Yeast mtDNA Research Articles

Identification of mitochondrial gene products by DNA-directed protein synthesis in vitro

1. 1. A cell-free system, derived from Escherichia coli is highly active in the linked transcription-translation of yeast mtDNA from both wild-type and petite strains. 2. 2. The products of synthesis are short ( M r < 10 000) hydrophobic polypeptides, which show a high tendency to aggregate in a specific fashion with E. coli and mitochondrial proteins. Aggregation is extremely persistent: alkali, sodium dodecyl sulphate urea , guanidinium · HCl and carboxymethylation reduce it, but do not eliminate it completely. 3. 3. Nevertheless, results of indirect immunoprecipitation tests suggest that antigenic determinants of cytochrome c oxidase are among the products synthesized. The immunoprecipitation appears specific by criteria including competition experiments and its absence when mtDNA from low complexity petites, retaining only the gene for 21 S rRNA and some flanking sequences, is used to programme protein synthesis. Electrophoretic analysis of material precipitated by anti-cytochrome c oxidase sera reveals four discrete polypeptides with molecular weights of 7400, 6400, 5000 and 4100, which probably represent polypeptide fragments carrying antigenic determinants of cytochrome c oxidase .

A conserved and unique (AT)-rich segment in yeast mitochondrial DNA

The mtDNA of the cytoplasmic petite mutant of yeast RD1A consists mainly of a perfect head-to-tail repetition of a known sequence of 66 consecutive AT and 2 GC base pairs. We have hybridized complementary RNA made on RD1A mtDNA with the mtDNAs of four different wild-type Saccharomyces strains that differ markedly in restriction fragmentation pattern. The tm's of the four heteroduplexes are identical to the tm of the homoduplex of RD1A mtDNA with complementary RNA of one repeat length. With all four wild-type mtDNAs this complementary RNA hybridizes mainly to a single restriction fragment of about 300 base pairs. This shows the conservation and individuality of at least one (AT)-rich segment in yeast mtDNA. The 300 base pair fragment has been mapped in the vicinity of the oxi-2 locus. The possible role of the (AT)-rich segment in the processing of the primary transcript of this region is discussed.

Replicative Deoxyribonucleic Acid Synthesis in Isolated Mitochondria from Saccharomyces cerevisiae

The characteristics of a system for the in vitro synthesis of mitochondrial deoxyribonucleic acid (mtDNA) in mitochondria isolated from Saccharomyces cerevisiae are described. In this system the exclusive product of the reaction is mtDNA. Under optimal conditions the initial rate of synthesis is close to the calculated in vivo rate; the rate is approximately linear for 20 min but then decreases gradually with time. DNA synthesis proceeds for at least 60 min and the de novo synthesis of an amount of mtDNA equivalent to 15% of the mtDNA initially present is achieved. The rate and extent of synthesis observed with mitochondria isolated from grande and petite (rho(-)) strains were similar. The mode of DNA synthesis is semiconservative; after density labeling with 5-bromodeoxyuridine triphosphate, in vitro, the majority of labeled DNA fragments of duplex molecular weight, 6 x 10(6), are of a density close to that calculated for hybrid yeast mtDNA. The density label is incorporated into one strand of the duplex molecules. These properties indicate that the synthesis resembles replicative rather than repair synthesis. This system therefore provides a convenient method for the study of mtDNA synthesis in S. cerevisiae. The observation that mtDNA synthesis is semiconservative in vitro suggests that the dispersive mode of synthesis observed in S. cerevisiae in vivo labeling studies is the result of some other process, possibly a high recombination rate.

Restriction cleavage map of mitochondrial DNA from the yeast Saccharomyces cerevisiae

Mitochondrial DNA (mtDNA) from the yeast Saccharomyces cerevisiae was cleaved by restriction endonucleases Eco RI, Hpa I, Bam HI, Hind III, Pst I, and Sal I, yielding 10, 7, 5, 6, 1, and 1 fragments, respectively. A physical ordering of the restriction sites on yeast mtDNA has been derived. Yeast mtDNA cannot be isolated as intact molecules, and it contains nicks and gaps which complicate the use of conventional fragment mapping procedures. Nevertheless, the position of each of the restriction sites was obtained primarily by reciprocal redigestion of isolated restriction fragments. This procedure was supplemented by co-digestion of mtDNA with a multisite enzyme and a single-site enzyme (i.e., Sal I or Pst I) which provided a unique orientation for overlapping fragments cleaved by Sal I or Pst I. The data obtained from these approaches were confirmed by analysis of double and triple enzyme digests. Analysis of partial digest fragments was used for positioning of the smallest Eco RI fragment. A comparison of mtDNA from four grande strains (MH41-7B, 19d, TR3-15A, and MH32-12D) revealed similar, but slightly varying restriction patterns, with an identical genome size for each of approximately 5 X 10(-7) d or 75 kb. A fifth grande strain, D273-10B from S. cerevisiae, revealed restriction patterns different from those of the above strains, with a smaller genome size of 70 kb.

Variant forms of mitochondrial translation products in yeast: evidence for location of determinants on mitochondrial DNA.

Products of mitochondrial protein synthesis in yeast have been labeled in vivo with 35SO42-. More than 20 polypeptide species fulfilling the criteria of mitochondrial translation products have been detected by analysis on sodium dodecyl sulfate-exponential polyacrylamide slab gels. A comparison of mitochondrial translation products in two wild-type strains has revealed variant forms of some polypeptide species which show genetic behavior consistent with the location of their structural genes on mtDNA. Our results demonstrate the feasibility of performing genetic analysis on putative gene products of mtDNA in wild-type yeast by direct examination of the segregation and recombination behavior of specific polypeptide species.

Restriction endonuclease analysis of mitochondrial DNA from grande and genetically characterized cytoplasmic petite clones of Saccharomyces cerevisiae.

Digestion of grande mitochondrial DNA (mtDNA) BY EcoRI restriction endonuclease gives rise to nine fragments with a total molecular weight of 51.8 x 10(6). HindIII digestion yields six fragments with a similar total molecular weight. Specific restriction fragments can be detected despite the fact that yeast mtDNA consists of a heterogeneous distribution of randomly broken molecules. Digestion patterns of 10 genetically characterized petite clones containing various combinations of five antiobiotic resistance markers indicate that the petite mtDNA predominantly represents deletion of the grande genome. The petite mtDNAs contained up to seven EcoRI restriction fragments which comigrate with grande restriction fragments, and at least one fragment that did not correspond to any in the grande. Some strains contained multiple fragments with mobility different from that of grande; these fragments were usually present in less than molar concentrations. The genetic markers were associated with individual sets of restriction fragments. However, several internal inconsistencies prevent the construction of a definitive genetic fragment map. These anomalies, together with the digestion patterns, provide strong evidence that, in addition to single contiguous deletion, other changes such as multiple deletion and heterogeneity of mtDNA populations are present in some of the petite mtDNAs.

Synthesis of mitochondrial proteins in an Escherichia coli cell-free system directed by yeast mitochondrial DNA.

The optimum conditions for transcription in vitro of yeast mtDNA into biologically relevant RNA by Escherichia coli RNA polymerase holoenzyme and yeast mitochondrial RNA polymerase was found to critically depend on salt concentration. RNA was transcribed (at 0.25 M KCl concentration) from high-molecular-weight mtDNA which was then translated in an E. coli (S-30) cell-free protein synthesising system. Efficient translation of mitochondrial RNA was achieved using conditions which had also been determined to be optimal in other systems. Identification of the polypeptides produced in the translation system was made using antiserum raised against mitochondrial membranes. Electrophoresis of the completely dissociated antigen-antibody complexes using dodecylsulphate-polyacrylamide gels revealed that the system in vitro produced polypeptides of similar molecular weight to those synthesised in vivo by cycloheximide-inhibited whole cells.

The organization of genes in yeast mitochondrial DNA

1. We have isolated large fragments of the mtDNA of the yeast Saccharomyces carlsbergensis and digested these with restriction endonucleases. The digestion products were separated by electrophoresis in agarose gels. 2. Endonucleases EcoRI, HindII + III, HpaI, HindIII and HapII yield 9, 11, 6, 0 and greater than 80 fragments, respectively. 3. By analysis of partial digestion products and by redigesting the fragments obtained with one endonuclease with a second, we have established the order of all EcoRI and HindII + III fragments. The map is circular and its contour length is 22.1 +/- 0.35 mum, in good agreement with earlier estimates of the size of yeast mtDNA, using electron microscopy and renaturation kinetics. 4. A comparison of the fragmentation pattern of mtDNAs from S. carlsbergensis and various strains of Saccharomyces cerevisiae with endonuclease HindII + III suggests that the overall gene order is similar.

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.

The nucleotide composition and pyrimidine clusters in DNA from beef heart mitochondria

Study of primary structure of mitochondrial DNA (mtDNA) helps elucidate the general problem of DNA specificity and the numerous questions pertaining to the origin, evolution and functions of this nucleic acid. What we know so far about the composition of mtDNA have been derived from p and T, determinations [ 11. We have very scanty knowledge about the presence, nature and distribution pattern of minor bases in mtDNA. We have detected a small DNA-methylase activity in a mitochondrial fraction from loach embryos. It was an indication that animal mtDNA may in principle be methylated [2]. A nearest-neighbour base sequences possess unique mtDNA from slime mould [3] and determination of pyrimidine clusters frequencies in yeast mtDNA [4] showed that their nucleotide sequences possess unique specific features. This may have aroused interest to studying mtDNA; however, no such information has come to our notice, especially with respect to animal mtDNA. This paper reports the results of a comparative study of the nucleotide composition (CC content), of the methylation character and the degree of pyrimidine clustering in DNA from beef heart mitochondria and nuclei.

Mitochondrial DNA from cytoplasmic petite mutants of yeast

1. We have isolated a series of cytoplasmic petite mutants from Saccharomyces cerevisiae, grown for 6 h in 100 μM ethidium and analysed total-cell DNA in CsCl equilibrium gradients to detect alterations in light-satellite DNA (mtDNA). 2. All three independently selected mutant clones had altered mtDNA 65 generations after mutagenesis. RD1 mtDNA contained two components with equilibrium densities of about 1.681 and 1.671 g/cm 3 (wild type, 1.684 g/cm 3); RD2 mtDNA was heterogeneous in density; RD3 mtDNA banded at about 1.680 g/cm 3 but this DNA was heterogeneous in melting and 25 % renatured very rapidly even in 0.02 M Na +. 3. In CsCl gradients containing ethidium RD1, mtDNA behaved as closed circular duplex DNA; RD1 mtDNA spread by the protein monolayer technique contained mainly short rod-like structures, interpreted as tightly twisted circles, and occasional open circles with contour lengths between 0.40 and 1.05 μm. No evidence for circular mtDNA in ethidium-CsCl gradients was obtained with 5 other mutants. 4. We conclude that the large-scale changes in mtDNA in ethidium-treated yeast are an early event in mutagenesis and that the selection process leading to clones with only one type of aberrant mtDNA may require over 65 generations.

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.

The number of 4-S RNA genes on yeast mitochondrial DNA

Summary Hybridization of mitochondrial 4-S RNA from Saccharomyces carisbergensis with mtDNA in the presence of excess unlabelled high-molecular-weight mtRNA gave a plateau of 0.9 μg RNA hybridized per 100 μg DNA. This indicates that yeast mtDNA contains at least 20 genes for tRNA.