Frequency Of Chromosome Loss Research Articles

Highly genomic instability of super-polyploid strains of Saccharomyces cerevisiae

Polyploid (2n, 3n, and 4n) genomes are known to be unstable in Saccharomyces cerevisiae. Here, we attempted construction of super-polypoid strains (defined as having higher ploidy than tetraploidy) up to 32n by using the matα2-PBT method that we newly developed and investigated their genomic stability. It is known that cell size increases as ploidy increases up to tetraploid. However, unexpectedly, there was no change in the average cell size of the super-polyploid strains compared with tetraploid or pentaploid strains. Smaller sized cells were observed at a rather higher frequency in super-polyploid cell populations compared with those of diploid, triploid and tetraploid strains, suggesting that ploidy reduction in super-polyploid strains occurs quickly at a relatively high frequency. Assuming that ploidy reduction occurs through chromosome loss (or non-disjunction) during mitotic growth, we also estimated the frequency of chromosome loss (or non-disjunction) in various polyploid strains. Our results indicated that the frequency of chromosome loss (or non-disjunction) is drastically increased (10-2-10-3/cells plated) in super-polyploid strains compared with that (10-4-10-5/cells plated) of conventional polyploid (2n-4n) strains. This is the first attempt of construction of super-polyploid strains and investigation of their genomic stability in S.cerevisiae. We believe that the matα2-PBT method will be an invaluable tool for investigating a variety of interesting issues regarding polyploidy and their genomic characterization in eukaryotes.

Depletion of Limiting rDNA Structural Complexes Triggers Chromosomal Instability and Replicative Aging of Saccharomyces cerevisiae.

Sir2 is a highly conserved NAD+-dependent histone deacetylase that functions in heterochromatin formation and promotes replicative life span (RLS) in the budding yeast, Saccharomyces cerevisiae Within the yeast rDNA locus, Sir2 is required for efficient cohesin recruitment and maintaining the stability of the tandem array. In addition to the previously reported depletion of Sir2 in replicatively aged cells, we discovered that subunits of the Sir2-containing complexes silent information regulator (SIR) and regulator of nucleolar silencing and telophase (RENT) were depleted. Several other rDNA structural protein complexes also exhibited age-related depletion, most notably the cohesin complex. We hypothesized that mitotic chromosome instability (CIN) due to cohesin depletion could be a driver of replicative aging. Chromatin immunoprecipitation assays of the residual cohesin (Mcd1-Myc) in moderately aged cells showed strong depletion from the rDNA and initial redistribution to the point centromeres, which was then lost in older cells. Despite the shift in cohesin distribution, sister chromatid cohesion was partially attenuated in aged cells and the frequency of chromosome loss was increased. This age-induced CIN was exacerbated in strains lacking Sir2 and its paralog, Hst1, but suppressed in strains that stabilize the rDNA array due to deletion of FOB1 or through caloric restriction. Furthermore, ectopic expression of MCD1 from a doxycycline-inducible promoter was sufficient to suppress rDNA instability in aged cells and to extend RLS. Taken together, we conclude that age-induced depletion of cohesin and multiple other nucleolar chromatin factors destabilize the rDNA locus, which then results in general CIN and aneuploidy that shortens RLS.

Sup35 prionization [PSI+] influence the frequency of the gene and chromosome mutations, accounted in the alpha-test in yeast Saccharomyces cerevisiae

Background. A lot of neurodegenerative diseases are coursed by amiloidization of proteins in nerve tissues. In the patients brains suffered from Alzheimer’s disease the high fraction of the nerve cells with abnormal chromosome amount was revealed. There are some data showing that prion form of protein PrP may prevent chromosome segregation in mitosis. But the direct association of prionisation and genome stability was not revealed. Materials and methods. We compared the yeast S. cerevisiae strain bearing the prion form of the termination translation factor Sup35, and the strain with non-prionized Sup35 in the alpha-test system. The model of the alpha-test is based on the mechanism of mating type switching in heterothallic yeast strains. The MAT locus that controls the mating type of yeast cell can be presented by two idiomorphs: the MATalpha and MATa that determine the alpha and a cell types, correspondingly. Only two cells with opposite mating types (alpha × a) could copulate. In the mixture of two yeast strains with alpha mating type the hybrids appears only if one of the parent cells had changed its mating type alpha → a. The mating type switching could course the following genetic events: the loss of the chromosome, gene conversion, recombination, loss of the arm of the chromosome, gene mutations and temporary lesions. These events could be distinguished by using the specially constructed alpha-test system. Results. The [PSI+] strain has showed 2-times decreased frequency of «illegitimate» hybridization in the alpha-test compared to [psi-] strain. But [PSI+] doesn’t influence the frequency of «legitimate» hybridization in the alpha × a crossing. The prion [PSI+] also 2-times reduces the frequency of chromosome loss and gene mutations and increases gene conversion 5-times. This results are also confirmed by the canavanine test. Conclusion. We investigated the effect of the Sup35 prionizaion on the genome stability. Unexpectedly in the [PSI+] strain the frequency of «illegitimate» hybridization was 2-times lower, and frequency of gene mutations and chromosome loss was also reduced. The mechanism of this effect is unclear and requires the further investigation.

Identification of Novel Chromosomal Aberrations Induced by (60)Co-γ-Irradiation in Wheat-Dasypyrum villosum Lines.

Mutations induced by radiation are widely used for developing new varieties of plants. To better understand the frequency and pattern of irradiation-induced chromosomal rearrangements, we irradiated the dry seeds of Chinese Spring (CS)-Dasypyrum villosum nullisomic-tetrasomic (6A/6D) addition (6V) line (2n = 44), WD14, with 60Co-γ-rays at dosages of 100, 200, and 300 Gy. The M0 and M1 generations were analyzed using Feulgen staining and non-denaturing fluorescence in situ hybridization (ND-FISH) by using oligonucleotide probes. Abnormal mitotic behavior and chromosomes with structural changes were observed in the M0 plants. In all, 39 M1 plants had structurally changed chromosomes, with the B genome showing the highest frequency of aberrations and tendency to recombine with chromosomes of the D genome. In addition, 19 M1 plants showed a variation in chromosome number. The frequency of chromosome loss was considerably higher for 6D than for the alien chromosome 6V, indicating that 6D is less stable after irradiation. Our findings suggested that the newly obtained γ-induced genetic materials might be beneficial for future wheat breeding programs and functional gene analyses.

Assaying benzene, a parquet varnish, and a synthetic thinner with respect to induction of in vivo chromosome loss in wing primordial cells of Drosophila

An assay detecting the in vivo loss of mwh+Y, a genetically engineered Y chromosome, in cells of the Drosophila wing primordia was published recently. Loss of the mwh+Y chromosome in any of the wing-disk cells – in a multiple wing hairs homozygous background – leads to the formation of an mwh mosaic spot (clone) in the emerging wing. The frequency and the size of the mwh clones allow detection and quantitative evaluation of environmental and/or genetic agents inducing chromosome loss. Using this novel technique, we analyzed the potential of vapors of benzene, a parquet varnish, and a synthetic thinner to induce chromosome loss. Exposure to 0.047μg/ml benzene vapor for one day or to 0.175μg/ml for four hours resulted in a significantly elevated mwh clone-frequency confirming the ability of benzene to induce chromosome loss. A one-day exposure to vapors of a parquet varnish or a 6-h exposure to vapors of a synthetic thinner slightly, yet significantly elevated the frequency of chromosome loss. Results of the present paper show the potential of vapors of the analyzed parquet varnish and synthetic thinner to induce chromosome loss, and illustrate the usefulness of the new technique.

Plc1p is required for proper chromatin structure and activity of the kinetochore in Saccharomyces cerevisiae by facilitating recruitment of the RSC complex

High-fidelity chromosome segregation during mitosis requires kinetochores, protein complexes that assemble on centromeric DNA and mediate chromosome attachment to spindle microtubules. In budding yeast, phosphoinositide-specific phospholipase C (Plc1p encoded by PLC1 gene) is important for function of kinetochores. Deletion of PLC1 results in alterations in chromatin structure of centromeres, reduced binding of microtubules to minichromosomes, and a higher frequency of chromosome loss. The mechanism of Plc1p's involvement in kinetochore activity was not initially obvious; however, a testable hypothesis emerged with the discovery of the role of inositol polyphosphates (InsPs), produced by a Plc1p-dependent pathway, in the regulation of chromatin-remodeling complexes. In addition, the remodels structure of chromatin (RSC) chromatin-remodeling complex was found to associate with kinetochores and to affect centromeric chromatin structure. We report here that Plc1p and InsPs are required for recruitment of the RSC complex to kinetochores, which is important for establishing proper chromatin structure of centromeres and centromere proximal regions. Mutations in PLC1 and components of the RSC complex exhibit strong genetic interactions and display synthetic growth defect, altered nuclear morphology, and higher frequency of minichromosome loss. The results thus provide a mechanistic explanation for the previously elusive role of Plc1p and InsPs in kinetochore function.

The enhancement of pericentromeric cohesin association by conserved kinetochore components promotes high-fidelity chromosome segregation and is sensitive to microtubule-based tension.

Sister chromatid cohesion, conferred by the evolutionarily conserved cohesin complex, is essential for proper chromosome segregation. Cohesin binds to discrete sites along chromosome arms, and is especially enriched surrounding centromeres, but past studies have not clearly defined the roles of arm and pericentromeric cohesion in chromosome segregation. To address this issue, we developed a technique that specifically reduced pericentromeric cohesin association on a single chromosome without affecting arm cohesin binding. Under these conditions, we observed more extensive stretching of centromeric chromatin and elevated frequencies of chromosome loss, suggesting that pericentromeric cohesin enrichment is essential for high-fidelity chromosome transmission. The magnitude of pericentromeric cohesin association was negatively correlated with tension between sister kinetochores, with the highest levels of association in cells lacking kinetochore-microtubule attachments. Pericentromeric cohesin recruitment required evolutionarily conserved components of the inner and central kinetochore. Together, these observations suggest that pericentromeric cohesin levels reflect the balance of opposing forces: the kinetochore-mediated enhancement of cohesin binding and the disruption of binding by mechanical tension at kinetochores. The involvement of conserved kinetochore components suggests that this pathway for pericentromeric cohesin enrichment may have been retained in higher eukaryotes to promote chromosome biorientation and accurate sister chromatid segregation.

Effect of the Major Repeat Sequence on Mitotic Recombination in Candida albicans

The major repeat sequence (MRS) is known to play a role in karyotypic variation in Candida albicans. The MRS affects karyotypic variation by expanding and contracting internal repeats, by altering the frequency of chromosome loss, and by serving as a hotspot for chromosome translocation. We proposed that the effects of the MRS on translocation could be better understood by examination of the effect of the MRS on a similar event, mitotic recombination between two chromosome homologs. We examined the frequency of mitotic recombination across an MRS of average size (approximately 50 kb) as well as the rate of recombination in a 325-kb stretch of DNA adjacent to the MRS. Our results indicate that mitotic recombination frequencies across the MRS were not enhanced compared to the frequencies measured across the 325-kb region adjacent to the MRS. Mitotic recombination events were found to occur throughout the 325-kb region analyzed as well as within the MRS itself. This analysis of mitotic recombination frequencies across a large portion of chromosome 5 is the first large-scale analysis of mitotic recombination done in C. albicans and indicates that mitotic recombination frequencies are similar to the rates found in Saccharomyces cerevisiae.

Measuring Nanometer Scale Gradients in Spindle Microtubule Dynamics Using Model Convolution Microscopy

A computational model for the budding yeast mitotic spindle predicts a spatial gradient in tubulin turnover that is produced by kinetochore-attached microtubule (kMT) plus-end polymerization and depolymerization dynamics. However, kMTs in yeast are often much shorter than the resolution limit of the light microscope, making visualization of this gradient difficult. To overcome this limitation, we combined digital imaging of fluorescence redistribution after photobleaching (FRAP) with model convolution methods to compare computer simulations at nanometer scale resolution to microscopic data. We measured a gradient in microtubule dynamics in yeast spindles at approximately 65-nm spatial intervals. Tubulin turnover is greatest near kinetochores and lowest near the spindle poles. A beta-tubulin mutant with decreased plus-end dynamics preserves the spatial gradient in tubulin turnover at a slower time scale, increases average kinetochore microtubule length approximately 14%, and decreases tension at kinetochores. The beta-tubulin mutant cells have an increased frequency of chromosome loss, suggesting that the accuracy of chromosome segregation is linked to robust kMT plus-end dynamics.

Expression of FLR1 Transporter Requires Phospholipase C and Is Repressed by Mediator

In budding yeast, phosphoinositide-specific phospholipase C (Plc1p encoded by PLC1 gene) is important for function of kinetochores. Deletion of PLC1 results in benomyl sensitivity, alterations in chromatin structure of centromeres, mitotic delay, and a higher frequency of chromosome loss. Here we intended to utilize benomyl sensitivity as a phenotype that would allow us to identify genes that are important for kinetochore function and are downstream of Plc1p. However, our screen identified SIN4, encoding a component of the Mediator complex of RNA polymerase II. Deletion of SIN4 gene (sin4Delta) does not suppress benomyl sensitivity of plc1Delta cells by improving the function of kinetochores. Instead, benomyl sensitivity of plc1Delta cells is caused by a defect in expression of FLR1, and the suppression of benomyl sensitivity in plc1Delta sin4Delta cells occurs by derepression of FLR1 transcription. FLR1 encodes a plasma membrane transporter that mediates resistance to benomyl. Several other mutations in the Mediator complex also result in significant derepression of FLR1 and greatly increased resistance to benomyl. Thus, benomyl sensitivity is not a phenotype exclusively associated with mitotic spindle defect. These results demonstrate that in addition to promoter-specific transcription factors that are components of the pleiotropic drug resistance network, expression of the membrane transporters can be regulated by Plc1p, a component of a signal transduction pathway, and by Mediator, a general transcription factor. The results thus suggest another layer of complexity in regulation of pleiotropic drug resistance.

Enhanced stimulation of chromosomal translocations by radiomimetic DNA damaging agents and camptothecin in Saccharomyces cerevisiae rad9 checkpoint mutants

Saccharomyces cerevisiae rad9 checkpoint mutants exhibit pleiotropic phenotypes, including higher frequencies of chromosome loss, radiation sensitivity, and decreased induction of DNA damage-inducible genes. We had previously shown that rad9 mutants exhibit higher frequencies of DNA damage-associated translocations but lower frequencies of DNA damage-associated sister chromatid exchange (SCE), compared to wild type. Herein, we have shown that differences between the frequencies of DNA damage-associated recombination in the rad9 mutant and wild type depend on the identity and the concentration of the DNA damaging agent. Translocation and SCE frequencies were measured in strains containing truncated his3 fragments, located either on chromosomes II and IV, or located in tandem on chromosome IV, respectively. DNA damage-associated frequencies of translocations after exposure to hydrogen peroxide (H 2O 2), bleomycin, phleomycin, cisplatin, and camptothecin are higher in the rad9 diploid than in wild type. However, translocation frequencies after exposure to 4-nitroquinoline 1-oxide (4-NQO) and N-methyl- N′-nitro- N-nitrosoguanidine (MNNG) are similar in rad9 and wild-type strains. We suggest that the deficiency in triggering G 2 arrest after exposure to specific DNA damaging agents results in the higher levels of DNA damage-associated translocations in rad9 mutants.

Functional characterization of CaCBF1, the Candida albicans homolog of centromere binding factor 1

The centromere binding factor 1 (Cbf1) is necessary for proper chromosome segregation and transcriptional activation of methionine biosynthesis genes in the yeast Saccharomyces cerevisiae and is essential for viability in the related yeasts Kluyveromyces lactis and Candida glabrata. To study the function of Cbf1p in Candida albicans, the major human fungal pathogen, we constructed strains in which both alleles of the CaCBF1 gene were deleted. The Δ cbf1 mutants exhibited a slow growth phenotype and were temperature-sensitive at 42 °C. In addition, the mutants were auxotrophic for sulfur amino acids and could grow on minimal medium only when it was supplemented with either methionine or cysteine, suggesting that CaCBF1 is necessary for the expression of genes involved in assimilation of inorganic sulfate. Deletion of CaCBF1 also resulted in morphological abnormalities, many cells being unusually large. All mutant phenotypes were complemented by reintroduction of a functional CaCBF1 copy. The Δ cbf1 mutants neither showed enhanced sensitivity to the microtubule destabilizing agent thiabendazole nor did they exhibit an increased frequency of chromosome loss. These results suggest that Cbf1p is not necessary for efficient chromosome segregation in C. albicans.

Genetic evidence for a role of phospholipase C at the budding yeast kinetochore.

Chromosome segregation during mitosis requires kinetochores, specialized organelles that mediate chromosome attachment to spindle microtubules. We have shown previously that in budding yeast, Plc1p (phosphoinositide-specific phospholipase C) localizes to centromeric loci, associates with the kinetochore proteins Ndc10p and Cep3p, and affects the function of kinetochores. Deletion of PLC1 results in nocodazole sensitivity, mitotic delay, and a higher frequency of chromosome loss. We report here that despite the nocodazole sensitivity of plc1Delta cells, Plc1p is not required for the spindle checkpoint. However, plc1Delta cells require a functional BUB1/BUB3-dependent spindle checkpoint for viability. PLC1 displays strong genetic interactions with genes encoding components of the inner kinetochore, including NDC10, SKP1, MIF2, CEP1, CEP3, and CTF13. Furthermore, plc1Delta cells display alterations in chromatin structure in the core centromere. Chromatin immunoprecipitation experiments indicate that Plc1p localizes to centromeric loci independently of microtubules, and accumulates at the centromeres during G(2)/M stage of cell cycle. These results are consistent with the view that Plc1p affects kinetochore function, possibly by modulating the structure of centromeric chromatin.

Spindle assembly checkpoint component CaMad2p is indispensable for Candida albicans survival and virulence in mice.

Here, we report an indispensable role for spindle assembly checkpoint (SAC) component CaMad2p in the survival and virulence of Candida albicans in mice. We hypothesized that cell cycle checkpoint functions, especially those monitoring the integrity of DNA and chromosome segregation, might be required for the pathogen to repair damage caused by host defence. To test this idea, we created SAC-defective mutants by deleting the CaMAD2 gene that encodes a key component of the SAC pathway. The CaMAD2 mutant appears normal in morphology, growth rate and growth mode switch in unperturbed conditions. However, it quickly loses viability when treated with nocodazole, which causes disassembly of mitotic spindles. The mutant also exhibits increased frequency of chromosome loss. The virulence of the mutant is greatly reduced in mice, presumably because of the inability of the mutant cells to stop the cell cycle when the host defence damages cellular components important for chromosome segregation. Supporting this hypothesis, unlike the wild-type cells that can proliferate within and eventually grow out of macrophages, most of the CaMAD2 null mutant cells are unable to survive. This study suggests that SAC is required for survival of C. albicans in the host and could thus be targeted for anti-C. albicans therapies.

Chromosome nondisjunction and loss induced by protons and X rays in primary human fibroblasts: role of centromeres in aneuploidy.

To study the origin of micronuclei induced in human primary fibroblasts by low-energy protons (7.7 and 28.5 keV/microm) and X rays, we have developed a combined antikinetochore-antibody (CREST) and FISH staining with pancentromeric probes. This technique allowed us to analyze the integrity of the kinetochore and centromeric DNA structures and to assess their role in induced aneuploidy. The effect of LET on radiation-induced chromosome nondisjunction was studied in binucleated cells with centromeric-specific DNA probes for chromosomes 7 and 11. Our results indicate that, though more than 90% of radiation-induced micronuclei were CREST(-)/FISH(-), 28.5 keV/microm protons and X rays were also able to induce statistically significant increases in the number of micronuclei that were CREST(-)/FISH(+) and CREST(+)/FISH(+), respectively. One interpretation of these results could be that the protons induced chromosome loss by kinetochore detachment or by breakage in the centromeric DNA region, whereas X rays induced aneuploidy through a non-DNA damage mechanism. Nondisjunction appears to be a far more important mechanism leading to radiation-induced aneuploidy. Irrespective of the higher frequency of micronuclei induced by 28.5 keV/microm protons, the frequency of chromosome loss was markedly higher for X rays than for 28.5 keV/microm protons, strengthening the hypothesis that non-DNA targets, such as components of the mitotic spindle apparatus, may be involved in aberrations in chromosome segregation after X irradiation.

Degradation of a cohesin subunit by the N-end rule pathway is essential for chromosome stability.

Cohesion between sister chromatids is established during DNA replication and depends on a protein complex called cohesin. At the metaphase-anaphase transition in the yeast Saccharomyces cerevisiae, the ESP1-encoded protease separin cleaves SCC1, a subunit of cohesin with a relative molecular mass of 63,000 (Mr 63K). The resulting 33K carboxy-terminal fragment of SCC1 bears an amino-terminal arginine-a destabilizing residue in the N-end rule. Here we show that the SCC1 fragment is short-lived (t1/2 approximately 2 min), being degraded by the ubiquitin/proteasome-dependent N-end rule pathway. Overexpression of a long-lived derivative of the SCC1 fragment is lethal. In ubr1Delta cells, which lack the N-end rule pathway, we found a highly increased frequency of chromosome loss. The bulk of increased chromosome loss in ubr1Delta cells is caused by metabolic stabilization of the ESP1-produced SCC1 fragment. This fragment is the first physiological substrate of the N-end rule pathway that is targeted through its N-terminal residue. A number of yeast proteins bear putative cleavage sites for the ESP1 separin, suggesting other physiological substrates and functions of the N-end rule pathway.

Checkpoint adaptation precedes spontaneous and damage-induced genomic instability in yeast.

Despite the fact that eukaryotic cells enlist checkpoints to block cell cycle progression when their DNA is damaged, cells still undergo frequent genetic rearrangements, both spontaneously and in response to genotoxic agents. We and others have previously characterized a phenomenon (adaptation) in which yeast cells that are arrested at a DNA damage checkpoint eventually override this arrest and reenter the cell cycle, despite the fact that they have not repaired the DNA damage that elicited the arrest. Here, we use mutants that are defective in checkpoint adaptation to show that adaptation is important for achieving the highest possible viability after exposure to DNA-damaging agents, but it also acts as an entrée into some forms of genomic instability. Specifically, the spontaneous and X-ray-induced frequencies of chromosome loss, translocations, and a repair process called break-induced replication occur at significantly reduced rates in adaptation-defective mutants. This indicates that these events occur after a cell has first arrested at the checkpoint and then adapted to that arrest. Because malignant progression frequently involves loss of genes that function in DNA repair, adaptation may promote tumorigenesis by allowing genomic instability to occur in the absence of repair.

Spontaneous loss of heterozygosity in diploid Saccharomyces cerevisiae cells.

To obtain a broad perspective of the events leading to spontaneous loss of heterozygosity (LOH), we have characterized the genetic alterations that functionally inactivated the URA3 marker hemizygously or heterozygously situated either on chromosome III or chromosome V in diploid Saccharomyces cerevisiae cells. Analysis of chromosome structure in a large number of LOH clones by pulsed-field gel electrophoresis and PCR showed that chromosome loss, allelic recombination, and chromosome aberration were the major classes of genetic alterations leading to LOH. The frequencies of chromosome loss and chromosome aberration were significantly affected when the marker was located in different chromosomes, suggesting that chromosome-specific elements may affect the processes that led to these alterations. Aberrant-sized chromosomes were detected readily in approximately 8% of LOH events when the URA3 marker was placed in chromosome III. Molecular mechanisms underlying the chromosome aberrations were further investigated by studying the fate of two other genetic markers on chromosome III. Chromosome aberration caused by intrachromosomal rearrangements was predominantly due to a deletion between the MAT and HMR loci that occurred at a frequency of 3.1 x 10(-6). Another type of chromosome aberration, which occurred at a frequency slightly higher than that of the intrachromosomal deletion, appeared to be caused by interchromosomal rearrangement, including unequal crossing over between homologous chromatids and translocation with another chromosome.

Differences in malsegregation rates obtained by scoring ana-telophases or binucleate cells.

In this work we have applied in situ hybridization with alphoid centromeric probes specific to chromosomes 7 and 11 to ana-telophase cells from human primary fibroblasts. The aim was to visualize the events leading to aneuploidy directly during anaphase, analyse the induction of aneuploidy during this mitotic stage and compare the frequencies of chromosome malsegregation observed in ana-telophases with the estimated malsegregation obtained in binucleate cells after a short cytochalasin B treatment. Significantly higher frequencies of chromosome loss and chromosome non-disjunction were observed in fibroblasts undergoing ana-telophase during recovery from a nocodazole-induced mitotic arrest compared with binucleate cells obtained by a further 30 min incubation with cytochalasin B. Using the same experimental schedule, analysis of hybridization signals in mononucleate cells showed higher frequencies of polyploid nuclei in cytochalasin B-treated cultures, indicating that part of the ana-telophases observed after release from the nocodazole-induced mitotic arrest may give rise to polyploid mononucleate cells instead of binucleate ones. A reduced distance between spindle poles was also measured in cells undergoing ana-telophase in the presence of cytochalasin B. Our study suggests that in nocodazole and cytochalasin B-treated cultures the shorter pole-to-pole distance may favour the reformation of a single membrane around telophase chromosomes, especially when several lagging chromosomes lie between the two future daughter nuclei. This would give rise to polyploid mononucleate cells at the ensuing interphase.

Regulation of Saccharomyces cerevisiae kinetochores by the type 1 phosphatase Glc7p.

We have investigated the role of protein phosphorylation in regulation of Saccharomyces cerevisiae kinetochores. By use of phosphatase inhibitors and a type 1 protein phosphatase mutant (glc7-10), we show that the microtubule binding activity, but not the centromeric DNA-binding activity, of the kinetochore complex is regulated by a balance between a protein kinase and the type 1 protein phosphatase (PP1) encoded by the GLC7 gene. glc7-10 mutant cells exhibit low kinetochore-microtubule binding activity in vitro and a high frequency of chromosome loss in vivo. Specifically, the Ndc10p component of the centromere DNA-binding CBF3 complex is altered by the glc7-10 mutation; Ndc10p is hyperphosphorylated in glc7-10 extracts. Furthermore, addition of recombinant Ndc10p reconstitutes the microtubule-binding activity of a glc7-10 extract to wild-type levels. Finally, the glc7-10-induced mitotic arrest is abolished in spindle checkpoint mutants, suggesting that defects in kinetochore-microtubule interactions caused by hyperphosphorylation of kinetochore proteins activate the spindle checkpoint.