Petite Mutants Of Yeast Research Articles

Selective covalent binding of an ethidium analog to mitochondrial DNA with production of petite mutants in yeast by photoaffinity labeling

A photosensitive azide derivative of ethidium bromide, 3(8)-amino-8(3)-azido-5-ethyl-6-phenyl phenanthridinium chloride, was prepared which binds covalently to DNA via a light-generated nitrene intermediate. When exposed to light in aqueous solvent the azide loses free nitrogen to yield a reactive nitrene, which reacts with water to form the hydroxylamine derivative. When the drug is photolyzed while tightly bound to DNA, rather than free in solution, a covalent complex is generated in situ due to the attack of the nitrene to form a covalent bond with the nucleic acid. A selectivity for cytoplasmic DNA reported previously for ethidium bromide was also shown for the azido analog by enhanced production of petite mutants in Saccharomyces following the photolysis of the azide in vivo. Isolated mitochondrial DNA following binding of the ethidium analog in vivo also appeared to retain a greater number of drug molecules per nucleotide than nuclear DNA. In vitro binding experiments did not suggest the same extent of preferential binding. This technique of photoaffinity labeling provides a means of studying the precise model of binding nucleic acid active drugs in relation to their biological effects.

The size of the repeating unit of the repetitive mitochondrial DNA from a “low-density” petite mutant of yeast

1. We have analysed further the properties of mtDNA of RD1A, an ethidium-induced cytoplasmic petite mutant of Saccharomyces cerevisiae. RD1A mtDNA contains 3 mole percent (G + C) the H-strand (the strand with the higher buoyant density in alkaline CsCl) contains 3 mole percent G and less than 0.1 mole percent C, the L-strand contains 3 mole percent C and less than 0.3 mole percent G. 2. Analysis of the pyrimidine tracts of RD1A mtDNA on DEAE-cellulose columns shows that the two longest tracts, T 6 and T 5 C 2, are present in approximately a 1 : 1 molar ratio, indicating that they occur once only in the proposed repeating unit. Direct sequence analysis of the T 5 C 2 tract shows that it is the unique sequence isomer T-T-T-C-C-T-T. Assuming the unique occurrence of T 6 and T 5 C 2 in the repeating unit of RD1A mtDNA, we calculate a minimal size of 68 nucleotides. 3. Degradation of the L-strand with the T-C base sequence specific, bacteriophage T 4-induced endonuclease IV yields material which migrates in a single, sharp peak in acrylamide gels; from its mobility in denaturing gels containing 4S and 5S RNA markers, the material is about 70 nucleotides long. 4. We conclude that the minimal repeating unit of RD1A mtDNA is approximately 70 nucleotides long.

Studies on mitochondrial gene purification using petite mutants of yeast: Characterization of mutants enriched in ribosomal RNA cistrons

Summary Normal yeast mtDNA contains one cistron each for the rRNA in the 37S and 50S subunits of mitochondrial ribosomes. Genetic information for 50S-rRNA is retained in mtDNA of petites carrying the loci for erythromycin or chloramphenicol resistance. In one strain retaining the erythromycin locus alone, 50S-rRNA hybridized to 14% of the mtDNA, representing a tenfold enrichment of these sequences compared with normal mtDNA. Where oligomycin or mikamycin loci are retained but chloramphenicol and erythromycin loci are deleted, little or no hybridization with 50S-rRNA is observed. Genes for 37S-rRNA are not retained in any of the petites investigated in this work. We conclude that the 50S-rRNA genes are located close to, or encompass, the chloramphenicol and erythromycin loci in mtDNA.

DNA-DNA Hybridization Studies of Mitochondrial DNA of Ethidium-Bromide-Induced Petite Mutants of Yeast

The mitochondrial DNA (mtDNA) of a grande strain and three petite mutant strains of yeast, Saccharomyces cerevisiae, has been analyzed by DNA-DNA filter hybridization and CsCl isopyknic density gradient centrifugation. All strains genetically transmitted either chloramphenicol or erythromycin resistance or both to sensitive nonpetite strains. The grande mtDNA had a buoyant density of 1.683 g/cm3, and the three petite mtDNAs had buoyant densities of 1.683, 1.682, and 1.689 g/cm3, respectively. The hybridization between 3H-labelled grande mtDNA and each of the three petite mtDNA filters was 42%, 11%, and 4% of isohybridization levels. Thermal denaturation of the hybrids indicated very specific hybridization. Homology between petite mtDNA was greatest in petites retaining common genetic markers for antibiotic resistance, and was least when different markers were retained. The results are consistent with deletion of different segments of grande mtDNA in the petite mutant.

Informational content of mitochondrial DNA from a “low density” petite mutant of yeast

Mitochondrial DNA from wild-type yeast Saccharomyces cerevisiae contains the genetic information for some mitochondrial tRNAs including tRNA ser and tRNA phe. In a “low density” petite mutant, mitochondrial DNA still retains the information for tRNA ser, while the information for tRNA phe is lost. The permanence of genetic information in this DNA containing only 3.6% G+C supports previous results concerning its intramolecular heterogeneity. An irregular distribution of G+C content along the molecule was further demonstrated by annealing experiments performed with DNA fragmented by sonication and fractionated on CsCl density gradient. These experiments show that the heavy fractions of the gradient preferentially anneal with mitochondrial seryl-tRNA.

The unusual properties of mtDNA from a “low-density” petite mutant of yeast

1. We have purified the mtDNA from the “low-density”, ethidium-induced petite mutant RD1A by repeated CsCl gradient centrifugation and hydroxylaptite chromatography. 2. The density in neutral CsCl of this DNA is 1.671 g/cm 3 and its T m in 0.2 M Na + is 69 °C. Base composition analysis gives A = T and G = C and G+C = 6 mole percent. 3. Quantitative renaturation studies show: (a) Denatured RD1A mtDNA renatures with second-order kinetics and contains no self-complementary single strands detectable by hydroxylapatite chromatography. (b) RD1A mtDNA must consist of a perfect repetition of a sequence of 300 nucleotides or less. (c) RD1A mtDNA is complementary to roughly 0.5 % of wild-type yeast mtDNA. 4. The behaviour of purified RD1A mtDNA on hydroxylapatite suggests that it largely consists of aggregates that are mechanically trapped on the column. Elution required a temperature near the T m of the DNA. 5. RD1A mtDNA sedimented through 3 M alkaline CsCl as heterogeneous linear DNA with a median molecular weight of about 1.7 · 10 6. 6. In neutral CsCl gradients containing ethidium the bulk of RD1A mtDNA showed a restricted uptake of ethidium; this was abolished by treatment with pancreatic deoxyribonuclease I. 7. Electron micrographs of RD1A mtDNA spread by the protein monolayer technique contained very large networks and an occasional small circle; spreading of the DNA under denaturing conditions confirmed that less than 0.1 weight percent is circular. 8. We propose that most of the isolated RD1A mtDNA is present in large fishnet concatenanes; the unwinding restriction imposed on DNA of which the strands are plaited into a fishnet concatenate explains the restricted ethidium uptake.

Hybridization of mitochondrial transfer RNA and mitochondrial DNA in petite mutants of yeast

The hybridization of mitochondrial leucyl- and mit-valyl-tRNA's with mitochondrial DNA of a series of petites has been used to characterize some of the changes in petite mit-DNA. The spontaneously mutated petite strain used (R1-6) was found to be heterogeneous with respect to hybridization with mitt-RNA's. Mit-DNA's isolated from subclones of the original strain differed in their ability to hybridize with grande mit-valyl- and mit-leucyl-tRNA's. Hybridization of mit-DNA of all petite subclones with grande mit-leucyl-tRNA resulted in apparent saturation levels that were equal, or usually two- to sixfold greater, than obtained with grande mit-DNA. In contrast, the ability to hybridize with grande mit-valyl-tRNA was lost by mit-DNA of some subclones, and partially retained in others. Mitochondrial leucyl- and valyl-tRNA's isolated from some petite strains retained their capacity to be aminoacylated in vitro, and were able to hybridize with mit-DNA of grande and some petite strains. The pattern of hybridization of petite mit-tRNA with mit-DNA from petite subclones was similar to that obtained with grande mit-RNA. Unlabeled mit-tRNA, but not supernatant tRNA, competed with the hybridization of both grande and petite tRNA's with grande and petite mit-DNA. Melting of the grande mit-tRNA petite mit-DNA hybrid resulted in a sharp curve with a T m equivalent to about 71 °C in 2 × standard saline citrate.

Influence of added nucleosides on acriflavin induction of petite mutants in Baker's yeast.

RECENT reports have suggested the occurrence of DNA in mitochondria of yeast1 and chick embryo2. These studies have added interest in view of the presence of DNA in the chloroplast fractions of algae and higher plants3, and its obvious implications for non-chromosomal inheritance4. The respiratory-deficient petite mutants of yeast possess altered mitochondria5, and they are easily induced by a variety of agents6. It is important to establish the chemical nature of the non-chromosomal determinants of this inheritance pattern, with DNA and RNA most probably involved, since they are the principal genetic materials. To approach the question of which nucleic acid might be the determinant for the respiratory characteristic, Moustacchi and Marcovich7 added uridine and thymidine along with the mutagenic base analogue 5-fluorouracil (5-FU). Their data showed that added thymidine was ineffective at all mutagen concentrations used, but that uridine significantly reduced the frequency of mutants induced at 5-FU concentrations of 0.1 and 0.2 µg/ml. There was no appreciable effect when uridine was added at 5-FU concentrations lower than 0.1 or higher than 0.2 µg/ml. When both nucleosides were added, the results paralleled those obtained with only thymidine added or with neither nucleoside present. They concluded that RNA was more likely than DNA to be the determinant involved, even though the uridine effect occurred only in a narrow portion of the concentration range of the mutagen. We have reinvestigated the problem, using the same experimental design, with the more commonly applied mutagen for petite induction, the acridine dye acriflavin. Unlike Moustacchi and Marcovich7, we found that both uridine and thymidine reduced the frequency of petite mutations induced by acriflavin. Our results, like theirs, also showed the nucleoside effect to be restricted to a narrow segment of the range of mutagen concentrations tested.