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PubMed1976

A novel class of Saccharomyces cerevisiae mutants specifically UV-sensitive to "petite" induction.

A mutant of Saccharomyces cerevisiae has been isolated which, though exhibiting a normal response to nuclear genetic damage by ultraviolet light (UV), is more sensitive than its wild type specifically in the production of the cytoplasmic (rho-) mutation by this agent. Some of the features of this mutation which has been designated uvsrho 5 are: i) The mutation is recessive, it exhibits a Mendelian, and hence presumably nuclear, pattern of segregation, but manifests its effects specifically and pleiotropically on mitochondrial functions. ii) Mutant cells resemble their wild type parents in a) growth characteristics on glucose; b) in their UV induced dose response to lethality or nuclear mutation and c) the ability of their mitochondrial genome, upon mating with appropriate testers, of transmitting and recombining various markers, albeit with enhanced efficiency. Similarly, d) they are able to modulate the expression of mitochondrial mutagenesis by ethidium bromide. Thus their mitochondrial DNA appears genetically as competent as that of the wild type. iii) Mutant cells differ from their wild type parents in a) growth characteristics on glycerol; b) susceptibility to induction of the mitochondrial (rho-) mutation by various mutagens, in that the rate of spontaneous mutation is slightly and that by UV is significantly enhanced, whild that by ethidium bromide is greatly diminished. Conversely, c) modulating influences resulting in the repair of initial damage are diminished fro UV and stimulated in the case of Berenil. iv) The amount of mitochondrial DNA per cell appears elevated in the mutant, relative to wild type, and its rate of degradation subsequent to a mutagenic exposure to either UV or ethidium bromide is diminished. v) A self-consistent scheme to account for this and all other information so far available for the induction and modulation of the (rho-) mutation is presented. In a previous study it was shown that some nuclear mutants of Saccharomyces cerevisiae, more sensitive to lethal damage induced by ultraviolet light (rad) than their parent wild type (RAD), also exhibit a concomitant modification in sensitivity to both nuclear and cytoplasmic genetic damage (Moustacchi, 1971). However, another class of rad mutants respond to the induction of the cytoplasmic "petite" also designated as rho- (or rho-) mutation by UV in a manner indistinguishable from that of the RAD strain. One possible interpretation of this last observation is that some of the steps in the expression of the UV damage on mitochondrial (mt)DNA may be governed by other nuclear and cytoplasmic genetic determinants, the products of which may then act specifically on mitochondrial lesions. If this assumption is correct, it should be possible to find mutants with a normal response to nuclear damage but specifically UV-sensitive towards induction of (rho-)...

DNA, Mitochondrial↗
PubMed1976

A map of the restriction targets in yeast 2 micron plasmid DNA cloned on bacteriophage lambda.

The 2 micron circular DNA from S. cerevisiae has been cloned on bacteriophage lambda. The two forms of circular DNA which exist in equilibrium due to recombination between inverted repeat sequences were separated as stable clones, and a map of targets for restriction endonucleases EcoRI, HindIII and HpaI was constructed. The circular DNAs isolated from a particular oligomycin resistant strain and its parent oligomycin snesitive strain were compared by restriction endonuclease analysis, and no difference was detected. The potential uses of cloned 2 micron DNA in determining the possible biological role of these plasmids are considered.

Coliphages↗
PubMed1975

Chloroplast genes in Chlamydomonas affecting organelle ribosomes. Genetic and biochemical analysis of analysis of antibiotic-resistant mutants at several gene loci.

Six chloroplast gene mutants of Chlamydomonas reinhardtii resistant to spectinomycin, erythromycin, or streptomycin have been assessed for antibiotic resistance of their chloroplast ribosomes. Four of these mutations clearly confer high levels of antibiotic resistance on the chloroplast ribosomes both in vivo. Although one mutant resistant to streptomycin and one resistant to spectinomycin have chloroplast ribosomes as sensitive to antibiotics as those of wild type in vivo, these mutations can be shown to alter the wildtype sensitivity of chloroplast ribosomes in polynucleotide-directed amino acid incorporation in vitro. Genetic analysis of these six chloroplast mutants and three similar mutants (Sager, 1972), two of which have been shown to affect chloroplast ribosomes (Mets and Bogorad, 1972; Schlanger and Sager, 1974), indicates that in Chlamydomonas at least three chloroplast gene loci can affect streptomycin resistance of chloroplast ribosomes and that two can affect erythromycin resistance. The three spectinomycin-resistant mutants examined appear to be alleles at a single chloroplast gene locus, but may represent mutations at two different sites within the same gene. Unlike wild type, the streptomycin and spectinomycin resistant mutants which have chloroplast ribosomes sensitive to antibiotics in vivo, grow well in the presence of antibiotic by respiring exogenously supplied acetate as a carbon source, and have normal levels of cytochrome oxidase activity and cyanide-sensitive respiration. We conclude that mitochondrial protein synthesis in these mutants is resistant to these antibiotics, whereas in wild type it is sensitive. To explain the behavior of these two chloroplast gene mutants as well as other one-step mutants which are resistant both photosynthetically and when respiring acetate in the dark, we have postulated that a mutation in a single chloroplast gene may result in alteration of both chloroplast and mitochondrial ribosomes. Mitochondrial resistance would appear to be the minimal necessary condition for survival of all such mutants, and antibiotic-resistant chloroplast ribosomes would be necessary for survival only under photosynthetic conditions.

Chlamydomonas↗
PubMed1976

Characterization of mutations in the penicillinase operon Staphylococcus aureus.

Mutant penicillinase plasmids, in which penicillinase synthesis is not inducible by penicillin or a penicillin analogue, were examined by biochemical and genetic analyses. In five of the six mutants tested, penicillinase synthesis could be induced by growth in the presence of 5-methyltryptophan. It is known that the tryptophan analogue 5-methyltryptophan is readily incorporated into protein by S. aureus and that staphylococcal penicillinase lacks tryptophan. 5-methyltryptophan seems to induce penicillinase synthesis in wild-type plasmids by becoming incorporated into the repressor and thereby inactivating the operator binding function of the penicillinase repressor. Therefore, induction of penicillinase synthesis in the mutant plasmids by 5-methyltryptophan strongly suggests that the noninducible phenotype of these five plasmids is due to a mutation that inactivates the effector binding site of the penicillinase repressor (i.e., the five mutant plasmids carry an iS genotype for the penicillinase repressor). This conclusion was supported by heterodiploid analysis. The mutant plasmid that did not respond to 5-methyltryptophan either produces an exceedingly low basal level of penicillinase or does not produce active enzyme. This plasmid seems to carry a mutation in the penicillinase structural gene or in the promoter for the structural gene. Thus, a genetic characterization of many mutations in the penicillinase operon can be accomplished easily and rapidly by biochemical analysis.

Extrachromosomal Inheritance↗
PubMed1977

Biogenesis of mitochondria 48: mikamycin resistance in Saccharomyces cerevisiae--a mitochondrial mutation conferring resistance to an antimycin A-like contaminant in mikamycin.

Commercial preparations of mikamycin have been shown to act as both inhibitors of mitochondrial protein synthesis and respiration. These preparations are shown to consist of two major streptogramin components (mikamycin A and mikamycin B) and a number of minor components. The major streptogramin components which inhibit mitochondrial protein synthesis in vitro are without effect in vivo due to whole cell impermeability to these compounds. A minor antimycin A-like component is the active compound in mikamycin preparations which inhibits growth of yeast cells on ethanol. The site of this inhibition is at the level of respiratory Comples III. The mitochondrial [mik 1-r] mutation confers resistance to this minor growth inhibitory component and cross resistance to antimycin A. For clarity the designation mik 1 has therefore been renamed ana 1 to denote the mitochondrial determinant conferring resistance to antimycin A. Genetic and physical mapping studies localise the ana 1 determinant in the region of mitochondrial DNA specifying cytochrome b. It is proposed that the ana 1 locus is part of a gene specifying a membrane component of Complex III.

Anti-Bacterial Agents↗
PubMed1977

The transposon Tn1 as a probe for studying ColE1 structure and function.

Insertion of the transposable genetic element Tn1 into different sites of plasmid ColE1 results in a number of mutnat phenotypes. Whereas all plasmid examined were present in normal amount, all showed reduced immunity to killing by colicin E1. Of six insertions isolated after conjugation, five fail to produce colicin, are conjugally proficient (transmissible), and map within a 500 nucleotide region of the genome. The other is conjugally deficient, produces colicin normally and maps close to two others with a similar phenotype isolated after transformation. Of four others isolated after transformation, two have similar properties to the original five transmissible plasmids. The other two are nontransmissible and produce colicin. Non-transmissibility is correlated with reduced relaxation complex. Patterns of protein synthesis in minicells by ColE1 and ColE1 :: Tn1 plasmids have been examined: all ColE1 plasmids containing Tn1 show an altered pattern of ColE1 protein synthesis in addition to three presumptive Tn1-specified proteins, one of which is shown to be beta-lactamase. ColE1 :: Tn1 plasmids can be inserted into the conjugative plasmid R64drd11 to form a cointegrate in which ColE1 and Tn1 function can be expressed.

Colicins↗
PubMed1977

Rapid screening for plasmid DNA.

A procedure is described for demonstrating plasmid DNA and its molecular weight, based on rate zonal centrifugation of unlabelled DNA in neutral sucrose gradients containing a low concentration of ethidium bromide. Each DNA species is then visualized as a discrete fluorescent band when the centrifuge tube is illuminated with ultra-violet light. Plasmids exist as closed circular and as relaxed circular molecules, which sediment separately, but during preparation of lysates, closed circular molecules are nicked so that each plasmid forms only a single band of relaxed circles within the gradient.

Centrifugation, Density Gradient↗
PubMed1977

Complementation of transfer deficient ColE1 mutants.

The transfer defect of some ColE1 mutants is complemented by ColE1 or ColK, but not by ColE2. This implies that at least one ColE1-specified protein or RNA is normally needed for ColE1 conjugal transfer. The gene(s) postulated for this function lies within a region whose length is at most 50% of the genome.

Ampicillin↗
PubMed1976

Characterization of the antibiotic resistance plasmid ERL1 from Streptococcus pyogenes.

The streptococcal plasmid ERL1 determining inducible resistance to erythromycin, lincomycin, and staphylomycin S was isolated by dye-buoyant density centrifugation and shown to have a molecular weight of about 17.5 Mdal, as revealed by sedimentation through neutral sucrose gradients. In SM60 cells entering the stationary phase its covalently closed circular form was present to the extent of 5 copies per chromosomal genome equivalent, ERL1 was subject to the DNA restriction and modification mechanism discovered in strain 56188. It did not appear to exercise restriction of phage DNA but mediated a partial release of the restricted growth of A25.

DNA, Bacterial↗
PubMed1992

Transmission of mitochondrial DNA in Ustilago violacea.

Mitochondrial DNA (mtDNA) restriction fragment length polymorphisms (RFLPs) were used as genetic markers for following mitochondrial transmission in the basidiomycete Ustilago violacea. Yeast-like cells of opposite mating types (a1 and a2) were mated on 2% water agar and were treated with alpha-tocopherol to induce formation of dikaryotic hyphae. Upon depletion of the alpha-tocopherol, the hyphae budded off haploid cells with parental nuclear genotypes. These cells were examined for mitochondrial RFLP phenotype. In progeny expressing the a1 mating type, mitochondria from either parent were observed equally frequently. In progeny with the a2 mating type, mitochondria were almost exclusively (94%) from the a2 parent.

Crosses, Genetic↗
PubMed1992

Cell type determines plastid transmission in tomato intergeneric somatic hybrids.

Mesophyll (M)- and suspension culture (S)-derived protoplasts of both Lycopersicon esculentum, tomato, and its wild relative Solanum lycopersicoides were fused as S + M, M + M and S + S combinations, respectively, to resolve the role of parental cell types in determining cpDNA transmission to intergeneric somatic hybrid plants. The mesophyll cpDNA was preferentially transmitted to 96% of the plants, each regenerated from a separate callus, in M + S and S + M fusion combinations. In contrast, for the M + M combination there was an equable distribution of either tomato cpDNA or that of S. lycopersicoides among the 34 hybrid plants. The number of plastids or proplastids in mesophyll or suspension protoplasts was not a factor regulating cpDNA transmission. Mesophyll or suspension protoplasts of both fusion partners had comparable frequencies of either plastid type with a mean of 23. The biased transmission of plastids from the mesophyll parent in somatic hybrid plants of S + M and M + S combinations appears to be due to differential multiplication of plastids, possibly conditioned by an unequal input of the nucleoids found in plastids versus proplastids. In the M + M fusion, plastid and nucleotide input and subsequent plastid multiplication are apparently equal, and when combined with random sorting out leads to an equal distribution of parental cpDNAs in the regenerated somatic hybrid plants. For the S+S combination, 22 somatic hybrid plants have exclusively tomato cpDNA, an outcome that is not readily explained by donor cell input.

Cell Fusion↗
PubMed1992

Subcellular fractionation of the two organelle DNAs of malaria parasites.

Malaria parasites contain two extrachromosomal DNAs, a 6 kb repetitive linear molecule which is assigned on the basis of its genetic content to the mitochondria, and a 35 kb transcriptionally active circular molecule whose intracellular location is not known. We used the polymerase chain reaction to detect and estimate the numbers of both molecules in sub-cellular fractions derived from the rodent parasite Plasmodium yoelii. The two DNA molecules were not coordinately partitioned by the fractionation process, the 6 kb molecule being more abundant, relative to the 35 kb circle, in a fraction enriched for mitochondria, the converse being true for a less dense fraction of unknown identity. This implies that the two molecules are located in different cellular compartments, and is consistent with other evidence suggesting they have different evolutionary origins.

Animals↗
PubMed1994

Extrachromosomal plasmids in the plant pathogenic fungus Rhizoctonia solani.

Extrachromosomal DNA elements were found in field isolates of Rhizoctonia solani belonging to anastomosis groups (AG) 1-5. An isolate of AG-5 (Rh41) contains a 3.6-kbp plasmid (pRS188) which has a similar A+T content to mitochondrial DNA. pRS188 is linear and has knob structures at its ends, as revealed by electron microscopy. Exonuclease digestions show that the linear ends of pRS188 are protected, and remain protected even after proteinase K digestion. pRS188 does not hybridise to nuclear or mitochondrial DNAs of its host isolate (Rh41), to total DNAs of other plasmid-less AG-5 isolates, or to total DNA of plasmid-harbouring isolates belonging to different AGs. Cellular-fractionation experiments suggest that pRS188 is associated with mitochondria, but it remains undecided whether this occurs inside or outside of the organelles. The nucleotide sequence of about 60% of the plasmid has been determined, revealing no open reading frame longer than 91 amino acids, and no known gene or genetic element is detected in the sequence contigs of 300-1572 bp length. Similar studies were performed with the plasmid pRS104 present in an isolate of AG-4 (Rh36), the sequence of which exhibits essentially the same features as pRS188 except that its A+T content resembles that of nuclear DNA. Pathogenicity tests reveal that the isolates Rh41 and R36 are as virulent as the plasmid-less isolates of AG-4 and -5, indicating that the plasmids do not play any role in pathogenicity.

Base Composition↗
PubMed1994

Homologous transformation of the edible basidiomycete Agrocybe aegerita with the URA1 gene: characterization of integrative events and of rearranged free plasmids in transformants.

The URA1 gene, encoding dihydroorotate dehydrogenase of the pyrimidine pathway, cloned into pUC18 (pUra1-1) was used to develop an homologous transformation system for the cultivated mushroom Agrocybe aegerita. Protoplasts of a ura1 auxotrophic strain were transformed by electroporation with efficiencies ranging from 1 to 26 transformants per micrograms of DNA. The phenotype of the stable Ura+ transformants suggested a strong nuclear heterogeneity further confirmed by Southern-blot analysis. All transformants acquired extrachromosomal forms derived from pUra1-1. Integration of pUra1-1 into chromosomal DNA occurred for some transformants. Plasmids containing the integrant of pUC18 recombined to different parts of the URA1 gene were rescued from A. aegerita transformants through transformation of E. coli. Their molecular analysis indicated that they represent products of the continuous excision of primary-integrated vector sequences rather than ARS-dependent autoreplicative forms.

Agaricales↗