Petite Mutants Of Yeast Research Articles

L-phenylacetylcarbinol production by yeast petite mutants

The yeast Saccharomyces cerevisiae is able to biotransform benzaldehyde into L-phenylacetylcarbinol (L-PAC), a key intermediate in the production of ephedrine and pseudoephedrine, by the action of pyruvate decarobxylase (PDC) enzyme. This biotransformation can alternatively be performed by acetohydroxyacid synthase (AHAS) which is a mitochondrial enzyme. In the yeast petite mutants, AHAS accumulates in the cytosol. In the current study, wild-type yeast cells and yeast petite mutants were examined for L-PAC biosynthesis. The results showed higher L-PAC titers in the yeast petite mutants. In addition, the effect of cell immobilization and carbon source (glucose or molasses) on L-PAC production was investigated. It was found that cell immobilization enhances L-PAC formation. The highest L-PAC concentration (2.4 g/l) was obtained at 2 g/l of benzaldehyde using the immobilized petite mutants grown on molasses.

What happens when Trypanosoma brucei leaves Africa

Julius Lukes and co-workers evaluated the evolutionary origin of Trypanosoma equiperdum and Trypanosoma evansi, parasites that cause horse and camel diseases. Although similar to T. brucei, the sleeping-sickness parasite, these trypanosomes do not cycle through the tsetse fly and have been able to spread beyond Africa. Transmission occurs sexually, or via blood-sucking flies or vampire bats. They concluded that these parasites, which resemble yeast petite mutants, are T. brucei sub-species, which have evolved recently through changes in mitochondrial DNA.

Uniparental inheritance of organelle genes

Uniparental inheritance of organelle genes

Induction of petite mutants in yeast Saccharomyces cerevisiae by the anticancer drug dequalinium

Dequalinium (DEQ), a drug with both antimicrobial and anticancer activity, induced the formation of petite (respiration-deficient) mutants in the yeast Saccharomyces cerevisiae. DEQ was found to be approximately 50-fold more potent than ethidium bromide (EB) at inducing petites. Analysis of the DEQ-induced petite mutants indicated a complete loss of mitochondrial DNA (<1 copy/cell). Prior to the loss of mtDNA, DEQ caused cleavage of the mtDNA into a population of fragments 30–40 kbp in size suggesting that this drug causes petites by inducing a breakdown of mtDNA. The selective effect of DEQ on yeast mtDNA may underlie the antifungal activity of this chemotherapeutic agent.

Interorganellar Communication: ALTERED NUCLEAR GENE EXPRESSION PROFILES IN A YEAST MITOCHONDRIAL DNA MUTANT

Communication between mitochondria and the nucleus is important for a variety of cellular processes such as carbohydrate and nitrogen metabolism, mating and sporulation, and cell growth and morphogenesis. It has long been known that the functional state of mitochondria can influence nuclear gene expression. For example, in yeast cells lacking the mitochondrial genome, the expression of several nuclear genes, such as CIT2 (citrate synthase), MRP13 (mitochondrial ribosomal protein), and DLD3 (d-lactate dehydrogenase) has been reported to be altered. Here we show by microarray analysis of the genome-wide transcription profile of Saccharomyces cerevisiae that yeast petite mutants lacking mitochondrial DNA induce genes coding for mitochondrial proteins, enzymes of the glycolytic pathway and of the citric acid cycle, cell wall components, membrane transporters, and genes normally induced by nutrient deprivation and a variety of stresses. Consistent with the observed induction of genes related to cell stress and those encoding membrane transporters, yeast petite cells showed increased resistance to severe heat shock and exhibited a pleiotropic drug resistance phenotype. The observed changes in nuclear gene expression in cells lacking mitochondrial DNA may have implications for the role of mitochondria in processes such as carcinogenesis and aging.

Amphimeric mitochondrial genomes of petite mutants of yeast. III. Generation by linking two secondary-structure-dependent illegitimate recombination events.

The generation of amphimeric mitochondrial petite genomes of yeast can be explained by a process that links together two illegitimate recombination events, each involving a pair of short inverted repeats. Following "diagonal" double-strand breaks and inter-strand ligations at both possible stem-and-loop structures, a subgenomic single-stranded DNA circle can be excised. This circle comprises four building blocks organized in the so-called datA arrangement where d and t correspond, respectively, to the segments looped out by the upstream and the downstream pair of inverted repeats, a to the sequence separating the two loops, and A to the inverted duplication of segment a. Depending on the different possible "diagonal" recombinations at the inverted repeats, any of four isomeric circles can be excised, representing in its double-stranded form the nascent basic unit of an amphimeric mitochondrial petite genome of yeast. These isomeric basic units differ in the relative orientation of their sequences d and t (called D and T, respectively, when inverted), and are designated datA, DatA, daTA, and DaTA. Any one of these may be replicated to form the previously described regularly arrayed multimeric flip-flop genomes. Our new understanding of the amphimeric mitochondrial petite genomes of yeast emphasizes the role that topological features of DNA can play in mitochondrial genome dynamics. It also permits the re-interpretation of various observations reported in the literature. Some of them, including EtBr-mutagenesis in yeast, are discussed.

Amphimeric mitochondrial genomes of petite mutants of yeast. I. Flip-flop amphimers make up the mitochondrial genomes of "palindromic" petite mutants of yeast.

The mitochondrial (mt) genomes of three spontaneous cytoplasmic "palindromic" petite mutants of yeast were studied by restriction-enzyme analysis. These mt genomes were shown to be made up of an amplified "master basic unit" consisting of two inverted segments (a and A) and of two different unique segments (d and t) separating them. The basic unit was called "amphimeric", this term having been first proposed for certain lambda-phage mutants. We propose that in the mt genomes of the petite mutants studied, the four possible variants of the amphimeric basic unit form two - "flip" and "flop" - tetra-amphimeric repeat units datA-datA-DaTA-DaTA and DatA-DatA-daTA-daTA, respectively. These repeat units make two types of "amphimeric" mt genomes which exist in equal proportions in the cell. In each mt genome, the duplicated segment regularly alternates in its direct and inverted orientation (a...A...a...A...), whereas the unique segments are arranged twice in tandem fashion and twice in inverted fashion (d...d...D...D...d...d...andt...t...T...T...t...t...). The only difference between flip and flop amphimeric mt petite genomes is the different relative orientation of the unique segments in the mono-amphimers. In the mono-amphimers of flip mt genomes, both unique segments are arranged in the same direction (d...t and D...T), whereas in the mono-amphimers of flop mt genomes, both unique segments are arranged in opposite directions (D...t and d...T). Control experiments on one spontaneous petite mutant (which was an ancestor of the mutants studied here) and on three independent, previously investigated, EtBr-induced mutants showed that all of them were, in fact, organized in the same way. Analysing our experimental data and the results published by others, we conclude that amphimeric organization is a general feature of mt petite genomes of yeast previously called "palindromic" or "rearranged".

Characterization of the Mitochondrial Genome ofClavicorona Pyxidata

A mitochondrial DNA restriction map was prepared for a strain of Clavicorona pyxidata using three enzymes, Hind III, Hpa II and Xba I. Hybridization with Suillus probes and yeast petite mutants est...

Characterization of the Mitochondrial Genome of Clavicorona pyxidata

A mitochondrial DNA restriction map was prepared for a strain of Clavicorona pyxidata using three enzymes, Hind III, Hpa II and Xba I. Hybridization with Suillus probes and yeast petite mutants established the location of ribosomal RNA genes, areas putatively associated with cytochrome c oxidase complex subunits I, II and III, cytochrome b and ATPase subunits 6 and 9. Genome size was estimated from restriction fragment digests at 56 kb.

Inversions in mitochondrial DNA of a petite mutant of yeast.

Mitochondrial DNA of an erythromycin-resistant petite mutant of yeast, E734, showed physical maps of inversion, which occurred between two cross-over sites in the fragments Hae A and Hae B. The pair of cross-over sites was inferred to be accommodated within a repeat unit of E734 mtDNA during petite mutation by joining two fragments excised from non-adjacent region of wild type mtDNA.

Induction of petite mutants in yeast by non-intercalative DNA-binding antitumour agents

A series of 17 bis-charged non-intercalative DNA-binding antitumour agents and 7 related inactive compounds have been tested for the induction of respiratory deficient (petite) mutants in Saccharomyces cerevisiae D5. Many compounds were strong inducers of petite mutants at concentrations which were not toxic to the growth of the yeast cells. Mutagenicity is only weakly correlated with in vitro inhibition of L1210 cell growth; however, mutagenicity, yeast toxicity and in vitro and in vivo antitumour activity are all correlated with selective binding to polydeoxy(adenylic-thymidylic) acid rather than polydeoxy(guanylic-cytidylic) acid, as measured by an ethidium competition assay. It is concluded that A-T-rich DNA may be a target for all the biological effects measured in this study. Furthermore, the possibility that the target for antitumour action may be tumour cell mitochondrial DNA is supported by these results.

The mitochondrial genomes of spontaneous orir petite mutants of yeast have rearranged repeat units organized as inverted tandem dimers

We have investigated the structure and organization of the mitochondrial genomes of two related ori r ( ori-rearranged) spontaneous petite mutants of Saccharomyces cerevisiae. In these mutant genomes every repeat unit contains an inverted terminal duplication harboring a second (inverted) ori sequence, and tandem pairs of repeat units alternate with tandem pairs in inverted orientation. We have shown that ori r genomes are organized as the genomes with inverted repeat units of ethidium bromide (EtBr)-induced petites, and we have clarified the mechanism by which such mutant mitochondrial genomes arise.

Identification of maxicircle DNA sequences in Leishmania tarentolae that are homologous to sequences of specific yeast mitochondrial structural genes

Sequences homologous to the yeast mitochondrial structural genes for cytochrome oxidase subunits I and II, ATPase 6 and cytochrome b were identified on the kinetoplast DNA maxicircle molecule by low stringency hybridization of maxicircle blots with heterologous probes derived from mitochondrial DNA of yeast petite mutants. No hybridization was observed with the yeast ATPase 9 gene probe. The relative extent of base sequence mismatch was determined by melting of the heterologous hybrids. Candidates for the transcripts of these presumptive structural genes were proposed with reference to the transcriptional map of the maxicircle of Leishmania tarentolae. These results provide the first indication that maxicircle DNA specifies information for a limited number of conserved mitochondrial gene products similar to those already described for other eukaryotic cells.

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

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.

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

We 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.

Characterization of variant neurospora crassa mitochondrial DNAs which contain tandem reiterations

Two variant mtDNA types (types Ila and HI-10) have been identified in individual subcultures of the extra-nuclear [ poky]mutant of Neurospora crassa. Eco RI digests of type Ila mtDNA are characterized by an extra band, a, Mr = 1.4 Mdal, which arises from tandemly inserted reiterations of a 1.4 Mdal sequence. Restriction enzyme analysis and Southern hybridization experiments show: that the 1.4 Mdal repeats are located at the junction of Eco RI-4 and -6, that the repeats contain sequences ordinarily present in Eco RI-4 and -6, that the repeats are oriented head-to-tail and that the number of repeats per molecule (n) varies from n = 0 to n = 8, with about half of the molecules containing no repeats. The 1.4 Mdal repeats appear to be actively maintained in type Ila mtDNA populations as a result of a specific alteration in mtDNA. Data are presented which suggest that this alteration may be located near small deletions and/or sequence changes in Eco RI-3 and -10, fragments almost exactly opposite the site of the repeats on the genome. The second variant, HI-10 mtDNA, arose in a heteroplasmic strain in which type Ila mtDNA was one component. The most striking feature of HI-10 mtDNA is the up to 5-fold amplification of an 18 Mdal segment extending from Eco RI-4 (the site of the 1.4 Mdal repeats) through the rRNA genes. Eco RI digests show that HI-10 possesses characteristic features of type Ila mtDNA, including the 1.4 Mdal repeats and the alteration in Eco RI-10. HI-10 mtDNA also shows a novel Eco RI fragment, β, Mr = 2.9 Mdal. The variant Neurospora mtDNAs may be generated by mechanisms analogous to those which give rise to defective mtDNAs of yeast petite mutants. The possible consequences of defective mtDNAs in obligately aerobic organisms are discussed.

Segments of mitochondrial DNA of yeast carrying antibiotic resistances

Mitochondrial DNA was isolated from an oligomycin-resistant petite mutant of yeast, Saccharomyces cerevisiae. It had repeated sequences of 3600 base pairs. This segment was about one twentieth of the whole mtDNA of wild type yeast, which had a size of 74 kilo base pairs.

The repeating nucleotide sequence in the repetitive mitochondrial DNA from a “low-density” petite mutant of yeast

The repeating nucleotide sequence of 68 base pairs in the mtDNA from an ethidium-induced cytoplasmic petite mutant of yeast has been determined. For sequence analysis specifically primed and terminated RNA copies, obtained by in vitro transcription of the separated strands, were use. The sequence consists of 66 consecutive AT base pairs flanked by two GC pairs and comprises nearly all of the mutant mitochondrial genome. The sequence, moreover, also represents the first part of wild-type mtDNA sequence so far.

Mitochondrial DNA replication in petite mutants of yeast: Resistance to inhibition by ethidium bromide, berenil and euflavine

Mitochondrial DNA (mtDNA) replication in petite mutants of Saccharomyces cerevisiae is generally less sensitive to inhibition by ethidium bromide than in grande (respiratory competent) cells. In every petite that we have examined, which retain a range of different grande mtDNA sequences, this general phenomenon has been demonstrated by measurements of the loss of mtDNA from cultures grown in the presence of the drug. The resistance is also demonstrable by direct analysis of drug inhibition of mtDNA replication in isolated mitochondria. Furthermore, the resistance to ethidium bromide is accompanied, in every case tested, by cross-resistance to berenil and euflavine, although variations in the levels of resistance are observed. In one petite the level of in vivo resistance to the three drugs was very similar (4-fold over the grande parent) whilst another petite was mildly resistant to ethidium bromide and berenil (each 1.6-fold over the parent) and strongly resistant (nearly 8-fold) to inhibition of mtDNA replication by euflavine. The level of resistance to ethidium bromide in several other petite clones tested was found to vary markedly. Using genetic techniques it is possible to identify those petites which display an enhanced resistance to ethidium bromide inhibition of mtDNA replication. It is considered that the general resistance of petites arises because a product of mitochondrial protein synthesis is normally involved in facilitating the inhibitory action of these drugs on mtDNA synthesis in grande cells. The various levels of resistance in petites may be modulated by the particular mtDNA sequences retained in each petite.

Site-specific recombination in “petite colony” mutants of Saccharomyces cerevisiae

We have studied the organization of parental and recombined repetitive molecules of mtDNA's from cytoplasmic petite mutants of S. cerevisiae. One parental petite mutant carries a segment conferring resistance to chloramphenicol. The other parental petite mutant carries a segment associated with the erythromycin resistance locus. It was found that both parental mtDNA's are arranged in palindromes. Recombinant mtDNA's display a highly ordered and unique arrangement of repeats. In such a recombinant molecule with a repeat length of 2.24 μm two inverted repeats of one parent (1.69 μm each) follow two inverted repeats of the other parent (0.5 μm each). The overall arrangement of recombinant mtDNA is a palindrome again. As judged by partial denaturation maps very little if any sequence homology exists between the parental sequences in agreement with previous results obtained by DNA-DNA hybridization and high resolution melting. Thus, recombination between different mtDNA sequences consists in end to end joining of two segments. In one recombinant the hybrid molecule is non distinguishable from the corresponding wild type sequence while in the other recombinant the repeat length is slightly smaller (2.12 μm). We interpret the structure of recombined molecules as indicating that the recombination occurs according to the Campbell (1962) model. We discuss the various facets of the “petite” phenomena such as the repetitive nature of their mtDNA and the formation of multimeric and additive series of circle lengths, in the light of this model. We stress the analogy between transposable or translocatable genetic elements in bacteria and the mitochondria DNA in yeast petite mutants.