Genetic analysis of mitochondrial rho-mutability in Saccharomyces. IV. Relation between spontaneous rho-mutability and mitotic stability in disomic Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae the disomy for chromosome XIV resembles the previously described disomy for chromosome IV in that it leads to a significant decrease in spontaneous rho- mutability. The nuclear srm1 mutation, reducing spontaneous rho- mutability, diminishes significantly the mitotic disome stability. So, the mechanisms of spontaneous rho- mutagenesis and mitotic disome stability seem to compete for the function affected by the srm1 mutation.     

Genetic analysis of the mitochondrial rho-mutability in Saccharomyces. II. Disomy for chromosome IV and spontaneous rho-mutability

The disomy for chromosome IV in the strains studied led to: reduction in the red pigmentation of ade1 mutant colonies; a decrease in spontaneous rho- mutant frequency, and impairment of sporulation in hybrids descended from disomic parents. The nuclear srm1 mutation decreasing the spontaneous rho- mutability promoted the spontaneous extra chromosome loss in the disomics for chromosome IV. This result suggests a close connection between the spontaneous rho- mutability and mitotic chromosome stability.     

Genetic analysis of mitochondrial rho-mutability in Saccharomyces yeasts. VI. Quantitative characteristics of the effect of mutation srm5 on the mitochondrial stability of natural and recombinant genetic structures

The srm5 mutation diminishes the spontaneous rho- mutation rate by an order of magnitude. Frequency of rho- mutations is 500 times lower in homozygous cultures, as compared with those of normal SRM+/SRM+ diploids. The rate of spontaneous loss of extra chromosome IV is about 25 times higher in srm5 disomes, as compared with SRM+ ones. Haploid srm1 srm5 transformants loose recombinant circular minichromosomes spontaneously about 4 times more frequently than srm1SRM5 cells. The data presented suggest that general control of mitotic stability of different (mitochondrial and nuclear, nuclear as well as recombinant) genetic structures operates in Sacch. cerevisiae. Autonomously replicating sequences (ARS elements) seem to be involved in this mechanism.     

Genetic analysis of mitochondrial rho-mutability in Saccharomyces. I. Polygenic determination of spontaneous rho-mutability: gene-modifiers and srm mutation

The phenotypic trait "starry colony" in Saccharomyces is associated with a high spontaneous rho- petite mutability. Genetic analysis of this trait has shown the high rho- mutability to be caused by several modifying genes present together in the cell genome. Every single modifying gene only produces a relatively small enhancement in the rho- mutability. Mutations in four nuclear srm (spontaneous rho- mutability) loci were isolated after mutagenic treatment of highly rho- mutable haploid cells. In contrast to the modifying genes, each of these mutations has a pronounced effect on the spontaneous rho- mutability, causing significant decrease in it.     

A nuclear mutation decreasing rho- production modifies the unstable nitrous-acid sensitivity of yeast

The nuclear mmgl mutation, which reduces rho- mutability in Saccharomyces cerevisiae, renders the rho+ cells less sensitive to inactivation by nitrous acid (NA) but has little or no effect on the NA sensitivity of the rho0 cells devoid of mitochondrial (mt) DNA. Therefore the cells' NA sensitivity seems to be influenced by an interaction of the mmgl mutation and the mt genome rather than the mmgl mutation itself. The clonal variation of NA sensitivity is high in MMG+ yeast and significantly reduced in rho0 mutants and mmgl cells. The results presented suggest that frequent spontaneous heritable changes of the mt genome occur in MMG+ cells, which, (i) unlike rho- mutations, do not damage the respiratory capacity, and (ii) manifest themselves in a high clonal variation of NA sensitivity.     

New Mutations of SRMGenes in Saccharomyces cerevisiae and Certain Characteristics of Their Phenotypic Effects

The effects of the previously identified mutations in nuclear genes SRM8, SRM12, SRM15, and SRM17on the maintenance of chromosomes and recombinant plasmids in Saccharomyces cerevisiaecells and on cell sensitivity to ionizing radiation were studied. The srm8mutation caused an increase in spontaneous chromosome loss in diploid cells. In yeast cells with the intact mitochondrial genome, all examined srmmutations decreased the mitotic stability of a centromeric recombinant plasmid with the chromosomal ARS element. Mutations srm12, srm15, and srm17also decreased the mitotic stability of a centromereless plasmid containing the same ARS element, whereas the srm8mutation did not markedly affect the maintenance of this plasmid. Mutations srm8, srm12, and srm17were shown to increase cell sensitivity to -rays. The SRM8gene was mapped, cloned, and found to correspond to the open reading frame YJLO76w in chromosome X.     

New mutation in Saccharomyces cerevisiae SRM genes and some features of their phenotypic effects

The effects of the previously identified mutations in nuclear genes SRM8, SRM12, SRM15, and SRM17 on the maintenance of chromosomes and recombinant plasmids in Saccharomyces cerevisiae cells and on cell sensitivity to ionizing radiation were studied. The srm8 mutation caused instability of chromosome maintenance in diploid cells. In yeast cells with the intact mitochondrial genome, all examined srm mutations decreased the mitotic stability of a centromeric recombinant plasmid with the chromosomal ARS element. Mutations srm12, srm15, and srm17 also decreased the mitotic stability of a centromereless plasmid containing the same ARS element, whereas the srm8 mutation did not markedly affect the maintenance of this plasmid. Mutations srm8, srm12, and srm17 were shown to increase cell sensitivity to gamma-ray irradiation. The SRM8 gene was mapped, cloned, and found to correspond to the open reading frame YJLO76w in chromosome X.     

Abnormal processing of transfected plasmid DNA in cells from patients with ataxia telangiectasia.

In order to assess spontaneous mutability and accuracy of DNA joining in ataxia telangiectasia, a disorder with spontaneous chromosome breakage, the replicating shuttle vector plasmid, pZ189, was transfected into SV40 virus-transformed fibroblasts from ataxia telangiectasia patients. The ataxia telangiectasia fibroblasts showed elevated frequency of micronuclei, a measure of chromosome breakage. The spontaneous mutation frequency was normal with circular plasmids passed through the ataxia telangiectasia line. These results were compared to those with transformed fibroblasts from a patient with xeroderma pigmentosum, and from a normal donor. Mutation analysis revealed spontaneous point mutations and deletions in the plasmids with all 3 cell lines, however, insertions or complex mutations were only detectable with the ataxia telangiectasia line. To assess DNA-joining ability, linear plasmids which require joining of the DNA ends by host cell enzymes for survival, were transfected into the cells. We found a 2.4-fold less efficient DNA joining in ataxia telangiectasia fibroblasts (p = 0.04) and a 2.0-fold higher mutation frequency (p less than 0.01) in the recircularized plasmids than with the normal line. Plasmid DNA joining and mutation frequency were normal with the xeroderma pigmentosum fibroblasts. These findings with the ataxia telangiectasia fibroblasts of abnormal types of spontaneous mutations in the transfected plasmid and inefficient, error-prone DNA joining may be related to the increased chromosome breakage in these cells. In contrast, an EB virus-transformed ataxia telangiectasia lymphoblast line with normal frequency of micronuclei showed normal types of spontaneous mutations in the transfected plasmid and normal frequency of DNA joining which was error-prone. These data indicate that mechanisms that produce chromosome breakage in ataxia telangiectasia cells can be reflected in processing of plasmid vectors.     

Spontaneous, ultraviolet and ionizing radiation mutagenesis in two auxotrophic strains of Salmonella typhimurium carrying an R plasmid.

Ultraviolet-induced, gamma-induced and spontaneous mutation yields were studied in two different auxotrophic strains of Salmonella typhimurium in the presence and absence of the UV-protecting drug resistance transfer factor R-Utrecht. One strain, carrying the hisC527 (amber) mutation, showed significantly increased spontaneous, UV- and gamma-induced mutability in the presence of the R-Utrecht plasmid. The other strain, carrying the trpD1 mutation (thought to be a missense mutation), also showed significantly increased UV mutability in the presence of the R-Utrecht plasmid. The other strain, carrying the trpD1 mutation (thought to be a missense mutation), also showed significantly increased UV mutability in the presence of the R factor, but appeared to show no significant increase in spontaneous mutability and only a very slight increase in gamma-mutability when carrying the R factor. These results demonstrate that the R-Utrecht plasmid, known to enhance UV-induced mutation yields in S. typhimurium, can also significantly enhance both spontaneous and gamma-induced mutation yields in this species. The latter effects are not so discernible with all markers, however, as shown by the results with strains carrying the trpD1 mutation. Enhancement of spontaneous mutability thus appears to be correlated with enhancement of gamma-mutability rather than UV mutability.     

The yeast HSM3 gene acts in one of the mismatch repair pathways.

Mutants with enhanced spontaneous mutability (hsm) to canavanine resistance were induced by N-methyl-N-nitrosourea in Saccharomyces cerevisiae. One bearing the hsm3-1 mutation was used for this study. This mutation does not increase sensitivity to the lethal action of different mutagens. The hsm3-1 mutation produces a mutator phenotype, enhancing the rates of spontaneous mutation to canavanine resistance and reversions of lys1-1 and his1-7. This mutation increases the rate of intragenic mitotic recombination at the ADE2 gene. The ability of the hsm3 mutant to correct DNA heteroduplex is reduced in comparison with the wild-type strain. All these phenotypes are similar to ones caused by pms1, mlhl and msh2 mutations. In contrast to these mutations, hsm3-1 increases the frequency of ade mutations induced by 6-HAP and UV light. Epistasis analysis of double mutants shows that the PMS1 and HSM3 genes control different mismatch repair systems. The HSM3 gene maps to the right arm of chromosome II, 25 cM distal to the HIS7 gene. Strains that bear a deleted open reading frame YBR272c have the genetic properties of the hsm3 mutant. The HSM3 product shows weak similarity to predicted products of the yeast MSH genes (homologs of the Escherichia coli mutS gene). The HSM3 gene may be a member of the yeast MutS homolog family, but its function in DNA metabolism differs from the functions of other yeast MutS homologs.     

Paternal uniparental disomy for chromosome 1 revealed by molecular analysis of a patient with pycnodysostosis.

Molecular analysis of a patient affected by the autosomal recessive skeletal dysplasia, pycnodysostosis (cathepsin K deficiency; MIM 265800), revealed homozygosity for a novel missense mutation (A277V). Since the A277V mutation was carried by the patient's father but not by his mother, who had two normal cathepsin K alleles, paternal uniparental disomy was suspected. Karyotyping of the patient and of both parents was normal, and high-resolution cytogenetic analyses of chromosome 1, to which cathepsin K is mapped, revealed no abnormalities. Evaluation of polymorphic DNA markers spanning chromosome 1 demonstrated that the patient had inherited two paternal chromosome 1 homologues, whereas alleles for markers from other chromosomes were inherited in a Mendelian fashion. The patient was homoallelic for informative markers mapping near the chromosome 1 centromere, but he was heteroallelic for markers near both telomeres, establishing that the paternal uniparental disomy with partial isodisomy was caused by a meiosis II nondisjunction event. Phenotypically, the patient had normal birth height and weight, had normal psychomotor development at age 7 years, and had only the usual features of pycnodysostosis. This patient represents the first case of paternal uniparental disomy of chromosome 1 and provides conclusive evidence that paternally derived genes on human chromosome 1 are not imprinted.     

Mutation analysis of UBE3A in Angelman syndrome patients.

Angelman syndrome (AS) is caused by chromosome 15q11-q13 deletions of maternal origin, by paternal uniparental disomy (UPD) 15, by imprinting defects, and by mutations in the UBE3A gene. UBE3A encodes a ubiquitin-protein ligase and shows brain-specific imprinting. Here we describe UBE3A coding-region mutations detected by SSCP analysis in 13 AS individuals or families. Two identical de novo 5-bp duplications in exon 16 were found. Among the other 11 unique mutations, 8 were small deletions or insertions predicted to cause frameshifts, 1 was a mutation to a stop codon, 1 was a missense mutation, and 1 was predicted to cause insertion of an isoleucine in the hect domain of the UBE3A protein, which functions in E2 binding and ubiquitin transfer. Eight of the cases were familial, and five were sporadic. In two familial cases and one sporadic case, mosaicism for UBE3A mutations was detected: in the mother of three AS sons, in the maternal grandfather of two AS first cousins, and in the mother of an AS daughter. The frequencies with which we detected mutations were 5 (14%) of 35 in sporadic cases and 8 (80%) of 10 in familial cases.     

Spontaneous and Ethyl Methanesulfonate-Induced Mutations Controlling Viability in DROSOPHILA MELANOGASTER. II. Homozygous Effect of Polygenic Mutations

Polygenic mutations affecting viability were accumulated on the second chromosome of Drosophila melanogaster by treating flies with EMS in successive generations. The treated chromosomes were later made homozygous and tested for their effects on viability by comparison of the frequency of such homozygotes with that of other genotypes in the same culture. The treated wild-type chromosomes were kept heterozygous in Pm/+ males by mating individual males in successive generations to Cy/Pm females. The number of generations of accumulation was 1 to 30 generations, depending on the concentration of EMS. A similar experiment for spontaneous polygenic mutations was also conducted by accumulating mutations for 40 generations. The lower limit of the spontaneous mutation rate of viability polygenes is estimated to be 0.06 per second chromosome per generation, which is about 12 times as high as the spontaneous recessive lethal mutation rate, 0.005. EMS-induced polygenic mutations increase linearly with the number of treated generations and with the concentration of EMS. The minimum mutation rate of viability polygenes is about 0.017 per 10(-4)m, which is only slightly larger than the lethal rate of 0.013 per 10(-4) m. The maximum estimate of the viability reduction of a single mutant is about 6 to 10 percent of the normal viability. The data are consistent with a constant average effect per mutant at all concentrations, but this is about three times as high as that for spontaneous mutants. It is obvious that one can obtain only a lower limit for the mutation rate, since some mutants may have effects so near to zero that they cannot be detected. The possibility of measuring something other than the lower limit is discussed. The ratio of the load due to detrimental mutants to that caused by lethals, the D/L ratio, is about 0.2 to 0.3 for EMS-induced mutants, as compared to about 0.5 for spontaneous mutants. This is to be expected if EMS treatment produces a large fraction of small deletions and other chromosome rearrangements which are more likely to be lethal.     

Mismatch repair and the regulation of phase variation in Neisseria meningitidis

Neisseria meningitidis controls the expression of several genes involved in host adaptation by a process known as phase variation. The phase variation frequency of haemoglobin (Hb) receptors among clinical isolates of serogroups A, B and C differed drastically, ranging from approximately 10(-6) to 10(-2) cfu-1. Frequencies of phase variation are a genetic trait of a particular strain, as two unlinked Hb receptors, hpuAB and hmbR, phase varied with similar frequencies within a given isolate. Based on these frequencies, six Neisserial clinical isolates could be grouped into three distinct classes; slow, medium and fast. An increase in phase variation frequency was accompanied by high rates of spontaneous mutation to rifampicin and nalidixic acid resistance in one medium and one fast strain. The remaining three medium strains displayed elevated levels of phase variation without increases in overall mutability, as they possessed low rates of spontaneous mutation to drug resistance. The mismatch repair system of N. meningitidis was found to play an important role in determining the overall mutability of the clinical isolates. Inactivation of mismatch repair in any strain, regardless of its original phenotype, increased mutability to a level seen in the fast strain. Insertional inactivation of mutS and mutL in the slow strain led to 500- and 250-fold increases in hmbR switching frequency respectively. Concurrently, the frequency of spontaneous point mutations of mutS and mutL mutants from the slow strain was increased 20- to 30-fold to the level seen in the high strain. The status of Dam methylation did not correlate with either the phase variation frequency of Hb receptors or the general mutability of Neisserial strains. Analysis of an expanded set of isolates identified defects in mismatch repair as the genetic basis for strains displaying both the fast Hb switching and high mutation rate phenotypes. In conclusion, elevated frequencies of phase variation were accompanied by increased overall mutability in some N. meningitidis isolates including strains shown to be mismatch repair defective. Other isolates have evolved mechanisms that seem to affect only the switching frequency of phase-variable genes without an accompanied increased accumulation of spontaneous mutations.     

Rifampicin supersensitivity of rho strains of E. coli, and suppression by sur mutation.

Summary Escherichia coli strains with mutations rho-115, rho-ts15, rho-101 (psu-1) or rho-102 (psu-2) are more sensitive (supersensitive) to rifampicin than isogenic parent strains, as measured by growth rate in broth and colony forming efficiency on solid media with 5, 10, or 20 g of rifampicin per ml. There is no change in sensitivity of rho mutants to the antibiotics penicillin, erythromycin, chloramphenicol, or the detergent desoxycholate. The rho-101 or rho-102 mutations confer rifampicin supersensitivity at 32°C but not 42°C. Mutants of a rho-115 strain that have lost polarity suppression can be isolated by selection for rifampicin resistance. This phenotype, Sur, is not due to reversion of the original rho gene mutation but to a second mutation perhaps in the gene for rho protein or the gene for the subunit of RNA polymerase. One class of Sur mutation, occurring in rho-115 cells isolated as resistant to 20 g of rifampicin per ml, is co-transducible with the marker ilv, and the gene order is rbs-ilv-sur-38. A model suggested by this map position is that the mutations rho-115 and sur-38 define the domain of rho protein which interacts with the subunit of RNA polymerase.     

Genetic study of saccharomycete yeast sensitivity to the lethal and mutagenic action of nitrous acid. III. Relation between mitochondrial genome variability and the unstable sensitivity of yeast cells to inactivation by nitrous acid

The sensitivity of the yeast Saccharomyces cerevisiae to nitrous acid (NA) is significantly influenced by various spontaneous mutations of the mitochondrial (mt) genome as well as by the nuclear mutation mmg 1 leading to a decrease in the spontaneous mutability of the mt genome. The mmg 1 locus and the mt genome most probably interact and this nucleo-cytoplasmic interaction plays a role in determining the NA sensitivity of yeast cells. A significant subclonal variation of the NA sensitivity has already been reported for the strains under study. Here we show this variability to decrease significantly when the cells are devoid of the mt DNA or carry the mmg 1 mutation. These data suggest a direct relation between the unstable NA sensitivity and the variability of the mt genome.     

Allele-specific replication of 15q11-q13 loci: a diagnostic test for detection of uniparental disomy.

Allele-specific replication differences have been observed in imprinted chromosomal regions. We have exploited this characteristic of an imprinted region by using FISH at D15S9 and SNRPN (small nuclear ribonucleo protein N) on interphase nuclei to distinguish between Angelman and Prader-Willi syndrome patient samples with uniparental disomy of chromosome 15q11-q13 (n = 11) from those with biparental inheritance (n = 13). The familial recurrence risks are low when the child has de novo uniparental disomy and may be as high as 50% when the child has biparental inheritance. The frequency of interphase cells with asynchronous replication was significantly lower in patients with uniparental disomy than in patients with biparental inheritance. Within the sample population of patients with biparental inheritance, those with altered methylation and presumably imprinting center mutations could not be distinguished from those with no currently detectable mutation. This test is cost effective because it is performed on interphase cells from the same hybridized cytological preparation in which a deletion is excluded, and additional specimens are not required to determine the parental origin of chromosome 15.     

Influencing the spontaneous and chemically induced mutability of E. coli through changing the genetic background

Summary The influence of a second auxotrophic marker to the spontaneous and chemical-induced mutability to prototrophy of a first auxotrophic marker in 7 monoauxotrophs and 31 biauxotrophs of E. coli K 12 was studied by growth layer technics. No case of influence of a second auxotrophy to the mutagen-induced mutability (7 mutagens tested) of the first auxotrophy among 172 possibilities was found. An influence to the spontaneous mutability seemed to be present in 4 cases out of 31. But 3 of them were shown to be imitated by influences of components of the medium to the growth of mutants or parent type. In one case a mutator mutation is responsible for the about 8 times higher rate of the mutation met 1+ in strain met 1/his 7 than in strain met 1. But backmutation and crossing experiments showed that the mutator (mum ⁺) was separate from the auxotrophic marker his 7 (recombination frequency 5/16). This mutator did not increase remarkably the spontaneous mutability of other markers tested (resistence to phages T1 or T4 or to Streptomycin 3, 5, 10, or 100 /ml, or to Chloramphenicol 2 /ml). It is assumed that the spontaneous mutations in the wild-type are at least partially different in their nature from the mutator promoted ones.     

Predicting the contribution of novel POLG mutations to human disease through analysis in yeast model

The yeast Saccharomyces cerevisiae was used to validate the pathogenic significance of eight human mutations in the gene encoding for the mitochondrial DNA polymerase gamma, namely G303R, S305R, R386H, R574W, P625R, D930N, K947R and P1073L, among which three are novel and four are of unclear pathological significance. Mitochondrial DNA extended and point mutability as well as dominance/recessivity of each mutation has been evaluated. The analysis in yeast revealed that two mutations, S305R and R386H, cannot be the sole cause of pathology observed in patients. These data led us to search for a second mutation in compound with S305R and we found a mutation, P1073L, missed in the first genetic analysis. Finally, a significant rescue of extended mutability has been observed for several dominant mutations by treatment with mitochondrial antioxidants.