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1.
Bavistan is known to be a potent inducer of chromosome malsegregation in Saccharomyces cerevisiae. The influence of different factors on the induction of chromosome malsegregation in S. cerevisiae D61.M was investigated. With both standard protocols used (16 h overnight incubation and cold treatment protocol) bavistan, in a concentration range of 2.5-20 micrograms/ml, induced malsegregants to the same extent. The frequencies of malsegregants obtained were not influenced by the plating volume used on selective medium. Induction of malsegregants and toxicity became stronger with increasing supplementation of the incubation medium with yeast extract and peptone. The effects of bavistan on chromosome malsegregation were more pronounced at 28 degrees C--the normal temperature for yeast growth--as compared to 33 and 37 degrees C. A study of the time dependence of the induction of chromosome loss showed that malsegregants can already be detected after 8 h and 1.5 h (second incubation period) using the incubation protocols without and with cold treatment, respectively. To clarify whether a selection towards malsegregants occurs, the growth of mixed cultures of red, cycloheximide-sensitive cells and white, cycloheximide-resistant, leucine-auxotrophic cells prepared at different ratios was compared. A strong selection towards red cells and against the malsegregants was observed. In addition, bavistan was tested for genotoxic activity in Salmonella (Ames test) and in yeast S. cerevisiae D7. No mutagenic activity was detected using S. cerevisiae D7 (gene conversion, reverse mutation, mitotic crossing-over) with and without rat-liver S9. In contrast bavistan induced histidine revertants in the frameshift strains TA1537, TA1538, TA97 and TA98 of Salmonella typhimurium after addition of an exogenous metabolic activation system.  相似文献   

2.
The yeast Saccharomyces cerevisiae is a unicellular fungus that can be cultured as a stable haploid or a stable diploid . Diploid cultures can be induced to undergo meiosis in a synchronous fashion under well-defined conditions. Consequently, yeasts can be used to study genetic effects both in mitotic and in meiotic cells. Haploid strains have been used to study the induction of point mutations. In addition to point mutation induction, diploid strains have been used for studying mitotic recombination, which is the expression of the cellular repair activities induced by inflicted damage. Chromosomal malsegregation in mitotic and meiotic cells can also be studied in appropriately marked strains. Yeast has a considerable potential for endogenous activation, provided the tests are performed with appropriate cells. Exogenous activation has been achieved with S9 rodent liver in test tubes as well as in the host-mediated assay, where cells are injected into rodents. Yeast cells can be recovered from various organs and tested for induced genetic effects. The most commonly used genetic end point has been mitotic recombination either as mitotic crossing-over or mitotic gene conversion. A number of different strains are used by different authors. This also applies to haploid strains used for monitoring induction of point mutations. Mitotic chromosome malsegregation has been studied mainly with strain D6 and meiotic malsegregation with strain DIS13 . Data were available on tests with 492 chemicals, of which 249 were positive, as reported in 173 articles or reports. The genetic test/carcinogenicity accuracy was 0.74, based on the carcinogen listing established in the Gene-Tox Program. The yeast tests supplement the bacterial tests for detecting agents that act via radical formation, antibacterial drugs, and other chemicals interfering with chromosome segregation and recombination processes.  相似文献   

3.
The diploid yeast strain BR1669 was used to study induction of mitotic and meiotic chromosome gain by selected chemical agents. The test relies on a gene dosage selection system in which hyperploidy is detected by the simultaneous increase in copy number of two alleles residing on the right arm of chromosome VIII: arg4-8 and cup1S (Rockmill and Fogel. 1988; Whittaker et al., 1988). Methyl methanesulfonate (MMS) induced mitotic, but not meiotic, chromosome gain. Methyl benzimidazol-2-yl carbamate (MBC) and ethyl methanesulfonate (EMS) induced both mitotic and meiotic chromosome gain. Propionitrile, a polar aprotic solvent, induced only mitotic chromosome gain; a reliable response was only achieved by overnight incubation of treated cultures at 0 degrees C. MBC is postulated to act by binding directly to tubulin. The requirement for low-temperature incubation suggests that propionitrile also induces aneuploidy by perturbation of microtubular dynamics. The alkylating agents MMS and EMS probably induce recombination which might in turn perturb chromosome segregation. Cyclophosphamide monohydrate and dimethyl sulfoxide (DMSO) failed to induce mitotic or meiotic chromosome gain.  相似文献   

4.
Phenobarbital (PB) specifically induces mitotic chromosomal malsegregation in the diploid Saccharomyces cerevisiae strain D61.M but no other genetic events such as mitotic recombination or point mutations. In accordance with the hypothesis that PB exerts its genotoxic activity by an interaction with tubulin, it stimulates the GTP-promoted assembly of porcine brain tubulin in vitro. This process is reversible thus excluding an unspecific denaturation of the tubulin protein by PB.  相似文献   

5.
MMS induced mitotic recombination but not mitotic chromosome loss when tested in pure form in strain D61.M of Saccharomyces cerevisiae, confirming previous results of Albertini (1991), whereas in Aspergillus nidulans it also induced chromosomal malsegregation in addition to mitotic recombination (Käfer, 1988). However, induction of mitotic chromosome loss was observed in combination with strong inducers of chromosome loss such as the aprotic polar solvents ethyl acetate and to a lesser extent methyl ethyl ketone but not with γ-valerolactone and propionitrile. In addition to this, 4 solvents, dimethyl formamide, dimethyl sulfoxide, dioxane and pyridine, enhanced the MMS-induced mitotic recombination in strain D61.M. An enhancement of MMS-induced mitotic recombination and reverse mutation could be demonstrated for ethyl acetate and γ-valerolactone in yeast strain D7.  相似文献   

6.
The purpose of this study was to examine the effect of extremely low frequency (ELF) magnetic fields on the induction of genetic damage. In general, mutational studies involving ELF magnetic fields have proven negative. However, studies examining sister-chromatid exchange and chromosome aberrations have yielded conflicting results. In this study, we have examined whether 60-Hz magnetic fields are capable of inducing mutation or mitotic recombination in the yeast Saccharomyces cerevisiae. In addition we determined whether magnetic fields were capable of altering the genetic response of S. cerevisiae to UV (254 nm). We measured the frequencies of induced mutation, gene conversion and reciprocal mitotic crossing-over for exposures to magnetic fields alone (1 mT) or in combination with various UV exposures (2-50 J/m2). These experiments were performed using a repair-proficient strain (RAD+), as well as a strain of yeast (rad3) which is incapable of excising UV-induced thymine dimers. Magnetic field exposures did not induce mutation, gene conversion or reciprocal mitotic crossing-over in either of these strains, nor did the fields influence the frequencies of UV-induced genetic events.  相似文献   

7.
Genetic effects of 5-azacytidine in Saccharomyces cerevisiae   总被引:3,自引:0,他引:3  
The base analog 5-azacytidine induced a variety of genetic and epigenetic effects in different organisms. It was tested in two diploid strains of the yeast Saccharomyces cerevisiae to study the induction of point mutation, mitotic reciprocal crossing-over, mitotic gene conversion (strain D7) and mitotic aneuploidy (strain D61.M). It was used on cells growing in its presence for 4-5 generations. There was a strong induction of both types of mitotic recombination and point mutation. However, there was no induction of mitotic chromosomal malsegregation under the same conditions.  相似文献   

8.
The chlorinated ethylenes 1,1-dichloroethylene (vinylidene chloride), trans-1,2-dichloroethylene, trichloroethylene, and tetrachloroethylene (perchloroethylene) were assayed for their ability to induce mitotic gene conversion and point mutation as well as mitotic aneuploidy in diploid strains of the yeast Saccharomyces cerevisiae. From strain D7 late logarithmic-phase cells grown in 20% glucose liquid medium, containing a high level of cytochrome P-450, as well as stationary-phase cells combined with an exogenous metabolic activating system (S9) were used, in order to activate the chlorinated compounds and to produce electrophilic mutagenic intermediates. Only 1,1-dichloroethylene exhibited a dose-dependent genetic activity, while the other ethylenes did not. The 2 ways of metabolic activation were compared and were found to cause approximately the same effect. In contrast to the findings with strain D7, vinylidene chloride, trans-1,2-dichloroethylene, and trichloroethylene induced, without metabolic activation, mitotic chromosomal malsegregation in strain D61.M. The presence of liver homogenate as an activating system did not enhance the respective frequencies of chromosome loss. In the case of tetrachloroethylene, sufficient data have not become available, since this compound showed a highly toxic effect towards yeast cells, decreasing the rate of surviving cells to less than 30% at a concentration of 9.8 mM.  相似文献   

9.
In order to evaluate the optimal experimental conditions and to identify the best growth phase for yeast genotoxicity studies, comparative experiments were performed with stationary and growing cells. Methyl methanesulfonate (MMS) and cyclophosphamide (CP) were used as chemical mutagens and strain D7 of Saccharomyces cerevisiae as detector of induced mitotic gene conversion (trp+ convertants) and point reverse mutation (ilv+ revertants) in log or stationary phase cells after either 4 or 16 h of treatment. The highest MMS-induced toxicity and genotoxicity were observed after 16 h of exposure in a suspension test with log phase cells, which is consistent with the greater permeability and sensitivity of growing yeast cells. The maximal induction of genetic effects and toxicity by CP was conversely obtained after 16 h of treatment in stationary phase cells. This may be ascribed to the greater ability of detoxication of growing cells as compared to resting cells. Our results suggest that in evaluating the mutagenicity of chemicals in yeast systems it is important to consider factors such as growth phase and exposure time.  相似文献   

10.
In Saccharomyces cerevisiae, Mre11p, Rad50p, and Xrs2p function as a multiprotein complex that has a central role in several DNA repair mechanisms. Though Mre11p has both single-stranded and double-stranded 3'-5' exonuclease activity in vitro, null mutants of MRE11, RAD50, and XRS2 exhibit reduced 5'-3' resection of HO-induced double-strand breaks (DSBs) in vivo. In this study, we analyzed four mre11 mutants harboring changes in the N-terminus of Mre11p where the four phosphoesterase motifs specify the in vitro nuclease activities of Mre11p and its homologues. We find that the 5'-3' resection defects in vivo do not correlate with several mitotic phenotypes: non-homologous end-joining (NHEJ), telomere length maintenance, and adaptation to the DNA damage-inducible G2/M checkpoint. Overexpression of the 5'-3' exonuclease Exo1p in a mre11Delta strain partially increased 5'-3' resection and partially suppressed both methyl methanesulfonate (MMS) hypersensitivity and adaptation phenotypes, but did not affect telomere length or NHEJ. Surprisingly, the co-expression of two alleles, mre11-58S and mre11-N113S, each of which confers MMS hypersensitivity and short telomeres, can fully complement the MMS sensitivity and shortened telomere length of mre11Delta cells. We propose that at least two separate activities associated with the N-terminus of Mre11p are required for its mitotic function.  相似文献   

11.
Y F Wei  B J Chen    L Samson 《Journal of bacteriology》1995,177(17):5009-5015
The alkB gene is one of a group of alkylation-inducible genes in Escherichia coli, and its product protects cells from SN2-type alkylating agents such as methyl methanesulfonate (MMS). However, the precise biochemical function of the AlkB protein remains unknown. Here, we describe the cloning, sequencing, and characterization of three Saccharomyces cerevisiae genes (YFW1, YFW12, and YFW16) that functionally complement E. coli alkB mutant cells. DNA sequence analysis showed that none of the three gene products have any amino acid sequence homology with the AlkB protein. The YFW1 and YFW12 proteins are highly serine and threonine rich, and YFW1 contains a stretch of 28 hydrophobic residues, indicating that it may be a membrane protein. The YFW16 gene turned out to be allelic with the S. cerevisiae STE11 gene. STE11 is a protein kinase known to be involved in pheromone signal transduction in S. cerevisiae; however, the kinase activity is not required for MMS resistance because mutant STE11 proteins lacking kinase activity could still complement E. coli alkB mutants. Despite the fact that YFW1, YFW12, and YFW16/STE11 each confer substantial MMS resistance upon E. coli alkB cells, S. cerevisiae null mutants for each gene were not MMS sensitive. Whether these three genes provide alkylation resistance in E. coli via an alkB-like mechanism remains to be determined, but protection appears to be specific for AlkB-deficient E. coli because none of the genes protect other alkylation-sensitive E. coli strains from killing by MMS.  相似文献   

12.
The genetic effects of the mitotic inhibitor methyl benzimidazole-2-yl-carbamate (MBC) have been studied in Saccharomyces cerevisiae. MBC had little or no effect on the frequency of mutation. In some experiments MBC caused an increase in the frequency of mitotic recombination; however, this effect was small and not reproducible. The primary genetic effect of MBC was to induce mitotic chromosome loss at a high frequency. Chromosome loss occurred at equal frequencies for all chromosomes tested (13 of 16). Cells which had lost multiple chromosomes were found more frequently than predicted if individual chromosome loss events were independent. The probability of loss for a particular chromosome increased with length of time cells were incubated with MBC. MBC treatment also increased the frequency at which polyploid cells were found. These results suggested that MBC acted to disrupt the structure or function of the mitotic spindle and cause chromosome nondisjunction.  相似文献   

13.
Tsutsui Y  Morishita T  Iwasaki H  Toh H  Shinagawa H 《Genetics》2000,154(4):1451-1461
To identify Schizosaccharomyces pombe genes involved in recombination repair, we identified seven mutants that were hypersensitive to both methyl methanesulfonate (MMS) and gamma-rays and that contained mutations that caused synthetic lethality when combined with a rad2 mutation. One of the mutants was used to clone the corresponding gene from a genomic library by complementation of the MMS-sensitive phenotype. The gene obtained encodes a protein of 354 amino acids whose sequence is 32% identical to that of the Rad57 protein of Saccharomyces cerevisiae. An rhp57 (RAD57 homolog of S. pombe) deletion strain was more sensitive to MMS, UV, and gamma-rays than the wild-type strain and showed a reduction in the frequency of mitotic homologous recombination. The MMS sensitivity was more severe at lower temperature and was suppressed by the presence of a multicopy plasmid bearing the rhp51 gene. An rhp51 rhp57 double mutant was as sensitive to UV and gamma-rays as an rhp51 single mutant, indicating that rhp51 function is epistatic to that of rhp57. These characteristics of the rhp57 mutants are very similar to those of S. cerevisiae rad57 mutants. Phylogenetic analysis suggests that Rhp57 and Rad57 are evolutionarily closest to human Xrcc3 of the RecA/Rad51 family of proteins.  相似文献   

14.
Triploid and tetraploid strains of Saccharomyces cerevisiae were constructed and the spontaneous loss during mitosis of one, two or three copies of chromosome VII was determined. In one strain, a triploid (VM2) in which expression of the recessive alleles can be observed only after loss of two copies of chromosome VII (3N-2), the spontaneous frequency of chromosome loss was lower than in the diploid D61.M. In another strain, a tetraploid (VM4) that also requires the loss of two copies of chromosome VII for observation (4N-2) of the recessive alleles, the spontaneous frequency was slightly higher than in the diploid D61.M. The spontaneous frequency of other genetic events (that is, mutation, recombination or chromosome breakage) were lower by 2-3 orders of magnitude than in the diploid strain D61.M. Induction of chromosome loss and other genetic events by nocodazole, ethyl acetate, hydroxyurea and ethyl methanesulfonate was determined in D61.M, VM2, and VM4, and the results were compared. Nocodazole and ethyl acetate induced chromosome loss in both the triploid and the tetraploid strains at lower concentrations than required in the diploid. These compounds also induced elevated frequencies of other genetic events in both the triploid and the tetraploid strains but not in the diploid. Hydroxyurea induced elevated frequencies of chromosome loss in the diploid and the tetraploid. Frequencies of chromosome loss in the triploid treated with hydroxyurea, although elevated, are based on observation of very few colonies of the correct phenotype. Ethyl methanesulfonate failed to induce chromosome loss in any of the three strains. Hydroxyurea and ethyl methanesulfonate did, however, induce very high frequencies of other genetic events.  相似文献   

15.
Large-scale transitions in genome size from tetraploid to diploid were observed during a previous 1800-generation evolution experiment in Saccharomyces cerevisiae. Whether the transitions occurred via a one-step process (tetraploid to diploid) or through multiple steps (through ploidy intermediates) remained unclear. To provide insight into the mechanism involved, we investigated whether triploid-sized cells sampled from the previous experiment could also undergo ploidy loss. A batch culture experiment was conducted for approximately 200 generations, starting from four triploid-sized colonies and one contemporaneous tetraploid-sized colony. Ploidy reduction towards diploidy was observed in both triploid and tetraploid lines. Comparative genomic hybridization indicated the presence of aneuploidy in both the founder and the evolved colonies. The specific aneuploidies involved suggest that chromosome loss was not haphazard but that nearly full sets of chromosomes were lost at once, with some additional chromosome mis-segregation events. These results suggest the existence of a mitotic mechanism allowing the elimination of an entire set of chromosomes in S. cerevisiae, thereby reducing the ploidy level.  相似文献   

16.
Sterling CH  Sweasy JB 《Genetics》2006,172(1):89-98
The DNA polymerase 4 protein (Pol4) of Saccharomyces cerevisiae is a member of the X family of DNA polymerases whose closest human relative appears to be DNA polymerase lambda. Results from previous genetic studies conflict over the role of Pol4 in vivo. Here we show that deletion of Pol4 in a diploid strain of the SK1 genetic background results in sensitivity to methyl methanesulfonate (MMS). However, deletion of Pol4 in other strain backgrounds and in haploid strains does not yield an observable phenotype. The MMS sensitivity of a Pol4-deficient strain can be rescued by deletion of YKu70. We also show that deletion of Pol4 results in a 6- to 14-fold increase in the MMS-induced mutation frequency and in a significant increase in AT-to-TA transversions. Our studies suggest that Pol4 is critical for accurate repair of DNA lesions induced by MMS.  相似文献   

17.
E K?fer 《Mutation research》1988,201(2):385-399
The possibility of more than 1 target for genotoxic effects of methyl methanesulphonate (MMS) was investigated, using mitotic test systems of the fungus Aspergillus. Haploid and diploid strains were exposed, either as dormant conidia or during mitosis, and analysed for induced aneuploidy and effects on genetic segregation. MMS treatment of haploid strains resulted in dose-dependent increases of stable mutants with altered phenotypes and semi-stable unbalanced aberrations (presumably duplications). In addition, but only in dividing cells, MMS induced unstable aneuploids. These mostly were hyperhaploid with few extra chromosomes and could be identified by comparison with standard disomic phenotypes. When well-marked diploids were treated 3 types of effect could be distinguished, using genetic and phenotypic criteria: (1) Clastogenic and mutagenic effects which caused dose-dependent increases of partial aneuploids with various abnormal phenotypes. These showed secondary genetic segregation of all types and produced euploid normal sectors by eliminating damaged chromosome segments. In addition, but only in dividing nuclei, MMS induced 2 types of segregation: (2) Reciprocal crossing-over at high frequency, recognisable as half or quarter colonies of mutant colour and in some cases as 'twin spots' (i.e., complementary pairs); (3) Trisomics and other aneuploids which showed characteristic phenotypes and expected segregation of markers: the types recovered indicate random malsegregation of chromosomes (occasional deviations resulted from coincidence with induced crossing-over). These results suggest that MMS may have 2 (or more) targets for genotoxic effects: DNA, as evident from induced mutations and aberrations, and from induced recombination in dividing cells; some non-DNA target (nucleotide or protein) essential for nuclear division and susceptible to alkylation, resulting in malsegregation and primary aneuploidy.  相似文献   

18.
Procedure for mutagenizing spores of Saccharomyces cerevisiae   总被引:1,自引:0,他引:1       下载免费PDF全文
A procedure for inducing mutants of a homothallic strain of Saccharomyces cerevisiae is described. The essential parts of the procedure are long incubation in Glusulase, which preferentially kills vegetative cells instead of spores, and treatment in 9% ethyl methanesulfonate, which also preferentially kills vegetative cells instead of spores. Consequently, the viable population is virtually 100% spores.  相似文献   

19.
Methyl methanesulfonate (MMS)-sensitive mutants of Saccharomyces cerevisiae belonging to four different complementation groups, when homozygous, increase the rate of spontaneous mitotic segregation to canavanine resistance from heterozygous sensitive (canr/+) diploids by 13-to 170-fold. The mms8-1 mutant is MMS and X-ray sensitive and increases the rate of spontaneous mitotic segregation 170-fold. The mms9-1 and mms13-1 mutants are sensitive to X rays and UV, respectively, in addition of MMS, and increase the rate of spontaneous mitotic segregation by 13-fold and 85-fold, respectively. The mutant mms21-1 is sensitive to MMS, X rays and UV and increases the rate of spontaneous mitotic segregation 23-fold.  相似文献   

20.
Essential oils (EOs) extracted from medicinal plants such as Origanum compactum, Artemisia herba alba and Cinnamomum camphora are known for their beneficial effects in humans. The present study was undertaken to investigate their possible antigenotoxic effects in an eukaryotic cell system, the yeast Saccharomyces cerevisiae. The EOs alone showed some cytotoxicity and cytoplasmic petite mutations, i.e. mitochondrial damage, but they were unable to induce nuclear genetic events. In combination with exposures to nuclear mutagens such as 254-nm UVC radiation, 8-methoxypsoralen (8-MOP) plus UVA radiation and methylmethane sulfonate (MMS), treatments with these EOs produced a striking increase in the amount of cytoplasmic petite mutations but caused a significant reduction in revertants and mitotic gene convertants induced among survivors of the diploid tester strain D7. In a corresponding rho0 strain, the level of nuclear genetic events induced by the nuclear mutagens UVC and 8-MOP plus UVA resulted in the same reduced level as the combined treatments with the EOs. This clearly suggests a close relationship between the enhancement of cytoplasmic petites (mitochondrial damage) in the presence of the EOs and the reduction of nuclear genetic events induced by UVC or 8-MOP plus UVA. After MMS plus EO treatment, induction of these latter events was comparable at least per surviving fraction in wildtype and rho0 cells, and apparently less dependent on cytoplasmic petite induction. Combined treatments with MMS and EOs clearly triggered switching towards late apoptosis/necrosis indicating an involvement of this phenomenon in EO-induced cell killing and concomitant decreases in nuclear genetic events. After UVC and 8-MOP plus UVA plus EO treatments, little apoptosis and necrosis were observed. The antigenotoxic effects of the EOs appeared to be predominantly linked to the induction of mitochondrial dysfunction.  相似文献   

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