Christa Heyting

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).

Two major components of synaptonemal complexes are specific for meiotic prophase nuclei

Monoclonal antibody II52F10 was elicited against purified synaptonemal complexes (SCs); it recognizes two major components of the lateral elements of SCs, namely an Mr=30 000 and an Mr=33000 protein. We studied the distribution of the antigens of II52F10 within tissues and cells of the male rat by immunoblot analysis and immuno-cytochemical techniques. Nuclear proteins from various cell types, including spermatogonia and spermatids, did not react with antibody II52F10 on immunoblots; the same holds for proteins from isolated mitotic chromosomes. As expected, an Mr=30 000 and an Mr=33 000 protein from spermatocyte nuclei did react with the antibody. In cryostat sections of liver, brain, muscle and gut we could not detect any reaction with II52F10. In the testis the reaction was confined to SCs or SC fragments. Partly on the basis of indirect evidence we identified the antigen-containing cells as zygotene up to and including post-diffuse diplotene spermatocytes. The persistence of some antigen-containing fragments in the earliest stages of spermatids could not be excluded. We conclude that the lateral elements (LEs) of SCs are not assembled by rearrangement of pre-existing components of the nucleus: at least two of their major components are newly synthesized, presumably during zygotene. Furthermore we conclude partly from indirect evidence that the major components of the LEs of SCs are not involved in the chromosome condensation processes that take place during the earliest stages of meiotic prophase.

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.

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.

Hybridization of N-acetoxy-N-acetyl-2-aminofluorene-labelled RNA to Q-banded metaphase chromosomes

This paper describes improvements of a recently developed immunocytochemical method for the detection of specific polynucleotide sequences within chromosomes, as well as conditions by which this method can be combined with chromosome banding. The immunocytochemical method involves modification of polynucleotide probes with N-acetoxy-N-acetyl-2-aminofluorene (AAAF)2), and hybridization of the modified probes with metaphase chromosomes; the hybrids are made visible immunocytochemically by means of an antiserum which recognizes AAAF-induced polynucleotide modifications. We have sorted out conditions which allow a high sensitivity of hybrid detection by the above procedure, in combination with chromosome banding. The best results are obtained if the ABC-technique is used for the visualization of the hybrids; the lower limit of detection is estimated to be a sequence of about 7000 nucleotides. The method can be combined with Q-banding of chromosomes, if this is performed not more than 1 day prior to hybridization, and if excitation of the Q-banded chromosomes is kept to a minimum.

The organization of repeating units in mitochondrial DNA from yeast petite mutants

SummaryWe have reinvestigated the linkage orientation of repeating units in mtDNAs of yeast ρ− petite mutants containing an inverted duplication. All five petite mtDNAs studied contain a continuous segment of wild-type mtDNA, part of which is duplicated and present in inverted form in the repeat. We show by restriction enzyme analysis that the non-duplicated segments between the inverted duplications are present in random orientation in all five petite mtDNAs. There is no segregation of sub-types with unique orientation. We attribute this to the high rate of intramolecular recombination between the inverted duplications. The results provide additional evidence for the high rate of recombination of yeast mtDNA even in haploid ρ− petite cells.We conclude that only two types of stable sequence organization exist in petite mtDNA: petites without an inverted duplication have repeats linked in straight head-to-tail arrangement (abcabc); petites with an inverted duplication have repeats in which the non-duplicated segments are present in random orientation.

[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.