Evolution of mitochondrial DNA in yeast: gene order and structural organization of the mitochondrial genome of Saccharomyces uvarum

We have determined the size, the restriction map and the gene order of the mitochondrial genome of the yeast Saccharomyces uvarum. Sequence analysis of the mitochondrial COXII gene confirmed the position of this yeast in the Saccharomyces cerevisiae-like group, near Saccharomyces cerevisiae and Saccharomyces douglasii. Most mitochondrial genes have been positioned on this approximately 57-kb long genome and three regions containing putative replication origins have been identified. The gene order of S. uvarum suggests that the mitochondrial genome of the S.cerevisiae-like yeasts could have evolved from an ancestral molecule, similar to that of S. uvarum, through specific genome rearrangements. Received: 22 April / 2 September 1997     

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

Summary We have reinvestigated the linkage orientation of repeating units in mtDNAs of yeast ^– 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 ^– 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.     

Physical mapping and genome organization of mitochondrial DNA from Candida maltosa

Summary Mitochondrial (mt) DNA of the ascomycetous yeast Candida maltosa was isolated and characterized. The mtDNA is circular and the size estimated from restriction analysis performed with 7 endonucleases was 52 kb pairs. A restriction map was constructed, using the cleavage data of four endonucleases. Using mt genes from Saccharomyces cerevisiae, six structural genes (large rRNA, apocytochrome b, cytochrome c oxidase subunit I and subunit 11, ATPase subunit 6 and subunit 9) were located on the C. maltosa chondriome by cross hybridization experiments. The comparison between the mt genomes of C. maltosa and six other yeasts showed differences in the overall genome organization.     

Clustering of tRNA genes in Paracentrotus lividus mitochondrial DNA

Summary We have determined the base sequence of the restriction fragment Bam1-2 (3,593) of Paracentrotus lividus (sea urchin) mtDNA. This fragment contains, in addition to genes previously identified (part of the 12S rRNA, ND1 and part of the ND2 mRNA), a cluster of 15 tRNA genes located between the 12S and ND1 genes. Also to be found in the tRNA gene cluster, between the tRNA^Thr and tRNA^Pro genes, is a sequence of 134 bp which constitutes the only non-coding region of this DNA so far identified. The distinctive organization of the tRNA genes and the extreme size reduction of the non-coding region suggest the existence of unique mechanisms for the regulation of gene expression in this organism.     

Complete nucleotide sequences and genome organization of four chimpanzee adenoviruses

The complete nucleotide sequences for four adenoviruses-Simian Adenoviruses 21, 22, 23, and 24, originally isolated from chimpanzees, were determined. The genome organization of the chimpanzee adenoviruses was found to be similar to that of other adenoviruses. The viral gene products of the adenoviruses Simian Adenoviruses 22, 23, and 24 are very closely related to those of the (previously sequenced) chimpanzee adenovirus Simian Adenovirus 25. Simian Adenovirus 21 is most similar to human subgroup B adenoviruses HAdV-3, HAdV-7, and HAdV-35. Analysis of the capsid proteins hexon and fiber of the chimpanzee adenoviruses also supports the placement of Simian Adenovirus 21 in subgroup B and Simian Adenoviruses 22, 23, and 24 in subgroup E.     

The organization of the genes for ribosomal RNA on mitochondrial DNA of Kluyveromyces lactis

Summary We have constructed a physical map of Kluyveromyces lactis mtDNA using the restriction enzymes HindII and HindIII. In contrast to Saccharomyces, the genes for the large and small ribosomal RNAs are much closer to each other, being separated by a maximal distance of 2,250 base pairs.Abbreviations bp base pair(s) - rRNA ribosomal RNA - SSC 0.15 M NaCl, 0.015 M sodium citrate (pH 7.0)     

Structural organization of the genome of the zygomycete Absidia glauca: evidence for high repetitive DNA content

Summary Total cell DNA of Absidia glauca has a GC-content of 44.6% ± 0.5% as determined from optical melting profiles which is in good accordance with values from equilibrium centrifugation in bisbenzimide containing CsCl gradients (46.2% = 1.1%), whereas mitochondrial DNA has a GC-content of only 30%. The genome size of Absidia glauca is approximately 36,000 kb, 8.6 times that of Escherichia coli. Three kinetically different fractions could be identified in reassociation experiments: a foldback-DNA fraction, comprising approximately 10% of the total DNA, repetitive DNA (25%) and single copy DNA (65%). This relatively high amount of repetitive DNA could partly be ascribed to ribosomal DNA (13%) and a new interspersed repetitive element (rAg1) which has been cloned in pBR325.     

Genome organization of mitochondrial DNA from the non-saccharomycete yeast Arxula adeninivorans LS3

Mitochondrial (mt) DNA of the asexual ascomycetous yeast Arxula adeninivorans LS3 was isolated and characterized. The mtDNA has a GC content of 30.3 mol%. It is circular and its size, as estimated by restriction analysis performed with nine endonucleases, was 35.5 kbp. Using mt gene-probes from Saccharomyces cerevisiae six structural genes (cob, cox1, cox2, oli1, oli2, and 21S rRNA) were located on the mitochondrial genome of A. adeninivorans. The comparison between the mt genomes of A. adeninivorans and other yeasts showed differences in genome organization.     

MboI,ThaI and HinfI endonuclease cleavage maps of the yeast mitochondrial DNA

Summary MboI, HinfI and ThaI cleavage maps have been constructed for the region of the mitochondrial DNA from S. cerevisiae where transfer RNA genes are principally located. About 40 cleavage sites have been localized between the C and P genetic markers. The MboI map covers about 50% of the total mitochondrial genome. For constructing maps we have used a series of rho^– deletion mutants whose mitochondrial DNAs have a typical single deletion structure as judged by previous genetic and physical analyses. The mutant DNAs carry known transfer RNA genes and genetic markers and, therefore, the comparison between genetic and restriction maps has allowed us to localize individual transfer RNA genes within defined physical segments.Abbreviations bp base pairs - mtDNA mitochondrial DNA - tRNA transfer RNA - rRNA ribosomal RNA - ThaI formerly TacI     

The mitochondrial genome of the aquatic phycomycete Allomyces macrogynus. Physical mapping and mitochondrial DNA instability

Summary A physical map of the mitochondrial genome of the aquatic phycomycete Allomyces macrogynus strain Burma 3–35 (35°C) has previously been published (Borkhardt and Delius 1983). This map has been extended in this study by locating 37 additional recognition sites for five new restriction enzymes in the mitochondrial genome. Homologous regions for the genes coding for cytochrome oxidase subunits 1, 2, and 3, apocytochrome b, ATPase subunits 6 and 9, the small and large ribosomal RNA, URF1, URF5, and perhaps urfa, a presumptive gene hitherto found only in the mitochondrial genome of the fission yeast Schizosaccharomyces pombe, were located in the mitochondrial genome of A. macrogynus by heterologous hybridizations with specific, mitochondria) gene probes from Saccharomyces cerevisiae, Aspergillus nidulans, Neurospora crassa, and S. pombe. The mitochondrial gene order in A. macrogynus was found to be identical to that of A. arbuscula; a gene order hitherto found only among members of the family Blastocladiaceae. Spontaneous insertion mutations have been found to occur quite frequently in the mitochondrial genome of A. macrogynus. In all mutated mitochondrial genomes so far studied, insertions have been located in a specific region located between the genes coding for the ATPase subunit 9 and the large ribosomal RNA. In two of the mutated mitochondrial genomes the insertional event(s) resulted in the presence of mitochondrial DNA molecules differing in size by multiples of approximately 70 base pairs.     

Isolation and characterization of mitochondrial DNA from the alkane yeast Saccharomycopsis lipolytica

Summary Mitochondrial (mt) DNA of the alkane yeast, Saccharomycopsis lipolytica, was isolated. Its buoyant density in CsCl was found to be of 1.687 g/cm³, indicating a GC content of 27.5% and its melting point T_m = 79.5 °C, indicating a GC content of 24.9%. The corresponding values for nuclear (n) DNA, are 1.709 g/cm³ (GC: 49.5%) and T_m = 90.5 (GC: 51.7%) respectively. Electron microscopy revealed that mtDNA has a circular structure with a contour length of about 14.5 µm corresponding to 45.5 kb per molecule. The size estimated from restriction analyses performed with 7 endonucleases was 48.35 kb/molecule. A restriction map was constructed, using the cleavage data of 4 endonucleases.     

Functional analysis of the yeast genome: use of two-dimensional gel electrophoresis to detect genes in randomly cloned DNA sequences

Summary Genes are overexpressed when present in yeast cells on multicopy plasmids. Taking advantage of the protein amplification which results from this overexpression, a method has been developed for large scale detection of yeast genes on randomly cloned DNA sequences. It is based on the analysis, by two-dimensional gel electrophoresis, of the proteins from yeast cells transformed with a yeast genomic DNA library constructed in a multicopy vector. We demonstrate here the applicability of this method for exploring the yeast genome. In addition, we report results which suggest that this method may also be useful for detecting regulatory genes.     

Spatial genome organization: contrasting views from chromosome conformation capture and fluorescence in situ hybridization

Although important for gene regulation, most studies of genome organization use either fluorescence in situ hybridization (FISH) or chromosome conformation capture (3C) methods. FISH directly visualizes the spatial relationship of sequences but is usually applied to a few loci at a time. The frequency at which sequences are ligated together by formaldehyde cross-linking can be measured genome-wide by 3C methods, with higher frequencies thought to reflect shorter distances. FISH and 3C should therefore give the same views of genome organization, but this has not been tested extensively. We investigated the murine HoxD locus with 3C carbon copy (5C) and FISH in different developmental and activity states and in the presence or absence of epigenetic regulators. We identified situations in which the two data sets are concordant but found other conditions under which chromatin topographies extrapolated from 5C or FISH data are not compatible. We suggest that products captured by 3C do not always reflect spatial proximity, with ligation occurring between sequences located hundreds of nanometers apart, influenced by nuclear environment and chromatin composition. We conclude that results obtained at high resolution with either 3C methods or FISH alone must be interpreted with caution and that views about genome organization should be validated by independent methods.     

Linear mitochondrial genome organization in vivo in the genus Pythium

Pulsed-field gel electrophoresis (PFGE) of isolates of Pythium oligandrum with linear mitochondrial genomes revealed a distinct band in ethidium bromide-stained gels similar in size to values estimated by restriction mapping of mitochondrial DNA (mtDNA). Southern analysis confirmed that these bands were mtDNA and indicated that linear genomes were present in unit-length size as well as multimers. Isolates of this species with circular mtDNA restriction maps also had low levels of linear mono- and multimers. visualized by Southern analysis of PFGE gels. Examination of 17 additional species revealed similar results; three species had distinct linear mtDNA bands in ethidium bromide-stained gels while the remainder had linear mono- and multi-mers in lower amounts detected only by Southern analysis. Sequence analysis of an isolate of P. oligandrum with a primarily circular mitochondrial genomic map and a low amount of linear molecules revealed that the small unique region of the circular map (which corresponded to the terminal region of linear genomes) was flanked by palindromic intrastrand complementary sequences separated by a unique 194-bp sequence. Sequences with similarity to ATPase9 coding regions from other organisms were located adjacent to this region. Sequences with similarity to mitochondrial origins of replication and autonomously replicating sequences were also located in this region: their potential involvement in the generation of linear molecules is discussed.     

The maize mitochondrial genome of the normal type and the cytoplasmic male sterile type T have very different organization

Summary The normal type (N) and cytoplasmic male sterile type T (cmsT) maize mitochondrial genomes are 570 kb and 540 kb in length respectively. Detailed hybridization studies have been undertaken to compare the sequence complexity and variation between the two genomes (genotype B37). They have approximately 500 kb of common sequences but there is considerable variation in sequence organization which can be accounted for by structural alterations from large and small permutations. The sequences not shared between the two genomes totaling 70 kb in N and 40 kb in cmsT can be accounted for by the presence of different repeated sequences, the absence of the integrated form of plasmids S1 and S2 in cmsT, the presence or absence of chloroplast sequences, and a number of sequences that are specific to N or to cmsT.     

Partial pedigree analysis of the segregation of yeast mitochondrial genes during vegetative reproduction

Summary A three-factor cross of Saccharomyces cerevisiae involving the cap1, ery1, and oli1 loci was done, with partial pedigree analyses of 117 zygotes. First, second, and third buds were removed and the genotypes of their diploid progeny determined, along with those of the residual zygote mother cell. Results were analyzed in terms of frequencies of individual alleles and of recombinant genotypes in the dividing cells. There is a gradual increase in the frequency of homoplasmic cells and in gene frequency variance during these three generations, as would result from stochastic partitioning of mtDNA molecules between mother and bud, probably coupled with random drift of gene frequencies in interphase cells. These phenomena are more pronounced for buds than for mothers, suggesting that buds receive a smaller sample of molecules. End buds are more likely to be homoplasmic and have a lower frequency of recombinant genotypes than do central buds; an end bud is particularly enriched in alleles contributed by the parent that formed that end of the zygote. Zygotes with first central buds produce clones with a higher recombination frequency than do those with first end buds. These results confirm previous studies and suggest that mixing of parental genotypes occurs first in the center of the zygote. If segregation were strictly random, the number of segregating units would have to be much smaller than the number of mtDNA molecules in the zygote. On the other hand, there is no evidence for a region of the molecule (attachment point) which segregates deterministically.     

Anatomy of amplified mitochondrial DNA in “Ragged” mutants of Aspergillus amstelodami: Excision points within protein genes and a common 215 bp segment containing a possible origin of replication

Summary The extranuclearly-inherited ragged growth phenotype (Rgd) of Aspergillus amstelodami is always accompanied by excision and head-to-tail amplification of mtDNA sequences. In one mutant strain (Rgd1) the amplified mtDNA segment (rgd1 DNA, monomeric length 0.9 kb) maps downstream of the large subunit ribosomal RNA gene (Region 1), whereas in all other strains analyzed the amplified sequences (rdg3-7DNA) are located in Region 2 between genes coding for cytochrome b and ATPase subunit 6. The various region 2 sequences differ in lengths (1.5 to 2.7 kb) but have in common a 215 bp sequence mapping between an. unidentified protein gene (corresponding to URF4 of human mtDNA) and an arginine tRNA gene. This common sequence may contain an origin of replication, because a looped-out hairpin structure similar to that of yeast and human mitochondrial origin sequences can be formed. Furthermore, Region 2 DNA suppresses replication of Region 1 DNA, indicating that the former group of molecules contains the more efficient origin. The nucleotide sequence of the rgd6 repeat unit starts and ends within protein genes of mtDNA, and no homologies were found between heads and tails or their flanking sequences.Abbreviations mtDNA DNA isolated from DNase — treated mitochondria - Rgd ragged mutant strain - rgdDNA highly-reiterated DNA sequences isolated simultaneously with the wild-type genome from mitochondria of ragged mutants - bp base pairs - kb kilobase pairs - URF unassigned reading frame