Johan P. M. Sanders

The organization of genes in yeast mitochondrial DNA

Summary1)We have constructed independent physical maps of the mtDNAs from three different wild-type Saccharomyces strains by double-digestion analysis and hybridization analysis, using restriction endonucleases EcoRI, HindII, HindIII, PstI, BamHI, Aval, HhaI, SalI and XhoI. Twentynine restriction enzyme sites have been localized on the mtDNA of Saccharomyces carlsbergensis, 47 on the mtDNA of Saccharomyces cerevisiae strain JS1-3D and 38 on the mtDNA of Saccharomyces cerevisiae strain KL14-4A.2)Although the three DNAs show considerable differences in their fragmentation patterns with most nucleases tested, the overall sequence organization of the three maps of 30–40 fragments is identical. Differences in the maps can be explained by extra restriction enzyme recognition sites, possibly located on inserted pieces of DNA and by insertions and deletions.3)Four major insertions (900, 1,500, 2,600 and 3,000 bp long) are found in KL14-4A mtDNA relative to Saccharomyces carlsbergensis mtDNA. These insertions are clustered in one quadrant of the mtDNA and account for the difference in size of these two mtDNAs (75,750 and 68,000 bp, respectively).

The organization of genes in yeast mitochondrial DNA. I. The genes for large and small ribosomal RNA are far apart

Summary We have determined the position of the two rRNA cistrons on the physical map of the mtDNA of Saccharomyces carlsbergensis obtained with restriction endonucleases. Hybridization of 125 I-labelled rRNA with DNA fragments of known location on the map shows that the two rRNA cistrons are at least 25 000 base pairs apart on this DNA of 70 000 base pairs.

Nature of the base sequence conserved in the mitochondrial DNA of a low-density petite

Abstract 1. 1. We have previously shown that the mtDNA of the ethidium-induced cytoplasmic petite mutant RD1A of the yeast Saccharomyces cerevisiae consists of perfect tandem repetitions of a very (A+T)-rich sequence of base pairs. 2. 2. The complementary strands of RD1A mtDNA were separated in alkaline CsCl and used for DNA · DNA hybridization with wild-type mtDNA and other petite mtDNAs. Hybridization was analysed either by standard filter techniques or with purified S1 nuclease from Aspergillus oryzae, which is specific for single-stranded DNA. The hybridization plateau of 0.3 % indicates that one copy of the repeating sequence of RD1A mtDNA is present in wild-type mtDNA, but the presence of up to 3 copies could not be excluded. 3. 3. The T m of the heteroduplex of wild-type and RD1A mtDNA on hydroxylapatite is 6 °C lower than that of the RD1A mtDNA homoduplex. This shows that some miscopying must have occurred during the initial mutagenic event that gave rise to RD1A mtDNA. The RD1A mtDNA sequence is also present in mtDNA of Saccharomyces carlsbergensis but in this case the T m of the heteroduplex is 13 °C below that of the homoduplex. 4. 4. No sequence homology was detected between mtDNA of RD1A and the repetitive (A+T)-rich mtDNAs of two other petite mutants, either in DNA · DNA hybridization or in DNA · RNA hybridization using complementary RNA made with RNA polymerase of Escherichia coli. This lack of homology was confirmed by the fingerprints of the complementary RNAs of the three DNAs. These results indicate that the initiation site for DNA synthesis does not form part of the repeating sequence of these mtDNAs. 5. 5. The analogy between the repetitive petite mtDNAs and the repetitive nuclear satellite DNAs of animal tissues, is stressed.

Fine structure of the 21S ribosomal RNA region on yeast mitochondrial DNA

Summary1We 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, SalI, 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 ω, 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.

Fine structure of the 21S ribosomal RNA region on yeast mitochondrial 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.

Properties of mitochondrial DNA from Kluyveromyces lactis

Abstract 1. We have isolated a closed circular DNA fraction from mitochondria purified from Kluyveromyces lactis by centrifuging a mitochondrial lysate to equilibrium in CsCl containing ethidium bromide. Electron micrographs of appropriate gradient fractions show predominantly circular duplex DNA with an average contour length ( ± S.D. ) of 11.4 (± 0.5) μ m . The circular DNA has the same buoyant density as mtDNA in NaI gradients, but represented only up to 6% of total mtDNA. 2. Denatured total mtDNA renatures with a kinetic complexity of about 20 · 106, a value consistent with the length observed in the electron microscope. 3. mtDNA from Kluyveromyces lactis hybridizes about twice as much mitochondrial rRNA isolated from Saccharomyces carlsbergensis than the equal amount of Saccharomyces carlsbergensis mtDNA (contour length 25 μm). 4. We conclude that intact mtDNA of Kluyveromyces lactis consists of a circular molecule with a contour length of 11.4 μm and a complexity equivalent to this size.

[18] Biochemical methods to locate genes on the physical map of yeast mitochondrial DNA

Publisher Summary This chapter discusses biochemical methods to locate genes on the physical map of yeast mitochondrial DNA. The mtDNA of the yeasts Saccharomyces carlsbergensis and Saccharomyces cerevisiae is a circular duplex DNA with a molecular weight of about 50 x 10 6 . Specific fragments of this DNA have been made with restriction endonucleases and the fragments have been positioned by conventional mapping techniques in a circular, “physical” fragment map. This map can be used to locate the position of various genes on the mtDNA. It reviews that genes for mtRNAs can be located on the map by hybridizing labeled RNAs to purified DNA fragments. Genes for mitochondrial tRNAs are located by loading the tRNAs with their cognate amino acid and hybridizing this aminoacyl tRNA labeled in the amino acid moiety to purified DNA fragments. Genetic markers can be located on the map by the use of cytoplasmic petite mutant mtDNA. Finally, the fragment map provides a method to analyze sequence variations in yeast mtDNA and to determine the positions of variable and constant sequences in this DNA in relation to structural and regulatory genes. Most of the technology involved in mtDNA mapping is routine in the mapping of viral and bacterial genes, and there is no use in repeating a description of this technology.

The construction of the physical maps of three different Saccharomyces mitochondrial DNAs

In this Appendix we shall present the construction of the physical maps of the mtDNAs isolated from Saccharomyces carIsbergensis NCYC74, Saccharomyces cerevisiae JS1-3D and KL14-4A. The three maps have been independently constructed, using the data from Tables 1, 2 and 4 of the preceding article. To save space we follow only a single line of reasoning for the construction of each map. The fragment orders deduced are usually based on several independent lines of reasoning, however, offer using additional evidence not included here. A more extensive version of this Appendix, which includes the additional evidence, is available on request. The new nomenclature of the fragments is given in the Methods of the preceding article.