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Escherichia coli EmrE is a small multidrug resistance protein encompassing four transmembrane (TM) sequences that oligomerizes to confer resistance to antimicrobials. Here we examined the effects on in vivo protein accumulation and ethidium resistance activity of single residue substitutions at conserved and variable positions in EmrE transmembrane segment 2 (TM2). We found that activity was reduced when conserved residues localized to one TM2 surface were replaced. Our findings suggest that conserved TM2 positions tolerate greater residue diversity than conserved sites in other EmrE TM sequences, potentially reflecting a source of substrate polyspecificity., (Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Disorders of mitochondrial DNA (mtDNA) maintenance have emerged as an important cause of human genetic disease, but demonstrating the functional consequences of de novo mutations remains a major challenge. We studied the rate of depletion and repopulation of mtDNA in human fibroblasts exposed to ethidium bromide in patients with heterozygous POLG mutations, POLG2 and TK2 mutations. Ethidium bromide induced mtDNA depletion occurred at the same rate in human fibroblasts from patients and healthy controls. By contrast, the restoration of mtDNA levels was markedly delayed in fibroblasts from patients with compound heterozygous POLG mutations. Specific POLG2 and TK2 mutations did not delay mtDNA repopulation rates. These observations are consistent with the hypothesis that mutations in POLG impair mtDNA repopulation within intact cells, and provide a potential method of demonstrating the functional consequences of putative pathogenic alleles causing a defect of mtDNA synthesis., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Saccharomyces cerevisiae is a good model with which to study the effects of morphologic differentiation on the ecological behaviour of fungi. In this work, 33 morphologic mutants of a natural strain of S. cerevisiae, obtained with UV mutagenesis, were selected for their streak shape and cell shape on rich medium. Two of them, showing both high sporulation proficiency and constitutive pseudohyphal growth, were analysed from a genetic and physiologic point of view. Each mutant carries a recessive monogenic mutation, and the two mutations reside in unlinked genes. Flocculation ability and responsiveness to different stimuli distinguished the two mutants. Growth at 37 degrees C affected the cell but not the colony morphology, suggesting that these two phenotypes are regulated differently. The effect of ethidium bromide, which affects mitochondrial DNA replication, suggested a possible "retrograde action" of mitochondria in pseudohyphal growth.

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 30-40kbp 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.

Myxococcus xanthus RppA and MmrA are homologous to methyl-accepting chemotaxis proteins (MCPs) and to multidrug transporters, respectively. We reported previously that rppA-mmrA double mutant exhibited reduced colony expansion, agglutination, and polysaccharide levels. We have demonstrated here that the rppA-mmrA mutant also exhibited reduced amino acid uptake. Furthermore, the double mutant appeared to be more susceptible to some antimicrobial agents, such as streptomycin, ethidium bromide and norfloxacin, than the wild-type. These phenotypes were not shown in the rppA or mmrA single mutant. These results indicate that M. xanthus RppA and MmrA are also involved in the uptake of amino acids and efflux of some antimicrobial agents.

Autosomal dominant and recessive forms of progressive external ophthalmoplegia (adPEO and arPEO) are mitochondrial disorders characterized by the presence of multiple deletions of mitochondrial DNA in affected tissues. Four adPEO-associated missense mutations have been identified in the ANT1 gene. In order to investigate their functional consequences on cellular physiology, we introduced three of them at equivalent positions in AAC2, the yeast orthologue of human ANT1. We demonstrate here that expression of the equivalent mutations in aac2-defective haploid strains of Saccharomyces cerevisiae results in (a) a marked growth defect on non-fermentable carbon sources, and (b) a concurrent reduction of the amount of mitochondrial cytochromes, cytochrome c oxidase activity and cellular respiration. The efficiency of ATP and ADP transport was variably affected by the different AAC2 mutations. However, irrespective of the absolute level of activity, the AAC2 pathogenic mutants showed a significant defect in ADP versus ATP transport compared with wild-type AAC2. In order to study whether a dominant phenotype, as in humans, could be observed, the aac2 mutant alleles were also inserted in combination with the endogenous wild-type AAC2 gene. The heteroallelic strains behaved as recessive for oxidative growth and petite-negative phenotype. In contrast, reduction in cytochrome content and increased mtDNA instability appeared to behave as dominant traits in heteroallelic strains. Our results indicate that S. cerevisiae is a suitable in vivo model to study the pathogenicity of the human ANT1 mutations and the pathophysiology leading to impairment of oxidative phosphorylation and damage of mtDNA integrity, as found in adPEO.

Background: The Saccharomyces cerevisiae strain, 22574d, lacks gamma amino butyric acid (GABA) transport activity and cannot grow on this amino acid as sole nitrogen source. Both transport of and growth on this amino acid are restored when the yeast is transformed with a form of mouse gamma actin containing an extended C-terminal sequence (M-g-A). The nature of the mutation in the transporter as well as the complementation mode are addressed., Material/methods: The cDNA sequence of the mutated transporter was achieved using reverse transcription, 3'-Race and cloning. For detection, Northern blotting and labeling with 32-P were used. The putative ability of the transporter to interact with gamma actin in vivo was examined by following the interaction in vitro of synthetic peptides corresponding to the C-termini of the gamma actin and the transporter., Results: Up to codon 394 the mutated and native transporters are identical. At the 3' end, the mutant is by extended by 32 codons from the delta region of a Ty1 transposon, giving an open reading frame of 426 codons, and a predicted structure of 9 of the 11 transmembrane domains. Peptides corresponding to the carboxy terminal regions of the truncated transporter and the elongated species of gamma actin show the potential to interact in vitro., Conclusions: The mutated GABA transporter mRNA contains an insert from the delta region of a Ty1 transposon. This insertion allows expression of a transporter capable of interacting with elongated gamma actin to rehabilitate the transporter.

The 7472insC mitochondrial DNA mutation in the tRNA(Ser(UCN)) gene is associated with sensorineural deafness combined, in some patients, with a wider neurological syndrome. In cultured cybrid cells it causes a 70% decrease in tRNA(Ser(UCN)) abundance and mild respiratory impairment, previously suggested to be due to decreased tRNA stability. When mitochondrial transcription was blocked by ethidium bromide treatment, the half-life of the mutant tRNA was not significantly different from that of wild-type tRNA(Ser(UCN)). Over-expression of mitochondrial translational elongation factor EF-Tu also had no effect on the mutant phenotype. However, during recovery from prolonged ethidium bromide treatment, the synthesis of the mutant tRNA(Ser(UCN)) was specifically impaired, without polarity effects on downstream tRNAs of the light strand transcription unit. We infer that the mutation acts posttranscriptionally to decrease tRNA(Ser(UCN)) abundance by affecting its synthesis rather than its stability. The extent of aminoacylation of the mutant tRNA was also decreased by approximately 25%. In contrast, the mutation had no detectable effect on tRNA(Ser(UCN)) base modification or structure other than the insertion of an extra guanosine templated by the mutation, which was structurally protected from nuclease digestion like the surrounding nucleotides. These findings indicate a common molecular process underlying sensorineural deafness caused by mitochondrial tRNA(Ser(UCN)) mutations.

The luminous intensity of dark variant (S1) separated from photobacterium phosphoreum (A2) was 1/10,000 less than that of wild-type. Ethidium bromide (EB) (0.6 mg/L), Mytomycin C (MC, 0.05 mg/L), 2-amino fluorene (2-AF, 1.0 mg/L) all could strongly induce reversion mutation for S1 within 24 h and increase reversion ratio significantly. The results of experiments indicated that these revertants had stable genetic characteristic and the mutation may take place at gene levels. The mutagenesis to S1 caused by EB, MC and 2-AF was detected and it may be used as a new rapid, simple and sensitive method for gene toxicant monitoring.

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.

Mutants defective in the synthesis of antitumor angucycline antibiotic landomycin E were obtained from the asporogenic and initial sporulating strains of Streptomyces globisporus 1912 by means of nitrosoguanidine treatment and UV-irradiation of the mycelium fragments and protoplasts. The frequency of induction of LanE mutants in the protoplasts of strain 3-1 was 3 times higher (0.55%) as compared to mycelium fragments (0.17%). After mutagenic treatment of spores the yield of such mutants was lower (0.044-0.071%). It was shown, that izoflavon daidzein was an effective and necessary regulator of the landomycin E synthesis. The collection of 53 landomycin defective mutants was divided into three groups on the basis of their ability to produce antibiotic and regulator: 1) mutants LanE-Dai+ (45 strains), which did not synthesize landomycin E, but produced daidzein; 2) mutants LanE+Dai- (4 strains), which could restore landomycin E synthesis after adding exogenic daidzein to the medium and 3) mutants LanE- Dai- which did not synthesize landomycin E in the presence of daidzein.

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.

Specific mgi mutations in the alpha, beta or gamma subunits of the mitochondrial F1-ATPase have previously been found to suppress rho0 lethality in the petite-negative yeast Kluyveromyces lactis. To determine whether the suppressive activity of the altered F1 is dependent on the F0 sector of ATP synthase, we isolated and disrupted the genes KlATP4, 5 and 7, the three nuclear genes encoding subunits b, OSCP and d. Strains disrupted for any one, or all three of these genes are respiration deficient and have reduced viability. However a strain devoid of the three nuclear genes is still unable to lose mitochondrial DNA, whereas a mgi mutant with the three genes inactivated remains petite-positive. In the latter case, rho0 mutants can be isolated, upon treatment with ethidium bromide, that lack six major F0 subunits, namely the nucleus-encoded subunits b, OSCP and d, and the mitochondrially encoded Atp6, 8 and 9p. Production of rho0 mutants indicates that an F1-complex carrying a mgi mutation can assemble in the absence of F0 subunits and that suppression of rho0 lethality is an intrinsic property of the altered F1 particle.

Five spontaneous mutants of Streptomyces lividans TK64 resistant to 5 or 15 microM ethidium bromide (EB) were isolated, and the corresponding mutations were mapped to two different chromosomal locations. Both types of mutations conferred unselected resistance to several basic dyes and norfloxacin. The strain with the low-level resistance exhibited wild-type levels of EB uptake and energy-dependent efflux, and the resistance mechanism is unclear. The highly resistant mutants, which additionally were resistant to phosphonium ions, had a reduced accumulation and an increased efflux of EB, reminiscent of a mammalian multidrug resistance efflux pump.

Defects in the RAD52 gene of the yeast Saccharomyces cerevisiae reduce the levels of the NUC2 endo-exonuclease by approximately 90% compared to the levels in wild-type strains. To examine the potential role of this nuclease in the induction of mitochondrial 'petite' mutations, congenic RAD52 and rad52-1 haploids were subjected to treatment with ethidium bromide, a well-known inducer of these mutations. The rad52 strain showed a much higher resistance to ethidium bromide-induced petite formation than the corresponding wild-type strain. Two approaches were taken to confirm that this finding reflected the nuclease deficiency, and not some other effect attributable to the rad52-1 mutation. First, a multicopy plasmid (YEp213-10) carrying NUC2 was transformed into a RAD52 strain. This resulted in an increased fraction of spontaneous petite mutations relative to that seen for the same strain without the plasmid and sensitized the strain carrying the plasmid to petite induction by ethidium bromide treatment. Second, a strain having a nuc2 allele that encodes a temperature-sensitive nuclease was treated with ethidium bromide at the restrictive and permissive temperatures. Petite induction was reduced under restrictive conditions. Enzyme assays revealed that the RAD52 (YEp213-10) strain had the highest level of antibody-precipitable NUC2 endo-exonuclease whereas the nuc2 and rad52 mutants had the lowest levels. Furthermore, addition of ethidium bromide to the reaction mixture stimulated the activity of the nuclease on double-stranded DNA. Petite induction by antifolate-mediated thymine nucleotide depletion was also inhibited by inactivation of RAD52 indicating that the effect of reduced NUC2 endo-exonuclease was not restricted to ethidium bromide treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Sodium dodecyl sulfate (SDS) was examined for its effect on petite and sectored colony induction in Saccharomyces cerevisiae by propidium iodide (PI) and ethidium bromide (EB). 4-h cultivation with 100 microM PI and 100 micrograms/ml SDS resulted in virtually all plated cells growing as sectored colonies with no decrease in viability. Sectored colonies are mixed colonies comprised of respiratory deficient and competent cells believed to be derived from an unstable respiratory deficient cell. Further cultivation with PI and SDS prior to plating led to induction of complete petite colonies with a rapid decrease in viable cells. PI alone at this concentration exhibited weak induction of sectored colonies (maximum 12.3% at 8 h) and petite colonies (maximum 10.8% at 12 h), but SDS alone caused induction of neither. 50 microM PI had almost the same activity as 100 microM except for a delay in the induction of sectored colonies in the initial stage, and a decreased rate of petite colony induction. The effects of 20 microM PI and SDS were much lower than that by 50 microM and no inhibition of growth was observed. 10 microM PI was quite inactive even in the presence of SDS. Under resting conditions, 10 approximately 100 microM PI and 100 micrograms/ml SDS induced about 60% sectored colonies at 12 h incubation and more than 60% petite colonies at 24 h. After 6 h incubation, decrease in survival was also observed.

The effect of ethidium bromide on the growth of a yeast mutant with an impaired mitochrondrial translocation system of adenine nucleotides (op-1 mutant) was investigated. It was found that the op-1 mutant stops growing both under growing and non-growing conditions after treatment with ethidium bromide and that the growth cannot be restored by adding low-molecular compounds to the growth medium. It was the aim of the experiments to clarify whether the cessation of growth of the op-1 mutant after induction of the rho- mutation can be simulated by inhibitors phenotypically changing the mitochondrial function. It appears likely that the op-1 mutant stops growing only after the rho- mutation has been induced, because the phenotypic simulation of the rho- mutation does not lead the cessation of growth of the op-1 mutant.

The induction of variants of L5178Y mouse-lymphoma cells resistant to 1-beta-D-arabinofuranosylcytosine (ara-C) has been investigated. The spontaneous mutation frequency was low, around 3 X 10(-8). Resistant variants were induced in a dose-dependent fashion following treatment with ethyl methanesulphonate (EMS), ethidium bromide and 2-acetylaminofluorene, but not with gamma-irradiation. With EMS as mutagen the frequency of ara-C-resistant variants was much lower than that obtained using other selective agents, whereas it was higher with the mutagens acetylaminofluorene and ethidium bromide. Out of 42 resistant clones examined, 38 showed normal sensitivity to thymidine and were destignated as Class 1, the remaining four (Class 2) were usually resistant to thymidine. Cells in Class 1 did not accumulate detectable amounts of ara-C and had negligible deoxycytidine kinase activity. They were about 3000-fold more resistant to the toxic effects of ara-C than were wild-type cells. Cells in Class 2 had a reduced ability to accumulate are-C and had 50% of the deoxycytidine kinase activity of wild-type cells. They were only 20 times more resistant to ara-c than were wild-type cells. The enzymatic defect in these cells is obscure but the phenotypic properties probably result from subtle alterations in the pool of deoxycytidine nucleotides.

The survival of the rho(+) factor and of Drug(R) mitochondrial genetic markers after exposure to ethidium bromide has been studied. A technique allowing the determination of Drug(R) genetic markers among a great number of both grande and petite colonies has been developed. The results have been analyzed by the target theory. The survival of the rho(+) factor is always less than the survival of any Drug(R) genetic marker. The survivals of C(R) and E(R) are similar to each other, while that of O(R) is greater than that of the other two Drug(R) markers. All possible combinations of Drug(R) markers have been found among the rho(-) petite cells induced, while the only type found among the grande colonies is the preexisting one. The loss of the C(R) and E(R) genetic markers was found to be the most frequently concomitant, while the correlation between the loss of the O(R) marker and the other two Drug(R) markers is less strong. Similar results have been obtained after U.V. irradiation. Interpretations concerning the structure of the yeast mitochondrial genome are given and hypotheses on the mechanism of petite mutation discussed.

In contrast to the wild-type, mutant [ANTr8] is able spontaneously to throw off stable respiratory deficient mutants. The frequency of these mutants is considerably enhanced by treatment with ethidium bromide (EB) or the azo-dye Janus green (JG). An unstable cell state with a petite-like phenotype is found in both mutant [ANTr8] and wild-type after EB-treatment. However, only in the mutant is this unstable cell state followed by the appearance of stable respiratory deficient (RD) mutants. Formation of microcolonies is observed both in [ANTr8] and wild-type. RD mutants were isolated after EB treatment. Three of them (mit-12, mit-25, and mit-30) were analyzed and mit-25 characterized in more detail.

Staphylococcus aureus strain S-6, which produces enterotoxin type B (SEB), and strain 10-275, a high toxin-producing mutant derived from S-6, display pronounced differences in dye sensitivity, osmotic stability, and bacitracin sensitivity. Such characteristics are consistent with the concept that strain 10-275 is a membrane mutant of strain S-6. Some membrane mutants of S. aureus strain 14458 exhibit about two- to three-fold increases in SEB production whereas other membrane mutants show about twofold increases in alpha-hemolysin production. It is suggested that specific and independent membrane mutations control the secretory processes resulting in the extracellular elaboration of these exoproteins.

Alterations in the physical characteristics of mitochondrial DNA accompanied increased spontaneous mutability to cytoplasmic respiratory-deficiency in yeast. Two systems were used to modify mutation rates, one physiological, the other genetic. Cells in log phase were shown to be more mutable than cells in stationary phase, and glucose-repressed cells were shown to be more mutable than unrepressed cells. A nuclear gene which acts as a mitochondrial mutator was found to increase spontaneous mutation rate by a factor of ten. An increase in endogenous formation of G+G-rich fragments of mt-DNA accompanied a physiological state conducive to higher mutability, and it is proposed that increased in vivo digestion of A+T-rich regions is involved in these alterations. Greater nuclease(s) activity accompanied the presence of the mutator gene, and it is proposed that this gene is concerned with the regulation of nuclease activity or with repair mechanisms.

The uptake of ethidium bromide by Escherichia coli K 12 cells has been studied by using 14C-labeled ethidium and spectrofluorometry on three E. coli strains: the first one (AB1157) has an ethidium-resistant phenotype; the second one derives from the first one after a single mutation (at 10 min on the E. coli genetic map) and has an ethidium-sensitive (Ebs) phenotype; the third one is the acrA strain which appeared to have the same phenotype as the Ebs strain. When the cells are in exponential growth, no ethidium enters wild-type cells, and a very limited amount of ethidium enters Ebs and acrA cells. Massive quantities of ethidium enter AB1157, Ebs, and acrA cells treated by uncouplers and respiring Ebs cells treated by the membrane ATPase-inhibitor dicyclohexylcarbodiimide. A small amount of ethidium enters cells treated in M9 succinate medium by metabolic inhibitors such as KCN or cells starved with oxygen in the same M9 medium. The amount of ethidium and ethidium dimer retained at equilibrium by either type of cell, and by cells infected by T5 phage, as well as the kinetics of influx and efflux, has been measured under a variety of situations (membrane energized or not, and/or membrane ATPase inhibited or not). Furthermore, it was shown that ethidium binds to both RNA and DNA when it enters CCCP-treated wild-type E. coli cells, whereas it binds mainly to DNA when it enters Ebs and acrA cells in exponential growth. As it will be discussed, it is difficult to account for the EthBr uptake by invoking only membrane functions and active transport. Therefore, it is proposed that the variations of the nucleic acid accessibility in E. coli cells might play a role in the control of this uptake. Accordingly, in ethidium-sensitive cells, the mutation would have caused a significant part of the chromosomal DNA (10-20%) to become accessible to ethidium. Hansen [Hansen M. T. (1982) Mutat. Res. 106, 209-216], after a study of the photobinding of psoralen to nucleic acids in the acrA mutant, also suggested that DNA environment was modified in acrA cells.

Acridine orange (AO), ethidium bromide (EB), ethyl methanesulfonate (EMS) and 8- ethoxycaffeine ( EOC ) were fed to larvae of Drosophila melanogaster in order to test their capacity for the induction of meiotic recombination in males. Our results show that AO and EB increase significantly the male recombination frequencies. No relationship between chromosome breakage ability and male recombination induction was found since EMS and EOC , two effective chromosome-breaking agents, were unable to increase the male recombination.

A strain of Saccharomyces cerevisiae (MH41-7B/011) was resistant to petite induction by ethidium bromide at 30 degrees, but was sensitive to induction by photolabeling with ethidium monoazide. These results suggested a defect in the mutant in metabolic activation of ethidium to account for its resistance. Synchronized cultures of both the mutant and the normal parent strains showed a substantial reduction in petite response to photolabeling in stationary phase cells which could not be accounted for by changes in cell penetration of the drug. The use of photolabeling with normal and mutant cells suggested that petite induction can be divided into early and late steps.

A genetic locus, aroT, located between chr and the trp operon in Salmonella typhimurium, and similar genes, aroR and aroS, near the trp locus of Escherichia coli, were found to be involved in the transport of aromatic amino acids. Genetic lesions at these loci cause a variable diminution in uptake and accumulation of aromatic amino acids, alanine and glycine compared with the wild type. The F'trp episome carries the aro R locus. Curing an E. coli strain of the F'trp episome which covers a chromosomal deletion from cysB through the trp operon and tonB regions, results in a 60 to 80% decrease in tryptophan uptake. The introduction of F'trp into a trp operon-deleted S. typhimurium of low transport ability restores transportability, suggesting that aroT in this organism may be homologous with aroR in E. coli. In E. coli, tryptophan accumulation is normally increased by prior growth in L-tryptophan, while in S. typhimurium it is repressed. In both genera, the trpR gene appears to have no effect on the tryptophan transport capabilities in response to changes in the concentration of L-tryptophan in the medium. Tryptophan transport in the S. typhimurium F'trp hybrid was subject to repression, while in the E. coli strain which carries F'trp covering the equivalent chromosomal delection, an increase in tryptophan accumulation was shown after growth in L-tryptophan supplemented medium.

A method for achieving strand specific nicking of DNA has been developed. Phosphorothioate groups were incorporated enzymatically into the (-)strand of M13 RF IV DNA. When such DNA is reacted with restriction endonucleases in the presence of ethidium bromide nicked DNA (RF II) is produced. All of the restriction enzymes tested linearised phosphorothioate-containing DNA in the absence of this dye. The strand specificity of the reaction was investigated by employing the ethidium bromide mediated nicking reaction in the phosphorothioate-based oligonucleotide-directed mutagenesis method. The mutational efficiencies obtained were in the region of 64-89%, indicating that these restriction enzymes hydrolyse the phosphodiester bond at the cleavage site of the unsubstituted (+)strand.

The production of petite mutations by different acridine analogs was studied in Saccharomyces cerevisiae. Compounds with amino substituents at the 2 and 3 positions of the acridine nucleus and methylation at position 10 were effective for petite induction in growing cells but not in resting cells, while those with chloro, nitro and methoxy substituents were not effective in either resting or growing cells. Photosensitive azido derivatives of the acridines were tested to evaluate the role of covalent drug attachment for mutagenesis in resting cells. Photolysis of resting cells with 9-axido, 3-azido-6-amino-, 9-azido-10-methyl-, or 3-azido-6-amino-10-methyl-acridine was highly toxic. 3-Azido-6-amino-acridine, and especially 3-azido-10-methyl-, and 3-azido-6-amino-10-methyl-acridine, were effective petite inducers in resting cells. Thus, the photosensitive (azido) group at position 9 produced only cell killing while the azido group at position 3 and/or 6 led to effective petite induction in resting cells. In contrast, petite induction was observed only for growing cells, for dark control experiments with these compounds or with the monoazide precursor compounds.

A series of mutants called ebi, less inducible by ethidium bromide than the parental strain for the rho+ leads to rho- mutation have been isolated after E.M.S. mutagenesis. Some of the ebi mutants also show an important accumulation of rho- cells, in the absence of ethidium bromide. Ebi mutations are nuclearly inherited as shown by meiotic segregation. The effects of these mutants on the transmission and recombination of mitochondrial genes among the diploid progeny of crosses have been studied. Some of the ebi mutants show a non coordinated transmission of the oli1 mitochondrial marker with respect to other mitochondrial markers unexpected for homosexual crosses. This bias which is independent from omega will be discussed in relation to the segregation and recombination. No significant decrease of the frequency of recombinants has been detected.

The germination and outgrowth of Saccharomyces cerevisiae ascospores were studied by determining the sensitivity of the ascospores to the action of chemical mutagens. Survival of the ascospores after N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) treatment was low during the first 2 h of germination and then increased and remained constant. Survival of the ascospores after 2-methoxy-6-chloro-9-(3-[ethyl-2-chloroethyl]aminopropylamino)acridine-2HC1 (ICR-170) treatment was constant from 0 to 5 h, but as the ascospores completed outgrowth at 6 h they became more sensitive to killing by ICR-170. Survival of the ascospores remained high during treatment with 2-methoxy-6-chloro-9-(3-[ethyl-2-hydroxyethyl]aminopropylamino)acridine-2HC1 (ICR-170-OH) or 2,7-diamino-10-ethyl-9-phenyl-phenanthridinium bromide. The main classes of mutations screened for were petites and auxotrophs. The induction of petites and auxotrophs by MNNG was independent of the stage of germination and outgrowth treated. Petite induction by ICR-170 was dependent upon the stage of germination and outgrowth treated. The early hours of germination (0 to 3 h) were not sensitive to petite induction. However, there was maximal petite induction at 5 h into germination and outgrowth, followed by a decline. During this same time period, ICR-170 induced less than 1% auxotrophic colonies. This finding is very unusual because ICR-170 induced 15% auxotrophic colonies in starved log-phase cultures of S. cerevisiae. The acridine ICR-170-OH induced no mutations during germination and outgrowth of the ascospores. Ethidium bromide induced petites, and the petite frequency became maximal at 5 h of germination and outgrowth, a result similar to that obtained with ICR-170.

Upon incubation with ethidium bromide (Etd Br) isolated mitochondria of Saccharomyces cerevisiae have been shown to be capable of performing five novel reactions: (a) a single scission of their DNA (mtDNA) coincident with (b) the incorporation of Etd Br into the product (mtDNA --> 2 mtDNA'-Etd Br); (c) an energy- (and probably ATP-) requiring degradation of mtDNA'-Etd Br to acid-labile products. These reactions acting in series generate (d) a DNase dependent on both Etd Br and an energy supply with mtDNA'-Etd Br as an obligatory intermediate. Coincident with (d) there occurs (e) an activation of adenosinetriphosphatase. Experiments with specific inhibitors suggest that the enzyme responsible is the mitochondrial adenosinetriphosphatase complex itself, and that it and the ability to carry out reactions (a) through (e) are tightly coupled to the energy-transducing functions of the particle.

Ethidium bromide (EB) and ultraviolet light (UV) in combination are known to produce a synergistic induction of "petite" mutants in yeast. Two other agents were combined with EB, 3-Carbethoxypsoralene (3 CPs) activated by 365 nm light or gamma rays. EB in combination with 3 CPs also resulted in an enhanced production of "petite" mutants. After the photoaddition of 3 CPs in exponential phase cells, recovery of the "petite" mutation during dark liquid holding was inhibited by the presence of EB producing an enhanced number of "petite" mutants. The behavior of mitochondrial antibiotic resistance markers after individual and combined treatments with EB and 3 CPs indicates a random loss of markers after EB and a preferential loss of a certain region for the 3 CPs photoaddition. The combination of the two agents leads to an additivity of total drug marker losses rather than a synergistic loss. The combination of EB with gamma rays produced no enhancement in "petite" induction. A combination of UV and 3 CPs showed a synergistic interaction for "petite" induction. These results indicate that the three agents, EB, UV and 3 CPs photoaddition may share a common repair step for mitochondrial lesions.

Frameshift mutations have been produced in specific repair-negative Salmonella tester strains by photoaffinity labeling technique using ethidium azide. Reversions requiring a +1 addition or a -2 deletion were specially sensitive. Mutagenesis was reduced by the simultaneous addition of non-mutagenic ethidium bromide, and was prevented by photolysis of the azide prior to culture addition. Identical tester strains active in DNA excision repaire were not mutagenized by the azide. These results are consistent with the interpretation that photolysis of the bound ethidium analog converts the drug from its noncovalent mode of binding (presumably intercalation) to a covalent complex with consequent production of frameshift mutations. Such photoaffinity labeling by drugs which bind to DNA not only confirms the importance of covalent drug attachment for frameshift mutagenesis, but also provides powerful techniques for studying the molecular deatils of a variety of genetic mechanisms.

The rate of structural chromosome mutations at metaphase of the first mitosis was determined in culture of embrionic mouse fibroblasts after UV-irradiation during the S-period (lambda = 265 nm at an incident dose of 40 erg/mm2). It is established that the mutation rate is higher at late metaphase than at early metaphase. After the cell treatment with intercalating compounds (actinomycin D, acridine orange or ethidium bromide) at metaphase, the rate of UV-induced chromosome aberrations was decreased (about 2-fold). It is concluded from the results obtained that the majority of aberrations arise during metaphase after UV-irradiation in the process of DNA synthesis. After the cell treatment with o-methylhydroxylamine (OMHA) during the S-period the rate of structural mutations was the same at late and early metaphases. This rate was not affected by the caffeine treatment at metaphase; during this stage the acentric chromosome fragments lie outside the equatorial plate, which is an indication that the OMHA-induced aberrations, in contrast to the UV-induced aberrations, are formed before the beginning of metaphase, possibly during the interphase. It is suggested that the chromosome condensation during metaphase is of importance in the formation of structural mutations.

The anticancer drugs, adriamycin and daunorubicin, as well as two other DNA reagents, ethidium bromide and 9-aminoacridine, all exert a differential inhibitory effect on nucleotide incorporation for purified DNA polymerases induced by mutant and wild-type bacteriophage T4. When compared with DNA polymerase of wild-type phage, antimutator enzymes are inhibited to a far greater extent and mutator enzymes to a lesser extent. In contrast, the polymerase-associated 3'-exonuclease activities of wild type and mutants are also inhibited by the compounds but nondifferentially.

Chloramphenicol-resistant mutants of Kluyveromyces lactis were isolated following manganese mutagenesis. Mutants were characterized biochemically in order to determine the effect of CAP on respiration. The genetic basis of the resistance was determined using mitotic segregation analysis of fused hybrids where the transmission of mitochondrial genes from a parent in each parasexual cross was impaired by treatment with ethidium bromide.

Petite strains in Saccharomyces exhibit enhanced spontaneous mutation rates of nuclear genes regardless of whether they are cytoplasmically or nuclearly inherited, or whether or not the cytoplasmic petite strains have mitochondrial DNA. In petite strains, the mutation rate for the nonsense allele lys1-1 is enhanced by a factor of 3-6 and for the missense allele his1-7 by a factor of 2 as compared with their grande counterparts. The reversion of a third allele, the putative frameshift mutation, hom3-10, is not enhanced in a petite background. The results indicate that the spontaneous mutation rate of an organism can be altered by indirect intracellular influences.

Genetic analysis of venturicidin-resistant mutants has revealed the presence of both nuclear and mitochondrial genes responsible for determining venturicidin sensitivity/resistance in Saccharomyces cerevisiae. Recombination studies show that the mutation with phenotype VENR is situated at mitochondrial locus OL I and is therefore extremely useful of future genetic manipulations as it gives a unique phenotype to this locus distinguishable from the second oligomycin locus OL II. The mutations with phenotype VENR OLYR are linked to oligomycin locus OL I and have been allocated a new mitochondrial locus, namely OL III. Three factor croses involving the venturicidin mutations at loci OL I and OL III have shown them to freely recombine with the other mitochondrial loci R I, R III and OL II. The mitochondrial genetic map is therefore represented as four 'recombinational linkage groups'. A fifth linkage group is also specified for mutants with phenotype VENR TETR, and is probably located on a separate DNA molecule from the four other groups.