Aneuploidy In Yeast Research Articles

Effect of yeast chromosome II aneuploidy on malate production in sake brewing

Chromosome aneuploidy is a common phenomenon in industrial yeast. Aneuploidy is considered one of the strategies to enhance the industrial properties of Saccharomyces cerevisiae strains. However, the effects of chromosomal aneuploidy on the brewing properties of sake have not been extensively studied. In this study, sake brewing was performed using a series of genome-wide segmental duplicated laboratory S.cerevisiae strains, and the effects of each segmentally duplicated region on sake brewing were investigated. We found that the duplication of specific chromosomal regions affected the production of organic acids and aromatic compounds in sake brewing. As organic acids significantly influence the taste of sake, we focused on the segmental duplication of chromosome II that alters malate levels. Sake yeast Kyokai No. 901 strains with segmental chromosome II duplication were constructed using a polymerase chain reaction-mediated chromosomal duplication method, and sake was brewed using the resultant aneuploid sake yeast strains. The results showed the possibility of developing sake yeast strains exhibiting low malate production without affecting ethanol production capacity. Our study revealed that aneuploidy in yeast alters the brewing properties; in particular, the aneuploidy of chromosome II alters malate production in sake brewing. In conclusion, aneuploidization can be a novel and useful tool to breed sake yeast strains with improved traits, possessing industrial significance.

Deposition of Centromeric Histone H3 Variant CENP-A/Cse4 into Chromatin Is Facilitated by Its C-Terminal Sumoylation.

Centromeric localization of CENP-A (Cse4 in Saccharomyces cerevisiae, CID in flies, CENP-A in humans) is essential for faithful chromosome segregation. Mislocalization of overexpressed CENP-A contributes to aneuploidy in yeast, flies, and humans, and is proposed to promote tumorigenesis in human cancers. Hence, defining molecular mechanisms that promote or prevent mislocalization of CENP-A is an area of active investigation. In budding yeast, evolutionarily conserved histone chaperones Scm3 and chromatin assembly factor-1 (CAF-1) promote localization of Cse4 to centromeric and noncentromeric regions, respectively. Ubiquitin ligases, such as Psh1 and Slx5, and histone chaperones (HIR complex) regulate proteolysis of overexpressed Cse4 and prevent its mislocalization to noncentromeric regions. In this study, we have identified sumoylation sites lysine (K) 215/216 in the C terminus of Cse4, and shown that sumoylation of Cse4 K215/216 facilitates its genome-wide deposition into chromatin when overexpressed. Our results showed reduced levels of sumoylation of mutant Cse4 K215/216R/A [K changed to arginine (R) or alanine (A)] and reduced interaction of mutant Cse4 K215/216R/A with Scm3 and CAF-1 when compared to wild-type Cse4 Consistent with these results, levels of Cse4 K215/216R/A in the chromatin fraction and localization to centromeric and noncentromeric regions were reduced. Furthermore, in contrast to GAL-CSE4, which exhibits Synthetic Dosage Lethality (SDL) in psh1∆, slx5∆, and hir2∆ strains, GAL-cse4K215/216R does not exhibit SDL in these strains. Taken together, our results show that deposition of Cse4 into chromatin is facilitated by its C-terminal sumoylation.

Aneuploidy in yeast: Segregation error or adaptation mechanism?

Aneuploidy is the loss or gain of chromosomes within a genome. It is often detrimental and has been associated with cell death and genetic disorders. However, aneuploidy can also be beneficial and provide a quick solution through changes in gene dosage when cells face environmental stress. Here, we review the prevalence of aneuploidy in Saccharomyces, Candida, and Cryptococcus yeasts (and their hybrid offspring) and analyse associations with chromosome size and specific stressors. We discuss how aneuploidy, a segregation error, may in fact provide a natural route for the diversification of microbes and enable important evolutionary innovations given the right ecological circumstances, such as the colonisation of new environments or the transition from commensal to pathogenic lifestyle. We also draw attention to a largely unstudied cross link between hybridisation and aneuploidy. Hybrid meiosis, involving two divergent genomes, can lead to drastically increased rates of aneuploidy in the offspring due to antirecombination and chromosomal missegregation. Because hybridisation and aneuploidy have both been shown to increase with environmental stress, we believe it important and timely to start exploring the evolutionary significance of their co‐occurrence.

N-terminal Sumoylation of Centromeric Histone H3 Variant Cse4 Regulates Its Proteolysis To Prevent Mislocalization to Non-centromeric Chromatin.

Stringent regulation of cellular levels of evolutionarily conserved centromeric histone H3 variant (CENP-A in humans, CID in flies, Cse4 in yeast) prevents its mislocalization to non-centromeric chromatin. Overexpression and mislocalization of CENP-A has been observed in cancers and leads to aneuploidy in yeast, flies, and human cells. Ubiquitin-mediated proteolysis of Cse4 by E3 ligases such as Psh1 and Sumo-Targeted Ubiquitin Ligase (STUbL) Slx5 prevent mislocalization of Cse4. Previously, we identified Siz1 and Siz2 as the major E3 ligases for sumoylation of Cse4. In this study, we have identified lysine 65 (K65) in Cse4 as a site that regulates sumoylation and ubiquitin-mediated proteolysis of Cse4 by Slx5. Strains expressing cse4 K65R exhibit reduced levels of sumoylated and ubiquitinated Cse4 in vivo. Furthermore, co-immunoprecipitation experiments reveal reduced interaction of cse4 K65R with Slx5, leading to increased stability and mislocalization of cse4 K65R under normal physiological conditions. Based on the increased stability of cse4 K65R in psh1∆ strains but not in slx5∆ strains, we conclude that Slx5 targets sumoylated Cse4 K65 for ubiquitination-mediated proteolysis independent of Psh1. In summary, we have identified and characterized the physiological role of Cse4 K65 in sumoylation, ubiquitin-mediated proteolysis, and localization of Cse4 for genome stability.

SUMO-Targeted Ubiquitin Ligase (STUbL) Slx5 regulates proteolysis of centromeric histone H3 variant Cse4 and prevents its mislocalization to euchromatin.

Centromeric histone H3, CENP-ACse4, is essential for faithful chromosome segregation. Stringent regulation of cellular levels of CENP-ACse4 restricts its localization to centromeres. Mislocalization of CENP-ACse4 is associated with aneuploidy in yeast, flies and tumorigenesis in human cells; thus, defining pathways that regulate CENP-A levels is critical for understanding how mislocalization of CENP-A contributes to aneuploidy in human cancers. Previous work in budding yeast has shown that ubiquitination of overexpressed Cse4 by Psh1, an E3 ligase, partially contributes to proteolysis of Cse4. Here, we provide the first evidence that Cse4 is sumoylated by E3 ligases Siz1 and Siz2 in vivo and in vitro. Ubiquitination of Cse4 by Small Ubiquitin-related Modifier (SUMO)-Targeted Ubiquitin Ligase (STUbL) Slx5 plays a critical role in proteolysis of Cse4 and prevents mislocalization of Cse4 to euchromatin under normal physiological conditions. Accumulation of sumoylated Cse4 species and increased stability of Cse4 in slx5∆ strains suggest that sumoylation precedes ubiquitin-mediated proteolysis of Cse4. Slx5-mediated Cse4 proteolysis is independent of Psh1 since slx5∆ psh1∆ strains exhibit higher levels of Cse4 stability and mislocalization compared to either slx5∆ or psh1∆ strains. Our results demonstrate a role for Slx5 in ubiquitin-mediated proteolysis of Cse4 to prevent its mislocalization and maintain genome stability.

Mitotic entry in the presence of DNA damage is a widespread property of aneuploidy in yeast.

Genetic instability is a hallmark of aneuploidy in budding and fission yeast. All aneuploid yeast strains analyzed to date harbor elevated levels of Rad52-GFP foci, a sign of DNA damage. Here we investigate how continuously elevated levels of DNA damage affect aneuploid cells. We show that Rad52-GFP foci form during S phase, consistent with the observation that DNA replication initiation and elongation are impaired in some aneuploid yeast strains. We furthermore find that although DNA damage is low in aneuploid cells, it nevertheless has dramatic consequences. Many aneuploid yeast strains adapt to DNA damage and undergo mitosis despite the presence of unrepaired DNA leading to cell death. Wild-type cells exposed to low levels of DNA damage exhibit a similar phenotype, indicating that adaptation to low levels of unrepaired DNA is a general property of the cell's response to DNA damage. Our results indicate that by causing low levels of DNA damage, whole-chromosome aneuploidies lead to DNA breaks that persist into mitosis. Such breaks provide the substrate for translocations and deletions that are a hallmark of cancer.

Quantitative proteomic analysis reveals posttranslational responses to aneuploidy in yeast.

Aneuploidy causes severe developmental defects and is a near universal feature of tumor cells. Despite its profound effects, the cellular processes affected by aneuploidy are not well characterized. Here, we examined the consequences of aneuploidy on the proteome of aneuploid budding yeast strains. We show that although protein levels largely scale with gene copy number, subunits of multi-protein complexes are notable exceptions. Posttranslational mechanisms attenuate their expression when their encoding genes are in excess. Our proteomic analyses further revealed a novel aneuploidy-associated protein expression signature characteristic of altered metabolism and redox homeostasis. Indeed aneuploid cells harbor increased levels of reactive oxygen species (ROS). Interestingly, increased protein turnover attenuates ROS levels and this novel aneuploidy-associated signature and improves the fitness of most aneuploid strains. Our results show that aneuploidy causes alterations in metabolism and redox homeostasis. Cells respond to these alterations through both transcriptional and posttranscriptional mechanisms.

Hydroquinone, a Benzene Metabolite, Induces Hog1-dependent Stress Response Signaling and Causes Aneuploidy in Saccharomyces cerevisiae

Previously, we have shown that phenyl hydroquinone, a hepatic metabolite of the Ames test-negative carcinogen o-phenylphenol, efficiently induced aneuploidy in Saccharomyces cerevisiae by arresting the cell cycle at the G2/M transition as a result of the activation of the Hog1 (p38 MAPK homolog)-Swe1 (Wee1 homolog) pathway. In this experiment, we examined the aneuploidy forming effects of hydroquinone, a benzene metabolite, since both phenyl hydroquinone and hydroquinone are Ames-test negative carcinogens and share similar molecular structures. As was seen in phenyl hydroquinone, hydroquinone induced aneuploidy in yeast by delaying the cell cycle at the G2/M transition. Deficiencies in SWE1 and HOG1 abolished the hydroquinone-induced delay at the G2/M transition and aneuploidy formation. Furthermore, Hog1 was phosphorylated by hydroquinone, which may stabilize Swe1. These data indicate that the hydroquinone-induced G2/M transition checkpoint, which is activated by the Hog1-Swe1 pathway, plays a role in the formation of aneuploidy.

1-Methyl-2-pyrrolidinone (NMP) does not induce structural and numerical chromosomal aberrations in vivo

1-Methyl-2-pyrrolidinone induces aneuploidy in yeast, but only under special treatment conditions. Other genotoxic effects have not been found in vitro, and in vivo no data are available in the literature. Therefore, NMP was investigated in the mouse micronucleus test and the Chinese hamster bone marrow test for structural and numerical chromosomal aberrations. These tests can detect both types of alterations as demonstrated by appropriate positive control substances (cyclophosphamide, vincristine sulfate and benomyl). NMP at single oral doses up to 3800 mg/kg body weight (∼ 80% of the LD 50) did not lead to an increase either in micronucleated erythrocytes or in structural or numerical chromosomal aberrations when bone marrow was sampled 16, 24 and 48 h after treatment in the micronucleus test or after 24 and 48 h for karyotype analysis.

Dichloroacetonitrile, a by-product of water chlorination, induces aneuploidy in Drosophila

Nitriles have been shown to be potent inducers of aneuploidy in yeast and Drosophila test systems. Haloacetonitriles are by-products of water chlorination that have been shown to be mutagenic and carcinogenic following topical application. In this report we show that dichloroacetonitrile, but not dibromoacetonitrile, is an effective inducer of aneuploidy in oocytes of Drosophila melanogaster. Following inhalation exposure of ZESTE adult females, dichloroacetonitrile (8.6 ppm) induced highly significant increments in the frequencies of sex chromosome loss and gain. Sodium cyanide was also found to be a highly effective inducer of germline aneuploidy, suggesting that cyanide toxicity may contribute to potency of nitriles as inducers of aneuploidy.

5 Final Report on the Safety Assessment of Ethyl Acetate and Butyl Acetate

Ethyl and Butyl Acetate are used as solvents in nail polish, nail polish removers, basecoats, and other manicuring preparations. Ethyl and Butyl Acetate were relatively nontoxic when administered orally, dermally, or by inhalation to rabbits, rats, mice, and guinea pigs. A nail polish containing 10% Ethyl Acetate and 25% Butyl Acetate was a moderate to severe irritant in unrinsed rabbit eyes and a mild irritant in rinsed rabbit eyes. Butyl Acetate was not a sensitizer in either mice or guinea pigs. Ethyl and Butyl Acetate were nonmutagenic when tested by the Ames procedure, Rec-assay, and micronucleus assay. Neither compound induces mitotic aneuploidy in yeast or chromosomal aberrations in Chinese hamster fibroblasts. Butyl Acetate was not teratogenic when inhaled. Ethyl Acetate and Butyl Acetate were mild skin irritants but not sensitizers to humans. Ethyl Acetate was neither phototoxic or photoallergenic in human tests. It is concluded that Ethyl Acetate and Butyl Acetate are safe as cosmetic ingredients in the present practices of use and concentration.

The detection of mitotic and meiotic aneuploidy in yeast using a gene dosage selection system.

A system is described in which spontaneous and chemically-induced mitotic and meiotic hyperploidy can be assayed in the same diploid culture of Saccharomyces cerevisiae. Monitoring gene dosage changes at two loci on chromosome VIII, the test utilizes a leaky temperature-sensitive allele arg4-8 and low level copper resistance conferred by the single copy allele cup1s. An extra chromosome VIII provides simultaneous increased dosage for both genes, resulting in colonies that are both prototrophic for arginine at 30 degrees C and copper resistant. During mitotic cell divisions in diploids, spontaneous chromosome VIII hyperploids (trisomes and tetrasomes) occur at a frequency of 6.4 x 10(-6) per viable cell. Among ascospores, the spontaneous chromosome VIII disome frequency is 5.5 x 10(-6) per viable spore. The tubulin-binding reagent methyl benzimidazol-2-yl carbamate (MBC) elicits enhanced levels of mitotic and meiotic aneuploidy relative to control levels. The system represents a novel model for examining chromosome behavior during mitosis and meiosis and provides a sensitive and quantifiable procedure for examining chemically induced aneuploidy.

Investigations of aneuploidy-inducing chemical combinations in Saccharomyces cerevisiae

For several years we have been investigating combinations of chemicals for their ability to induce aneuploidy. Earlier published results indicated that combinations of certain chemicals showed a potentiation effect while other combinations did not. We have continued to explore this phenomenon and report additional findings in this communication. Combinations of ethyl acetate and methyl ethyl ketone showed a potentiation effect as did 1-methyl-2-pyrrolidinone-nocodazole combinations. Combinations that did not show a potentiation effect were 2-pyrrolidinone-nocodazole and 1-methyl-2-pyrrolidinone-ethyl acetate. We also found that nocodazole, which is a potent inducer of aneuploidy in yeast extract-peptone-dextrose (YEDP) medium but not in synthetic complete (SC) medium, showed a potentiation effect with ethyl acetate in SC medium. This effect in SC medium is similar to that previously reported for nocodazole with ethyl acetate in YEPD medium. When nocodazole was dissolved in 1-methyl-2-pyrrolidinone as a concentrated stock solution, a potentiation effect occurred even at low concentrations of the solvent.

Differential staining of chromosomes and spindle cannot be used as an assay to determine the effect of cancer promoters on primary cultures of human fibroblasts

The differential staining technique allows simultaneous visualization of chromosomes and spindle fibers. The relative sensitivity of this test for chemicals with high tumor-promoting activity (TPA and iodoacetic acid) or for low tumor-promoting chemicals (4β-PDD and α-phorbol) was analyzed. Moreover, to ensure a complementary reference system, metals with possible mutagenic (As > Hg > Cd) and carcinogenic (As > Cd = Hg) properties were tested in parallel. As and Hg, which are known as spindle poisons, do induce, as expected, a disaggregation of the spindle fibers; with Cd, no clear-cut disappearance of the spindle is observed. As far as the cancer promoters are concerned, there is no evidence that their ability to induce mitotic aneuploidy in yeast would be confirmed by the induction of spindle disappearance and/or abnormal chromosome segregation in primary cultures of human fibroblasts.

Genetic change may be caused by interference with protein-protein interactions

Several aprotic polar solvents were shown to induce mitotic aneuploidy in yeast: diethyl ketone, γ-valerolactone, pyridine, pivalinic acid nitrile, phenylacetonitrile and fumaric acid dinitrile. Only fumaric acid dinitrile also strongly induced other types of genetic effects including mitotic crossing-over, mitotic gene conversion and point mutation. The other substances only induced aneuploidy and this only over a very narrow dose range. The treatment protocol used suggested that these chemicals acted via interference with tubulin assembly and disassembly causing a malfunctioning of spindle fiber microtubules. This hypothesis was tested using twice recycled porcine brain tubulin. Diethyl ketone, γ-valerolactone, pyridine and phenylacetonitrile inhibited GTP-promoted assembly of porcine brain tubulin in vitro in the concentration range needed for the induction of mitotic aneuploidy in yeast. Pivalinic acid nitrile accelerated tubulin aggregation whereas fumaric acid dinitrile had no effect even at concentrations 18 times higher than the lowest tested concentration effective in yeast. The in vitro experiments with porcine brain tubulin further suggest that genetic change can result from interference with specific protein-protein interactions. Fumaric acid dinitrile was the only exception since it did induce aneuploidy but had no effects on the assembly of porcine brain tubulin. This could be caused either by interference with protein-protein interactions other than between molecules during assembly and disassembly of microtubules or species-specific differences in susceptibility between yeast spindle and porcine brain tubulin.

Aprotic polar solvents inducing chromosomal malsegregation in yeast interfere with the assembly of porcine brain tubulin in vitro

A number of aprotic solvents which had previously been found to induce mitotic aneuploidy in yeast were tested for their effects on re-assembly of twice recycled tubulin from pig brain. Some of the solvents which were strong aneuploidy-inducing mutagens in yeast slowed down tubulin assembly in vitro at concentrations lower than those required for aneuploidy induction. Ethyl acetate, methyl acetate, diethyl ketone and acetonitrile fell into this category. Other strong aneuploidy-inducing agents like acetone and 2-methoxyethyl acetate accelerated tubulin assembly. Non-genetically active methyl isopropyl ketone and isopropyl acetate both accelerated assembly, whereas methyl n-propyl ketone and n-propyl acetate were weak inducers of aneuploidy and slowed down the rate and extent of assembly. Those chemicals which slowed down the assembly rate also reduced the extent of assembly. Most chemicals which accelerated assembly also led to an increased extent of assembly, with the exception of isopropyl acetate. At the higher concentrations, however, a maximum assembly rate was reached which was followed by a slow decline. Although a perfect correlation between effects on the induction of chromosomal malsegregation and the interference with tubulin assembly in vitro was not seen, the experiments with tubulin were carried out using this class of chemicals because some of them strongly induced mitotic aneuploidy under conditions which suggested tubulin to be the prime target. The lack of a perfect coincidence might be due to species differences between the porcine brain and the yeast spindle tubulin, or the test for aneuploidy induction may have been negative because the concentrations required for an effect on yeast tubulin may be greater than the general lethal toxicity limit. Bearing this reservation in mind, the results suggest that the yeast aneuploidy test has a considerable predictive value for mammalian mutagenicity.

Induction of aneuploidy by oncodazole (nocodazole), an anti-tubulin agent, and acetone

Oncodazole (nocodazole) is a compound which interacts with yeast and bovine tubulin. We have shown that it induces aneuploidy in the yeast Saccharomyces cerevisiae at very low concentrations. In the course of a search for an appropriate solvent for oncodazole we observed that acetone also induces mitotic aneuploidy in yeast. This effect of acetone was greatly enhanced when the treatment of growing cells at 28°C was interrupted by a period of holding at ice-bath temperature.

Assay of the induction of mitotic crossing-over and aneuploidy in yeast by BC, 4CMB and 4HMB

Both BC and 4CMB but not 4HMB were shown to be capable of inducing mitotic crossing-over in exponential phase cells of the D6 strain of the yeast Saccharomyces cerevisiae. In contrast, none of the 3 test compounds were capable of inducing mitotic chromosome aneuploidy.

Tumour promoters induce mitotic aneuploidy in yeast.

Tumour promoters, which complete the process of carcinogenesis initiated by subthreshold doses of carcinogens, can effect changes in gene regulation and cellular differentiation1,2. Whether tumour promoters also induce genotypic changes is unclear. 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent tumour promoter, is inactive in gene mutation assays3–5, but has been reported by some6,7, though not others8–10, to induce sister chromatid exchanges, which may be cytological indications of recombinational events11, in mammalian cells. However, TPA alone is not recombinogenic in yeast12. We now report that in a genetic system using the yeast Saccharomyces cerevisiae13–15, TPA fails to induce recombination but induces mitotic aneuploidy—a change in chromosome number which may lead to hemizygosity of recessive mutations or to phenotypic changes due to an alteration in gene balance. The ability of other agents to induce aneuploidy in this system also correlates with their tumour-promoting activity.

The tolerance of aneuploidy in yeast.

SUMMARYSingle ascospore cultures from triploids were screened as a potential source of disomic tester stocks for the purpose of genetical mapping of chromosomes in yeast. A high tolerance of aneuploidy was found, and strains disomic for only one chromosome were rare. There was a high occurrence of strains disomic at specific chromosomes and in certain multiple combinations of disomic chromosomes.