The oli1 gene and flanking sequences in mitochondrial DNA of Saccharomyces cerevisiae: the complete nucleotide sequence of a 1.35 kilobase petite mitochondrial DNA genome covering the oli1 gene

Summary As part of our genetic and molecular analysis of mutants of Saccharomyces cerevisiae affected in the oli1 gene (coding for mitochondrial ATPase subunit 9) we have determined the complete nucleotide sequence of the mtDNA genome of a petite (23-3) carrying this gene. Petite 23-3 (1,355 base pairs) retains a continuous segment of the relevant wild-type (J69-1B) mtDNA genome extending 983 nucleotides upstream, and 126 nucleotides downstream, of the 231 nucleotide oli1 coding region. There is a 15-nucleotide excision sequence in petite 23-3 mtDNA which occurs as a direct repeat in the wild-type mtDNA sequence flanking the unique petite mtDNA segment (interestingly, this excision sequence in petite 23-3 carries a single base substitution relative to the parental wild-type sequence). The putative replication origin of petite 23-3 is considered to lie in its single G,C rich cluster, which differs in just one nucleotide from the standard ori ^s sequence. The DNA sequences in the intergenic regions flanking the oli1 gene of strain J69-1B (and its derivatives) have been systematically compared to those of the corresponding regions of mtDNA in strains derived from the D273-10B parent (sequences from the laboratory of A. Tzagoloff). The nature and distribution of the sequence divergencies (base substitutions, base deletions or insertions, and more extensive rearrangements) are considered in the context of functions associated with mitochondrial gene expression which are ascribed to specialized sequences in the intergenic regions of the yeast mitochondrial genome.     

ARS activity along the yeast mitochondrial apocytochrome b region: correlation with the location of petite genomes and consensus sequences

Summary Seven MboI fragments spanning the mitochondrial apocytochrome b gene in Saccharomyces cerevisiae strain D273-10B were cloned in the BamHI site of the integrative yeast vector YIp5 and the capacity for autonomous replication was subsequently assayed in yeast. The positive correlation found between the ars-like activity in four fragments and the presence of regions common to multiple ethidium bromide-induced petite (rho^–) genomes suggests that the mitochondrial sequences possibly active as origins of replication in low-complexity neutral or weakly suppressive rho^– mutants could be functionally related to the yeast nuclear replicator 11 nucleotide motif defined by Broach et al. (1983).Abbreviations mtDNA mitochondrial DNA - bp base pairs - kbp kilobase pairs     

Physical map of the COB region in mitochondrial DNA of Saccharomyces cerevisiae

Summary A physical map of the COB region in mtDNA of yeast has been established. This region harbours a split gene coding for apocytochrome b. It includes restriction sites of seven endonucleases (EcoRI, HindII, HindIII, HaeIII, HpaII, AluI and BamHI). Various mtDNA sequences of this region, retained in a series of genetically characterized rho^– clones have been allocated to this map. The combination of this physical map with a genetic map of the rho^– clones revealed that 1) cob^– mutational sites spread over 8,400 bp, 2) mutations in sequences coding for apocytochrome b map in five distinct segments which are separated by intervening sequences with minimum lengths from 350 to 1,900 bp.Abbreviations mtDNA mitochondrial DNA - b bases - bp basepairs     

Physical mapping of genetic determinants on yeast mitochondrial DNA affecting the apparent size of the Var 1 polypeptide

Summary The var1 locus on yeast mitochondria) DNA is defined by a set of alleles that affect the apparent size (M_r 40,000–44,000) of a mitochondria) translation product (var1), a protein of the mitochondria) 38S ribosomal subunit. Using petite deletion and restriction endonuclease site mapping, we have defined the physical location of all var1 alleles to a specific restriction fragment of roughly 2.1 kbp located between the antibiotic resistance loci ery1 and olil. Surprisingly, from base sequence studies of this region by Tzagoloff et al. (1980), it appears that the DNA fragment we have mapped contains little or none of the structural gene for the var1 protein since the fragment is composed primarily of long stretches of dA + dT interspersed with short clusters high in dG + dC. Nevertheless, by a type of complementation test termed zygotic gene rescue (Strausberg and Butow, 1977), we show that with petites retaining that restriction fragment and short flanking sequences, var1 polypeptide characteristic of the strain from which the petite was derived, is expressed in zygotes formed between the petite and a wild-type tester. Thus the ability of the var1 determinant to act in trans suggests that control of expression of different var1 species involves intermolecular interactions, perhaps at the level of RNA splicing. Our results are discussed in terms of several possible models for the organization and control of the var1 structural gene.     

Deletions and rearrangements in Kluyveromyces lactis mitochondrial DNA

Summary Three classes of respiratory deficient mutants have been isolated from a fusant between Kluyveromyces lactis and Saccharomyces cerevisiae that contains only K. lactis mtDNA. One class (15 isolates), resemble ⁰ mutants of S. cerevisiae as they lack detectable mtDNA. A second class (16 isolates), resemble point mutations (mit ^–) or nuclear lesions (pet ^–) of S. cerevisiae as no detectable change is found in their mtDNA. The third class (five isolates), with deletions and rearrangements in their mtDNA are comparable to S. cerevisiae petite (^–) mutants. Surprisingly, three of the five deletion mutants have lost the same 8.0 kb sector of the mtDNA that encompasses the entire cytochrome oxidase subunit 2 gene and the majority of the adjacent cytochrome oxidase subunit 1 gene. In the other strains, deletions are accompanied by complex rearrangements together with substoiciometric bands and in one instance an amplified sector of 800 bp. By contrast to G+C rich short direct repeats forming deletion sites in S. cerevisiae mtDNA, excision of the 8.0 kb sector in K. lactis mtDNA occurs at an 11 bp A+T rich direct repeat CTAATATATAT. The recovery of three strains manifesting this deletion suggests there are limited sites for intramolecular recombination leading to excision in K. lactis mtDNA.     

About biased and non-biased transmission of chloroplast genes following artificial fusion of gametes in Chlamydomonas reinhardtii

Summary Artificial polyethyleneglycol induced fusions of gametes of opposite mating-types carrying chloroplast markers give rise to fusion products transmitting either both markers or the marker from the mt ⁺ or from the mt ^– parent exclusively. The frequencies of the three classes of products were approximately equal in our experiments (Matagne 1981). Similar experiments performed by Matsuda et al. (1983) gave different results, namely a preferential transmission of chloroplast gene from the mt ⁺ parent, very similar to that observed in vegetative zygotes obtained in sexual crosses. Results described here show that in experimental conditions used by Matsuda et al., sexual copulation does occur, leading to formation of zygotes which were misinterpreted as artificial fusion products and gave a biased transmission of chloroplast genes.     

Assembly of the mitochondrial membrane system. Organization of yeast mitochondrial DNA in the Oli1 region

Summary The mitochondrial DNA (mtDNA) of a cytoplasmic petite mutant (DS401) of Saccharomyces cerevisiae genetically marked for the ATPase proteolipid, serine tRNA and varl genes has been characterized by restriction endonuclease analysis and DNA sequencing. The DS401 mtDNA segment is 5.3 kb long spanning the region between 79.1 and 86.8 units of the wild type genome. Most of the DS401 mtDNA consists of A+T rich sequences. In addition, however, there are ten short sequences with a high content of G+C and two sequences that have been identified as the ATPase proteolipid and the serine tRNA genes. The two genes map at 81 and 83 units and are transcribed from the same DNA strand. Even though there are other possible coding sequences in the DNA segment, none are sufficiently long to code for a gene product of the size of the varl protein. Based on the relative organization of the G+C rich clusters and genes, a model has been proposed for the processing of mitochondria) RNA. This model postulates the existence of mitochondrial double strand specific RNases that cleave the RNA at the G+C clusters.     

Characterization of a novel open reading frame,urf a,in the mitochondrial genome of fission yeast: Correlation of urf a mutations with a mitochondrial mutator phenotype and a possible role of frameshifting in urf a expression

Summary Between the genes for tRNA^gln and tRNA^ile an open reading frame of 227 amino acids has been identified which is unique among known mitochondrial genomes and which has been termed urf a (Lang et al. 1983; Kornrumpf et al. 1984). It uses the mitochondrial genetic code, i.e., it contains a TGA codon, whereas all other protein-encoding genes, and all but one intronic open reading frame, use the standard genetic code (UGG for tryptophan). A previous paper has demonstrated that mutator strains show an increased formation of mitochondrial drug-resistant and respiration-deficient mutants (including deletions). In this paper we show that the mutator activity is correlated with mutations in urf a. A detailed analysis of one urf a mutant is presented (ana ^r -6), where the deletion of an A residue leads to a frameshift mutation and consequently to premature termination of the putative protein. The phenotype of colonies originating from a single mutant clone varies from no growth up to full growth on non-fermentable substrate. This phenomenon of phenotypic segregation can be explained by the ability of the cell to perform translational frameshifting. A detailed analysis of the DNA sequence and the putative urf a protein will be presented and a possible function of the protein will be discussed.Dedicated to Professor Fritz Kaudewitz on the occasion of his 70th birthday on March 11, 1991.     

Repair properties in yeast mitochondrial DNA mutators

Summary After ethy1methanesulfonate mutagenesis of the strain Saccharomyces cerevisiae D273-1013, out of 100,000 survivors, 1,000 were selected for their high production of petite mutants at 36 °C. Among these 1,000 mutators, 5 also showed an increased frequency of spontaneous point mutations measured at 25 °C. Further analysis revealed that in all mutators, except 2, petite accumulation proceeded at 25 °C as well as 36 °C. In these 2 mutants, the production of petite mutants was much higher at 36 °C than at 25 °C. In one of them, however, the mutator and the thermosensitive petite phenotypes were due to mutations in two unlinked nuclear genes. In the other mutants, both traits were the result of a mutation in a single nuclear gene. The mutators fell into three complementation groups (tpm1, tpm2, mup1). No complementation was observed between tpm1 mutants and the gam4 mutant previously described by Foury and Goffeau (1979). From the latter and the present works, only four complementation groups (gam1, gam2, gam4 or tpml, mupl) have been identified and it is likely that the number of genes controlling specifically the spontaneous mutability of the mtDNA is low. The mutators exhibited a variety of responses to damaging agents such as UV light and ethidium bromide; especially in a representative mutant from the complementation group tpm1, the induction of ^– mutants was sensitive to UV light and resistant to ethidium bromide. In addition, we found that in the mutants from this complementation group, the synthesis of mtDNA in isolated mitochondria was low; however their mitochondrial DNA polymerase activity was similar to that of the wild type strain. A relationship might exist between the mutator phenotype and the low mtDNA synthesis in the tpm1 mutants.     

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.     

Cloning of a DNA fragment from Cephalosporium acremonium which functions as an autonomous replication sequence in yeast

Summary A fragment of DNA which functions as an autonomous replication sequence in yeast was cloned from Cephalosporium acremonium. Mitochondrial DNA (mtDNA) was isolated from an industrial strain of C. acremonium (08G-250-21) highly developed for the production of the antibiotic, cephalosporin C. Size, 27 kb, and restriction pattern indicated this DNA was identical to mtDNA previously isolated (Minuth et al. 1982) from an ancestral strain (ATTC 14553) which produces very low amounts of cephalosporin C. A 1.9 kb Pst1 fragment of the Cephalosporium mtDNA was inserted into a Pst1 site of the yeast integrative plasmid, Ylp5, to produce a 7.5 kb plasmid, designated pPS1. The structure of pPS1 was verified by restriction analysis and hybridization.PS1 transformed Saccharomyces cerevisiae (DBY-746) to uracil prototrophy at a frequency of 272 transformants/g DNA. Transformation frequencies of 715 transformants/g DNA and zero were obtained for the replicative plasmid, YRp7, and the integrative plasmid YIp5, respectively. Southern hybridization and transformation of E. coli by DNA from yeast transformed by pPS1 verified that pPS1 replicates autonomously in yeast.The uracil-independent pPS1-yeast transformants were mitotically unstable. The average retention of pPS1 after three days growth in selective and non-selective medium was 4.5% and 0.4%, respectively, compared to retentions of 4.6% and 0.5% for YRp7. The properties of pPS1 were compared to those of a related plasmid, pCP2. pCP2 was constructed (Tudzynski et al. 1982) by inserting the C. acremonium 1.9 kb Pst1 fragment into the yeast integrative plasmid, pDAM1.     

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.     

High-resolution analysis of locomotor activity rhythms indisconnected,a visual-system mutant ofDrosophila melanogaster

Free-running locomotor activity and eclosion rhythms ofDrosophila melanogaster, mutant at thedisconnected (disco) locus, are substantially different from the wild-type phenotype. Initial periodogram analysis revealed little or no rhythmicity (Dushayet al., 1989). We have reanalyzed the locomotor activity data using high-resolution signal analysis (maximum-entropy spectral analysis, or MESA). These analyses, corroborated by autocorrelograms, uncovered significant residual circadian rhythmicity and strong ultradian rhythms in most of the animals tested. In this regard thedisco mutants are much like flies expressing mutant alleles of theperiod gene, as well as wild-type flies reared throughout life in constant darkness. We hypothesize that light normally triggers the coupling of multiple ultradian oscillators into a functional circadian clock and that this process is disrupted indisco flies as a result of the neural lesion.This work was supported in part by NIH Grant FM-33205.     

A “Stopper” mutant of Neurospora crassa containing two populations of aberrant mitochondrial DNA

Summary [E35], an extranuclear mutant of Neurospora crassa has all the phenotypic characteristics of the stopper mutants (De Vries et al. 1980). In the present work, the mitochondrial DNA as well as the mitochondrial translation products are characterized further. The primary mutational event appears to have been the deletion of about 4 kbp from the wild-type genome. Moreover, after prolonged vegetative growth the mutant accumulates an 8-m circular mtDNA, which was demonstrated both by electronmicroscopy and by restriction enzyme analysis. Hence, the mutant contains two populations of aberrant mitochondrial DNA, the smaller of which is an amplification of the rRNA-tRNA part of the larger. We propose that the primary deletion has generated a signal in the larger DNA which can cause premature termination of replication at the deletion site, and subsequent circularization of the unfinished daughter molecule. Finally, the deleted part may contain a determinant for synthesis of a protein of 11 kDal. The function of this protein, which is not a subunit of the F₀ ATPase, is not yet known.Abbreviations (k)bp (kilo)basepairs - kDal kilodalton - mt mitochondrial     

Restriction map of the mitochondrial DNA of the true slime mould,Physarum polycephalum: linear form and long tandem duplication

Summary The mitochondrial DNA (mtDNA) of the true slime mould, Physarum polycephalum strain CH934xCH938, was isolated and characterized by restriction mapping. Cloned fragments of the mtDNA were assembled and used to construct the restriction map. This map showed that the mtDNA was a linear molecule of 86.0 kb with a tandem duplication of 19.6 kb. The terminal fragments were identified by sensitivity to Bal31 exonuclease. One of the duplications was located at the right end and the other was located 5 kb from the left end. Each duplicated segment contained 26 restriction sites for ten enzymes and these restriction sites were completely conserved in each duplication. Genes for the large and small rRNAs were mapped to positions about 30 kb from the right end of the mtDNA by hybridization with its own rRNAs. With the exception of a probe for the gene for the large rRNA in Tetrahymena pyriformis mtDNA, various probes from the mtDNAs of Saccharomyces cerevisiae and T. pyriformis showed no significant hybridization to any of the restriction fragments of the mtDNA from P. polycephalum.     

Regulation of synthesis and secretion of acid and alkaline phosphatases in Neurospora crassa

Summary We show that N. crassa represses the production of acid phosphatase at pH higher than 8.0, irrespective of the carbon source used, whereas production was stimulated by sucrose at slightly acidic pH. The same profile of acid phosphatase production was observed in the pho-2A, pho-3A, nuc-1A, nuc-2A and preg ^c mutant strains. We also show that acid phosphatase synthesized by the preg ^c mutant strain grown on high phosphate medium has pronounced differences when compared to the enzyme synthesized by the wild-type strain grown on low phosphate medium in terms of heat stability, steady-state kinetic properties and DEAE-cellulose chromatography. In addition, the synthesis and/or secretion of only phosphate-repressible alkaline phosphatase is affected by mutations in acu-1, and acu-5 and acu-7 genes. These results, which indicate distinct pathways for the synthesis and secretion of acid and alkaline phosphatases in N. crassa, contradict the dosage titration model proposed by Metzenberg et al. (1974) whereby the synthesis of these enzymes should occur through a single hierarchical regulatory circuit as a response to phosphate starvation.     

Mitochondrial DNA mutation and heteroplasmy in type I Leber hereditary optic neuropathy.

Leber hereditary optic neuropathy (LHON) is a maternally inherited disorder characterized by bilateral acute or subacute loss of central vision, primarily in young males. A G----A single base mutation at 11778nt of the mitochondrial genome which eliminates a SfaNI restriction site [Wallace et al., 1988; Holt et al., 1989; Hotta et al., 1989; Singh et al., 1989; Vilkki et al., 1989; Yoneda et al., 1989; Stone et al., 1990; Lott et al., 1990.] has been found in more than 60% of the families with LHON studied. We studied 25 persons from 4 families with LHON using SfaNI and Mae III digestion of a 201 base pair polymerase chain reaction (PCR) product encompassing the 11778nt mutation. The loss of the SfaNI site and the acquisition of a Mae III site at 11778nt were identified in all maternal relatives of the LHON families studied. The mutation was heteroplasmic in all affected individuals, female carriers, and males at-risk. The heteroplasmy of mitochondrial DNA (mtDNA) was also identified by direct DNA sequencing of PCR amplified by direct DNA sequencing of PCR amplified mtDNA digested by SfaNI or Mae III. It appears that the proportion of the mutant mtDNA correlates with the severity of the disease.     

Mutations in four chloroplast loci of Chlamydomonas reinhadtii affecting the photosystem I reaction centers

Summary In this work seven chloroplast mutations conferring a deficiency in photosystem I reaction centers have been mapped at four chloroplast loci in Chlamydomonas reinhardtii. Recombination frequencies were estimated from diploid progeny of vegetative zygotes. These four loci were scattered throughout the chloroplast genome. The three mutations at locus I were found to be tightly linked to a mutation in the rbcL gene coding for the large subunit of ribulose-1,5-bisphosphate carboxylase (Dron et al. 1983). As the psaA2 gene coding for one apoprotein of the chlorophyll-complex CPI, identified by its homology with the corresponding maize gene (Fish et al. 1985), has been found close to the rbcL gene (Dron et al. 1982), the psaA2 gene could be at locus 1.     

An Improved Assay Shows No Association Between the CYP2A6 Gene and Cigarette Smoking Behavior

Pianezza et al. (1998) reported an association between the CYP2A6 gene, which codes for the enzyme that metabolizes nicotine to cotinine, and cigarette smoking behavior. We attempted to replicate their findings by analyzing the CYP2A6 gene in a population of 385 individuals using the same two-step PCR assay described by Pianezza et al. but found no association between genotype and either smoking status or cigarette consumption. We then developed a single-step PCR method that is specific for the CYP2A6 locus and eliminates a high rate of false-positive mutations detected by the two-step assay. Although this assay gave a much lower frequency of mutant alleles, there was again no association of CYP2A6 genotype with smoking behavior.     

Does mitochondrial DNA length influence the frequency of spontaneous petite mutants in yeasts?

Summary Results from the theory of random walks applied to the random excision hypothesis for production of petite mutants in yeast suggest that frequency of excision should increase as a linear function of mitochondrial DNA length (see appendix). For a series of petite positive yeasts we have determined the spontaneous petite frequency (ranging from about 0.003% to 9%) and length of mtDNA (ranging from about 19 Kbp to c. 108 Kbp) and found that, while the frequency of petite mutants does generally increase with mtDNA length, the relationship is far from linear. Although these results are inconclusive concerning the random excision hypothesis they do indicate that amongst related yeasts other factors have a greater influence than mtDNA length in determining the frequency of petite mutants.