 S RNA of Chironomus thummi larvae was purified from total phenol extracted RNA by gel filtration and labelled to about 10(7) dpm/mug with carrier-free iodine-125. After hybridization in situ of 125I-5 S RNA and autoradiography only region B3c-e (containing two "normal" and two very faint bands) of chromosome II of salivary gland cells was highly labelled. In chromosomes of an animal showing pairing discontinuities a clearly "heterozygous" labelling of the 5 S RNA region was found. Region B3c-e shows no clearcut morphological signs of puffing or autoradiographically detectable 3H-uridine incorportion in spite of a continuous synthesis of 5 S RNA in salivary gland cells.

Animals↗