Detection of induced mitotic chromosome loss in Saccharomyces cerevisiae--an interlaboratory assessment of 12 chemicals

Induced mitotic chromosome loss was assayed using diploid yeast strain S. cerevisiae D61.M. The test relies upon the uncovering and expression of multiple recessive markers reflecting the presumptive loss of the chromosome VII homologue carrying the corresponding wild-type alleles. An interlaboratory study was performed in which 12 chemicals were tested under code in 2 laboratories. The results generated by the Berkeley and the Darmstadt laboratories were in close agreement. The solvents benzonitrile and methyl ethyl ketone induced significantly elevated chromosome loss levels. However, a treatment regime that included overnight storage at 0 degree C was required to optimize chromosome loss induction. Hence, these agents are postulated to induce chromosome loss via perturbation of microtubular assembly. Fumaronitrile yielded inconsistent results: induction of chromosome loss and respiratory deficiency was observed in both laboratories, but the response was much more pronounced in the Darmstadt trial than that observed in Berkeley. The mammalian carcinogens, benzene, acrylonitrile, trichloroethylene, 1,1,1-trichloroethane and 1,1,1,2-tetrachloroethane failed to induce chromosome loss but elicited high levels of respiratory deficiency, reflecting anti-mitochondrial activity. Trifluralin, cyclophosphamide monohydrate, diazepam and diethylstilbestrol dipropionate failed to induce any detectable genetic effects. These data suggest that the D61.M system is a reproducible method for detecting induced chromosome loss in yeast.     

Comparison of chemically induced chromosome loss in a diploid, triploid, and tetraploid strain of Saccharomyces cerevisiae.

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.     

Recessive Lethal Mutations and the Maintenance of Duplication-Bearing Strains of Dictyostelium discoideum

Recessive lethal mutations have been isolated and used to maintain n + 1 aneuploid strains of Dictyostelium discoideum carrying a duplication of part or all of linkage group VII. The recessive lethal mutations, relA351 and relB352, arose spontaneously in diploids; no mutagenic treatment was used in the isolation of these mutations. The probable gene order on linkage group VII is: centromere, relB couA, bsgB, cobA, relA. Maintenance of aneuploids disomic for linkage group VII was made possible by complementation of a rel mutation on each linkage group VII homologue by the corresponding wild-type allele on the other linkage group VII homologue. The duplication-bearing disomic strains were slow-growing and produced faster-growing sectors on the colony edge. Haploid sectors probably arise by a combination of mitotic recombination and subsequent loss of one homologue, diploid sectors may be formed by chromosome doubling to 2n + 2, followed by chromosome loss to return to 2n, and aneuploid sectors may arise by deletion or new mutation.     

The influence of solvent stress on MMS-induced genetic change in Saccharomyces cerevisiae

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.     

The influence of solvent stress on MMS-induced genetic change in Saccharomyces cerevisiae

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.     

Observations on chromosome loss detection by multiple recessive marker expression in strain D61.M of Saccharomyces cerevisiae

Since chromosomes of fungi are difficult to observe directly, strains have been developed in which chromosome loss can be detected by the use of genetic markers. In the diploid D61.M strain of Saccharomyces cerevisiae, the loss of a copy of chromosome VII that carries 3 dominant wild-type alleles is measured by expression of 3 recessive mutant alleles carried on the other remaining copy of chromosome VII. We have tested the hypothesis that expression of the 3 recessive alleles might be due to 3 simultaneous independent genetic events other than chromosome loss, such as mutation or recombination. We have measured, when possible, the frequencies of expression for each of these recessive alleles, independently and in combination one with another, under both selective and non-selective conditions. Our results show that simultaneous expression of these 3 recessive alleles is attributable to chromosome loss (greater than 98%). Similarly, at least 99% of the nocodazole-induced events are attributable to chromosome loss. In contrast, most if not all of the apparent chromosome loss induced by ethyl methanesulfonate is due to multiple events of mutation or recombination.     

Induction of chromosome loss by mixtures of organic solvents including neurotoxins

Twenty-three aprotic polar solvents - 3 nitriles, 8 organic esters, 10 ketones and 2 lactones - and LiCl were tested in combination with propionitrile alone or a mixture of ethyl acetate and propionitrile for the induction of mitotic chromosome loss in the D61.M strain of the yeast Saccharomyces cerevisiae. Propionitrile and ethyl acetate are very potent inducers of chromosome loss. Mixtures of propionitrile and ethyl acetate induced chromosome loss at much higher frequencies than was observed with the pure chemicals. To test the potentiating effects of propionitrile or mixtures of propionitrile with ethyl acetate on other chemicals, they were used in concentrations that were at or below the level for induction of chromosome loss. Twenty chemicals when tested in pure form were negative or only marginally active in the test for chromosome loss. Except for amyl propionate and benzyl acetate, the same chemicals showed strong induction in combination treatments with the potentiating chemicals. All the ketones including the neurotoxic methyl ethyl ketone, 2-hexanone and 2.5-hexanedione induced high frequencies of chromosome loss. Only methyl ethyl ketone is capable of inducing high levels of chromosome loss when tested in the pure form at much higher concentrations. 1-Methyl-2-pyrrolidinone and gamma-valerolactone had previously been shown to induce chromosome loss only when the treatment at a growth-supporting temperature was interrupted by a cold shock within a narrow range of low temperatures which prevented growth. Both gave very strong induction in combination treatment performed at a continuous growth-supporting temperature. LiCl is a weak inducer of chromosome loss: strong induction can be achieved in combination treatments.     

The detection of monosomic colonies produced by mitotic chromosome non-disjunction in the yeast Saccharomyces cerevisiae

A diploid yeast strain, D6, is described which monitors mitotic non-disjunction by the phenotypic expression of a set of coupled and recessive markers flanking the centromere of chromosome VII. These markers are not expressed in the heterozygous condition prevailing in D6. The left arm of chromosome VII carries a tightly centromere linked marker, leu1 (leucine requirement), distal to leu1 in this order: trp5 (trytophan requirement), cyh2 (recessive resistance to cycloheximide) and met 13 (requirement for methionine). The right arm is marked with ade3 (simultaneous requirement for adenine and histidine). D6 is homozygous for ade2 and consequently, forms red rather than the normally white colonies. It shows no requirement for the above amino acids and it is sensitive to cycloheximide.Unmasking of all the markers on chromosome VII leads to colonies that are white because ade3 sets a block preceding the ade2 block (which causes the accumulation of a precursor of the red pigment), they require leucine, tryptophan and methionine, and grow on media with cycloheximide. Cells are plated on a cycloheximide medium where red and white colonies are formed. Colonies of spontaneous origin were tested. The majority of the white colonies expressed all the recessive markers whereas only few of the red colonies expressed all the markers on the left arm of chromosome VII.Basically expression of recessive markers on both sides of the centromere can be explained as a result of two coincident events of mitotic crossing over. However, the frequency of colonies expressing centromere linked leu1 was 14 times higher among the white types than the red ones. This suggested that the white, cycloheximide resistant, leucine requiring colonies arose by mitotic non-disjunction and not only by two coincident mitotic crossing over events.Presumptive spontaneous monosomic segregants were placed on sporulation medium. Only 8 out of 30 isolates sporulated, which showed that these eight segregants were diploid at the time of sporulation. They could have arisen by two coincident crossover events or through restoration of a normal disomic condition after non-disjunction had occurred. The genetic data thus leaves us with only its statistical argument in favour of non-disjunction. Further confirmation of monosomic nature of the white cycloheximide resistant colonies was provided by the estimates of their DNA contents. Compared to the stock wild type diploids the presumptive monosomics showed a reduction in DNA content.We have utilized D6 to investigate the possible induction of mitotic non-disjunction after treatment with gamma rays, heat shock at 52°C and ultraviolet irradiation. In all cases white, cycloheximide resistant colonies were produced at levels significantly higher than that found in untreated cultures. In order to detect the production of monosomic cells, treated cultures were grown for 48 h in non-selective medium after exposure to allow for “expression” of the monosomic condition.     

Long-Tract Mitotic Gene Conversion in Yeast: Evidence for a Triparental Contribution during Spontaneous Recombination

In Saccharomyces cerevisiae, spontaneous mitotic gene conversion at one site is statistically correlated with recombination at other loci. In general, coincident conversion frequencies are highest for tightly linked markers and decline as a function of intermarker distance. Paradoxically, a significant fraction of mitotic gene convertants exhibits concomitant nonreciprocal segregation for multiple and widely spaced markers. We have undertaken a detailed genetic analysis of this class of mitotic recombinants. Our results indicate that mitotic gene conversion in yeast is frequently associated with nonreciprocal segregation of markers centromere-distal to the selected site of conversion. In addition, distal markers are often found to be mosaic within the product colonies. These observations, and others described here, suggest that a percentage of gene conversion in vegetative yeast cells is coupled to a chromosome break and repair mechanism. This hypothesis was further tested using a strain trisomic for chromosome VII which was specially marked to detect homolog-dependent repair events. An association between mitotic gene conversion events and the production of broken chromosomes which are repaired by a homologous-pairing-copy mechanism was supported.     

Characterization of a Mutation in Yeast Causing Nonrandom Chromosome Loss during Mitosis

Diploid strains of the yeast Saccharomyces cerevisiae homozygous for a recessive chromosome loss mutation (chl) exhibit a high degree of mitotic instability. Cells become monosomic for chromosome III at a frequency of approximately one percent of all cell divisions. Chromosome loss at this high frequency is also found for chromosome I, and at lesser frequencies for chromosomes VIII and XVI. In contrast, little or no chromosome loss is found for six other linkage groups tested (II, V, VI, VII, XI and XVII). The chl mutation also induces a ten-fold increase in both intergenic and intragenic mitotic recombination on all ten linkage groups tested. The chl mutation does not cause an increase in spontaneous mutations, nor are mutant strains sensitive to UV or γ irradiation. The effects of chl during meiosis are observed primarily in reduced spore viability. A decrease in chromosome III linkage relationships is also found.     

Detection of the loss of 1 chromosome from pair III in Saccharomyces cerevisiae yeasts

A diploid strain of Saccharomyces cerevisiae, T6 is described which monitors both mitotic crossing over and induction of aneuploidy. The chromosome III carries recessive markers: rgh12 of "rough colony" phenotype closely linked to centromere, the left arm is marked with his4, the right arm is marked both with thr4 and the locus of mating type alpha. Expression of all the markers on chromosome III leads to formation of colonies which are rough, require histidine and threonine, and are of alpha mating type. These colonies arise as a result of the loss of a chromosome during mitosis, which makes the strain allow detection of monosomic cells formation. Chromosome XV carries two phenotypically distinguishable and recessive alleles of the gene ade2: ade2-192 (causes red coloration of colonies) and ade2-G45 (causes pink coloration of colonies). Mitotic crossing over generates two reciprocal products which can be revealed together in colonies as pink and red sectors in double-spotted colonies. Both double-spotted and monosomic colonies have been obtained after treatment with gamma-rays. The frequency of mitotic crossing over after irradiation by 1000-3000 Gray increased up to 2-3.2% (the spontaneous level was 0.006%), the frequency of aneuploidy was 0.12 to 0.57% at plating immediately after irradiation (the spontaneous monosomics were not observed among 1.5 X 10(5) cells scored). Induction of mitotic crossing over and aneuploidy by benomyl was rather slight (up to 0.05 and 0.006%, respectively).     

Differential Mitotic Stability of Yeast Disomes Derived from Triploid Meiosis

The frequencies of recovered disomy among the meiotic segregants of yeast (Saccharomyces cerevisiae) triploids were assessed under conditions in which all 17 yeast chromosomes were monitored simultaneously. The studies employed inbred triploids, in which all homologous centromeres were identical by descent, and single haploid testers carrying genetic markers for all 17 linkage groups. The principal results include: (1) Ascospores from triploid meiosis germinate at frequencies comparable to those from normal diploids, but most fail to produce visible colonies due to the growth-retarding effects of high multiple disomy. (2) The probability of disome formation during triploid meiosis is the same for all chromosomes; disomy for any given chromosome does not exclude simultaneous disomy for any other chromosome. (3) The 17 yeast chromosomes fall into three frequency classes in terms of disome recovery. The results support the idea that multiply disomic meiotic segregants of the triploid experience repeated, nonrandom, post-germination mitotic chromosome losses (N + 1 leads to N) and that the observed variations in individual disome recovery are wholly attributable to inherent differences in disome mitotic stability.     

Genetics of Yeast Glucokinase

Mutants of Saccharomyces cerevisiae lacking glucokinase (EC 2.7.1.2) have no discernible phenotypic difference from the wild-type strain; in a hexokinaseless background, however, they are unable to grow on any sugar except galactose. Reversion studies with glucokinase mutants indicate that the yeast S. cerevisiae has no other enzyme for phosphorylating glucose except the two hexokinases, P1 and P2, and glucokinase. Spontaneous revertants of hxk1 hxk2 glk1 strains collected on glucose regain any one of these three enzymes. The majority of glucokinase revertants synthesize species of enzyme activity that are kinetically or otherwise indistinguishable from the wild-type enzyme. In a few cases the reverted enzyme is very perceptibly altered in properties with a Km for glucose two orders of magnitude higher than that of the enzyme from the wild-type parent. These recessive, noncomplementing mutants, thus, define a single structural gene GLK1 of glucokinase. Yeast diploids lacking all of the three enzymes for glucose phosphorylation fail to sporulate. Heterozygosity of either of the hexokinase genes HXK1 or HXK2, but not GLK1, restores sporulation. The location of GLK1 on chromosome III was indicated by loss of this chromosome when hexokinaseless diploids heterozygous for glk1 were selected for resistance to 2-deoxyglucose; the homologue of chromosome III carrying GLK1, the mating-type allele and other nutritional markers on this chromosome was lost. Meiotic mapping of glucokinase executed with heterozygosity of one of the hexokinases indicated that the gene GLK1 defining the structure of glucokinase protein is located on the left arm of chromosome III 24 cM to the left of his4 in the order: leu2--his4--glk1. --Only two of 206 independent glucokinase mutants are nonsense ochre, both of which map at one end of the gene. In hxk1 only one of 130 isolates is a nonsense mutation, whereas in hxk2 none has been found among 220 independent mutants. These results raise the possibility that the protein products of these genes have some other essential function. --An earlier mapping result for hxk2 has been corrected. The new location is on the left arm of chromosome VII, 17 cM distal to ade5 in the order: lys5--ade5--hxk2.     

Somatic events unmask recessive cancer genes to initiate malignancy

A heritable mutation predisposes an individual to certain childhood malignancies, such as retinoblastoma and Wilms' tumor. The chromosomal locations of the genes responsible for the predisposition are known by linkage with chromosomal deletions and enzyme markers. A study of these tumors in comparison to the normal constitutional cells of the patients, using enzyme and DNA markers near the predisposing genes, has shown that these genes are recessive to normal wild-type alleles at the cellular level. Expression of the recessive phenotype (malignancy) involves the same genetic events that were observed in Chinese hamster cell hybrids carrying recessive drug resistance genes. In both the experimental and clinical situations, the wild-type allele is most commonly eliminated by chromosome loss with duplication of the mutant chromosome. Simple chromosome loss and mitotic recombination have been documented in both systems. In the remaining 30% of cases, inactivation or microdeletion of the wild-type allele are assumed to be responsible for expression of the recessive phenotype. Osteosarcoma is a common second tumor in patients who have had retinoblastoma. Studies with markers in osteosarcoma show that these tumors also result from unmasking of the recessive phenotype by loss of the normal allele at the retinoblastoma locus, whether or not the patient had retinoblastoma. Subsequent chromosomal rearrangements and amplification of oncogenes that occur in these homozygous tumors provide progressive growth advantage. In other malignancies, in which studies have so far focused on oncogene amplification and chromosomal rearrangements, unmasking of recessive mutations may also be the critical initiating events.     

Association of Disomic Chromosome Loss with Ems-Induced Conversion in Yeast

Experimental tests with the yeast Saccharomyces cerevisiae of a previously proposed model suggesting a causal relationship between disomic chromosome loss (n + 1 → n) and centromere-adjacent mitotic gene conversion were performed. Disomic haploid cells heteroallelic at two loci on the left arm of chromosome III were exposed to ethyl methanesulfonate (EMS) under nonlethal conditions; EMS-induced prototrophic gene convertants were selected and tested for coincident chromosome loss. The principal results are: (1) The frequency of chromosome loss among EMS-induced gene convertants selected to arise near the centromere is markedly enhanced over basal levels and remains constant, independent of EMS exposure. There is little such enhancement among EMS-induced convertants selected to arise far from the centromere. (2) Chromosome loss is almost completely associated with induced conversion of the centromere-proximal allele at the centromere-adjacent heteroallelic locus. This result is identical to (and confirms) results found previously for spontaneous loss-associated conversion. (3) The conversion polarity at the centromere-adjacent locus among unselected (nonloss-associated) induced or spontaneous mitotic convertants is identical to that among meiotic convertants and markedly favors the contromere-distal allele. These findings are wholly consistent with, and strengthen, the hypothesis that structural involvement of centromeric regions in nearby recombinational events may interfere with proper segregational function and lead to mitotic chromosome loss.     

Formaldehyde, glyoxal, urethane, methyl carbamate, 2,3-butanedione, 2,3-hexanedione, ethyl acrylate, dibromoacetonitrile and 2-hydroxypropionitrile induce chromosome loss in Saccharomyces cerevisiae

Induction of mitotic chromosome loss could be demonstrated for the dialdehyde glyoxal, the diketones 2,3-butanedione and 2,3-hexanedione, ethyl and methyl carbamate, ethyl acrylate, dibromoacetonitrile, 2-hydroxypropionitrile and formaldehyde, but only when they were combined with subacute concentrations of propionitrile, which is a strong inducer of chromosomal malsegregation. The same chemicals did not induced mitotic chromosome loss when applied in pure form. However, glyoxal, ethyl acrylate, dibromoacetonitrile and formaldehyde when applied in pure form also induced mitotic recombination. Respiratory deficiency was induced, in the absence of propionitrile, by these recombinogenic agents and also by 2,3-hexanedione and 2-hydroxypropionitrile which are not recombinogenic.     

Formaldehyde, glyoxal, urethane, methyl carbamate, 2,3-butanedione, 2,3-hexanedione, ethyl acrylate, dibromoacetonitrile and 2-hydroxypropionitrile induce chromosome loss in Saccharomyces cerevisiae.

Induction of mitotic chromosome loss could be demonstrated for the dialdehyde glyoxal, the diketones 2,3-butanedione and 2,3-hexanedione, ethyl and methyl carbamate, ethyl acrylate, dibromoacetonitrile, 2-hydroxypropionitrile and formaldehyde, but only when they were combined with subacute concentrations of propionitrile, which is a strong inducer of chromosomal malsegregation. The same chemicals did not induced mitotic chromosome loss when applied in pure form. However, glyoxal, ethyl acrylate, dibromoacetonitrile and formaldehyde when applied in pure form also induced mitotic recombination. Respiratory deficiency was induced, in the absence of propionitrile, by these recombinogenic agents and also by 2,3-hexanedione and 2-hydroxypropionitrile which are not recombinogenic.     

Genotoxic evaluation of selective serotonin-reuptake inhibitors by use of the somatic mutation and recombination test in Drosophila melanogaster

This study evaluated different concentrations of selective serotonin-reuptake inhibitors (citalopram and sertraline) for genotoxicity by use of the somatic mutation and recombination test (SMART) in Drosophila melanogaster. Three-day-old larvae, trans-heterozygous for the multiple wing hairs (mwh) and flare (flr3) genes were treated with these two compounds. Two recessive markers were located on the left arm of chromosome 3, i.e. 'multiple wing hairs' (mwh) in map position 0.3 and 'flare-3' (flr3) at 38.8, while the centromere was located in position 47.7. SMART is based on the loss of heterozygosity, which may occur through various mechanisms, such as mitotic recombination, mutation, deletion, half-translocation, chromosome loss, and non-disjunction. Genetic changes occurring in somatic cells of the wing's imaginal discs, cause the formation of mutant clones on the wing blade. The results of this study show that citalopram had a genotoxic effect in the Drosophila SMART. Sertraline, however, did not show any genotoxic effect in balancer heterozygous wings. This study concluded that more information is needed to be certain regarding the mutagenic effects of sertraline.     

Isolation of two genes that affect mitotic chromosome transmission in S. cerevisiae

Two DNA sequences that reduce mitotic fidelity of chromosome transmission have been identified: MIF1 and MIF2. MIF1 is a unique sequence located on the right arm of chromosome XII that stimulates loss and recombination for both chromosomes V and VII when present in a high copy number plasmid. MIF1 is not essential for cell division but is necessary for the normal fidelity of chromosome transmission. MIF2 is a unique sequence located 15 cM distal to HIS6 on chromosome IX that induces a high frequency of chromosome VII loss and a lower frequency of chromosome V loss when present in high copy number; it has no effect on mitotic recombination. Disruption of the genomic MIF2 locus was lethal and cells lacking this function arrested division with a terminal phenotype characteristic of a block in DNA replication or nuclear division.