A controversy concerning the structure of the mitochondrial genome of a yeast petite mutant: resolution by sequence analysis

Summary Sequence analysis was used to define the repeat unit that constitutes the mitochondrial genome of a petite (rho ^–) mutant of the yeast Saccharomyces cerevisiae. This mutant has retained and amplified in tandem a 2,547 by segment encompassing the second exon of the oxi3 gene excised from wild-type mtDNA between two direct repeats of 11 nucleotides. The identity of the mtDNA segment retained in this petite has recently been questioned (van der Veen et al., 1988). The results presented here confirm the identity of this mtDNA segment to be that determined previously by restriction mapping (Carignani et al., 1983).     

Polymorphic variations in the ori sequences from the mitochondrial genomes of different wild-type yeast strains

Summary We determined the restriction maps and primary structures of two as yet poorly characterized regions of the mitochondrial genomes of different wildtype strains of Saccharomyces cerevisiae. These regions respectively comprised the ori1 sequence and the newly identified ori8 sequence. Ori1 and ori8, together with their flanking sequences, exhibit a large polymorphism, resulting from specific variations due to insertions or deletions of optional GC clusters at different locations. The mechanisms underlying such sequence rearrangements are discussed.     

The in vivo effect of acriflavine on mitochondrial functions in the petite negative yeast Hansenula saturnus

Summary In the petite negative yeast Hansenula saturnus, acriflavine determined a decrease of cell yield and of the total Q_O2,the disappearance of the cytochromes aa ₃ and b and the inhibition of in vivo mitochondrial protein synthesis without affecting the cell survival. The restriction enzymes analysis of mitDNA shows that no specific fragmentation occurred after acriflavine treatment.     

Effect of ethidium bromide on transmission of mitochondrial genomes and DNA synthesis in the petite negative yeast Schizosaccharomyces pomhe

Summary Treatment of haploid strains of the petite negative yeast Schizosaccharomyces pomhe with ethidium bromide prior to mating with untreated cells reduces transmission of mitochondrial markers from the treated strains. This effect is fully reversible after 20 generations of growth in drug free medium before mating. In contrast to the petite positive yeast Saccharomyces cerevisiae, where nuclear DNA synthesis is not affected but mitochondrial DNA is degraded in the presence of 20 g/ml ethidium bromide, the same concentration decreases both nuclear and mitochondrial DNA synthesis in Schizosaccharomyces pomhe. After removal of the drug, nuclear DNA synthesis increases faster than its mitochondrial counterpart in Schizosaccharomyces pomhe.     

Preparation of yeast mitochondrial DNA for direct sequence analysis

We describe two simple protocols for preparation of templates for direct sequencing of yeast mitochondrial DNA (mtDNA) by automatic DNA analyzers. The protocols work with a range of yeast species and yield a sufficient quantity and quality of the template DNA. In combination with primer-walking strategy, they can be used either as an alternative or a complementary approach to shot-gun sequencing of random fragment DNA libraries. We demonstrate that the templates are suitable for re-sequencing of the mtDNA for comparative analyses of intraspecific variability of yeast strains as well as for primary determination of the complete mitochondrial genome sequence.     

Comparison of Tetrahymena ARS sequence function in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe

Summary We have isolated several Tetrahymena thermophila chromosomal DNA fragments which function as autonomously replicating sequences (ARS) in the heterologous Saccharomyces cerevisiae and Schizosaccharomyces pombe selection systems. The Tetrahymena ARS sequences were first isolated in S. cerevisiae and were derived from non-ribosomal micro- and macronuclear DNA. Sequence analysis of the ARS elements identified either perfect or close matches with the 11 by S. cerevisiae ARS core consensus sequence. Subcloning studies of two Tetrahymena ARS elements defined functional regions ranging in size from 50 to 300 bp. Testing of the ARS elements in S. pombe revealed that most of the T. thermophila inserts confer ARS function in both yeasts, at least in the sense of promoting a high transformation frequency to plasmids which contain them. However, the actual sequences responsible for ARS activity were not always identical in the two yeasts.     

Comparative analysis of the mitochondrial genomes of Chlamydomonas eugametos and Chlamydomonas moewusii

Summary We report the cloning and physical mapping of the mitochondrial genome of Chlamydomonas eugametos together with a comparison of the overall sequence structure of this DNA with the mitochondrial genome of Chlamydomonas moewusii, its closely related and interfertile relative. The C. eugametos mitochondrial DNA (mtDNA) has a 24 kb circular map and is thus 2 kb larger than the 22 kb circular mitochondrial genome of C. moewusii. Restriction mapping and heterologous fragment hybridization experiments indicate that the C. eugametos and C. moewusii mtDNAs are colinear. Nine cross-hybridizing restriction fragments common to the C. eugametos and C. moewusii mtDNAs, and spanning the entirety of these genomes, show length differences between homologous fragments which vary from 0.1 to 2.3 kb. A 600 bp subfragment of C. moewusii mtDNA, within one of these conserved fragments, showed no hybridization with the C. eugametos mtDNA. Of the 73 restriction sites identified in the C. eugametos and C. moewusii mtDNAs, five are specific to C. moewusii, eight are specific to C. eugametos and 30 are common to both species. Hybridization experiments with gene probes derived from protein-coding and ribosomal RNA-coding regions of wheat and Chlamydomonas reinhardtii mtDNAs support the view that the small and large subunit ribosomal RNA-coding regions of the C. eugametos and C. moewusii mtDNAs are interrupted and interspersed with each other and with protein-coding regions, as are the ribosomal RNA-coding regions of C. reinhardtii mtDNA; however, the specific arrangement of these coding elements in the C. eugametos and C. moewusii mtDNAs appears different from that of C. reinhardtii mtDNA.     

Reorientation of the distal region in linkage group IIR of fission yeast

The genetic map of the fission yeast Schizosaccharomyces pombe has been revised in the distal region of chromosome arm IIR. The spo4 locus, hitherto considered the outermost marker, has been moved to an intermediate position. As a result, and in accordance with recent physical mapping data, the order of the entire distal subgroup of some 12 genetic markers is reversed relative to previously published gene maps.     

Chloroplast origins of DNA replication are distinct from chloroplast ARS sequences in two green algae

Summary A 5.3 kb chloroplast restriction fragment of Chlamydomonas reinhardii containing an origin of DNA replication and a sequence capable of promoting autonomous replication in C. reinhardii (ARC sequence) also carries an ARS sequence (autonomous replication in yeast). The ARC and ARS elements have been physically mapped and shown to be distinct from the origin of DNA replication. Similarly, restriction fragments containing the origin of chloroplast DNA replication from Euglena gracilis are unable to promote autonomous replication in yeast.     

The mcm2-1 mutation of yeast causes DNA damage with a RAD9 requirement for repair

The minichromosome maintenance mutation, mcm2-1, has been found to synthesize damaged DNA at 35°C. Growth at this temperature rendered the mutant strain more sensitive to killing by ultraviolet irradiation. DNA damage could also be detected by pulsed-field gel electrophoresis, where a higher fraction of the DNA loaded was retained in the inserts at the wells. During the exponential phase of growth at this temperature about 50% of the cells had large buds, with the nucleus at or near the neck of the bud in most cases. The incorporation of the rad9 deletion in the mcm2-1-carrying strain caused a reduction in the percentage of large-budded cells and a moderate loss of cell viability. The results are consistent with mcm2-1 causing DNA damage leading to the arrest of cells in the S/G₂ phase of the cell cycle, which was partially dependent on the RAD9 gene product.     

A 61-kb ring chromosome shows an ARS-dependent increase in its mitotic stability in the mcm2 mutant of yeast

We have studied the effects of ARS addition and deletion on the maintenance of a 61-kb ring derivative of chromosome III in a minichromosome maintenance mutant of yeast carrying the mcm2-1 mutation. When this ring chromosome, CIIIR, had either of its two strong origins deleted, the resultant chromosome showed a much greater instability in the mutant as compared to that of the wild-type strain. Integration of more ARSs improved the maintenance of CIIIR in the mutant but not in the wild-type strain. Increase in the size of CIIIR, without any ARS addition, did not improve the stability in either strain. A spontaneous revertant for improved growth at 35°C also co-reverted for minichromosome and CIIIR maintenance. The results suggest that ARS malfunctioning leads to minichromosome and chromosome loss from mutant cells, affecting their growth at higher temperatures.     

Mitochondrial genetics of Coprinus: Recombination of mitochondrial genomes

Summary The formation of the sexual mycelium or dikaryon in the basidiomycete Coprinus cinereus involves exchange and migration of nuclei without accompanying exchange of mitochondria. The dikaryotic growth which appears around the periphery of mated monokaryons has exclusively the mitochondrial genome of the recipient cells. Recombination of mitochondrial genomes is not, however, precluded during dikaryosis. Using monokaryons with different mitochondrial gene mutations, [acu-10] causing cytochrome aa ₃ deficiency and[cap-1.1] conferring resistance to chloramphenicol, it was shown that recombinant mitochondria arise in the zone of contact of mated monokaryons.     

Evidence that the single CDC8 gene which encodes thymidylate kinase is involved in DNA replication,recombination and mutation in yeast

Summary The conditional cdc8 mutant is known to be defective, under restrictive conditions, in the elongation of DNA during synthesis. In yeast the CDC8 gene encodes thymidylate kinase. We show here that UV-induced gene conversion and gene mutation events require the participation of this CDC8 gene. Thus, the same thymidylate kinase is incolved both in DNA replication and in UV-induced gene conversion and gene mutation in yeast.     

Interactions between the yeast mitochondrial and nuclear genomes: isogenic suppressive and hypersuppressive petites differ in their resistance to the alkaloid lycorine

Summary In a previous paper we have shown that the alkaloid lycorine inhibits growth of rho ⁺, mit ^- and rho ^-, strains of Saccharomyces cerevisiae, whereas strains devoid of mitochondrial DNA (rho ^o) are resistant to more than 200 g/ml of the alkaloid. In this report we show that hypersuppressive petites are almost as resistant as rho ^o mutants, whereas isogenic rho ^- petites, which have retained tained longer segments of the genome, are sensitive to the drug.     

Organization and chromosomal location of repetitive DNA sequences in three species of squamate reptiles

Repetitive DNA sequences were isolated from the genomes of species representing three major clades of squamate reptiles. A repetitive sequence (Cn4C7) was isolated from the New Mexican whiptail lizard,Cnemidophorus neomexicanus. This sequence is distributed throughout the chromosomes, but is more concentrated in the telomeric region. Cn4C7 also hybridizes to the chromosomes of otherCnemidophorus. Some evidence was found for concerted evolution of this repeat in hybrid unisexual lineages. In the lesser earless lizard,Holbrookia maculata, the predominant repeat in the genome is represented by a sequence (Hm1E11) which is restricted to the area flanking the centromere in all species ofHolbrookia. Two families of repetitive sequences (one dispersed, and the other telomeric) were isolated from the western diamondback rattlesnake,Crotalus atrox. The type and distribution of repetitive sequences in squamates is often taxon-specific, and may be useful as characters for elucidating taxonomic relationships.