Gene Copy-Number Variation in Haploid and Diploid Strains of the Yeast Saccharomyces cerevisiae

The kinetochore is the macromolecular protein complex that mediates chromosome segregation. The Dsn1 component is crucial for kinetochore assembly and is phosphorylated by the Aurora B kinase. We found that Aurora B phosphorylation of Dsn1 promotes the interaction between outer and inner kinetochore proteins in budding yeast.     

The Fermentative Formation of CDP-Choline by Haploid Cells of Yeasts and the Change of the Temperature-sensitivity of a Mutant of a Yeast,Saccharomyces rouxii

Phosphorylation of CMP and formation of CDP-choline were tested with various haploid cells of yeasts. Most of them had more or less the ability, but a mutant (Lys–M7, alpha type) of Saccharomyces rouxii was found to lack the ability. Further study revealed the change of the temperature-sensitivity of the mutant, which could not produce CDP-choline when treated at 37°C, whereas it could at 16°C. The growth of the mutant was more sensitive to temperatures than that of the wild strain. The former did not grow at 36.3°C, while the latter grew.     

Rare-Cell Fusion Events between Diploid and Haploid Strains of the Sexually Agglutinative Yeast HANSENULA WINGEI

Diploids of the yeast Hansenula wingei are nonagglutinative and do not form zygotes in mixed cultures with either sexually agglutinative haploid mating type. However, a low frequency of diploid x haploid cell fusions (about 10^-3) is detectable by prototrophic selection. This frequency of rare diploid x haploid matings is not increased after the diploid culture is induced for sexual agglutination. Therefore, we conclude that genes that repress mating are different from those that repress sexual agglutination.——Six prototrophs isolated from one diploid x haploid cross had an average DNA value (µg DNA per 10⁸ cells) of 6.19, compared to 2.53 and 4.35 for the haploid and diploid strains, respectively. Four prototrophs were clearly cell-fusion products because they contained genes from both the diploid and the haploid partners. However, genetic analysis of the prototrophs yielded results inconsistent with triploid meiosis; all six isolates yielded a 2:2 segregation for the mating-type alleles and linked genes.——Mitotic segregation of monosomic (2n-1) cells lacking one homolog of the chromosome carrying the mating-type locus is proposed to explain the rare production of sexually active cells in the diploid cultures. Fusion between such monosomic cells and normal haploids is thought to have produced 3n-1 cells, disomic for the chromosome carrying the mating-type locus. We conclude that in the diploid strain we studied, the physiological mechanisms repressing sexual agglutination and conjugation function efficiently, but events occuring during mitosis lead to a low frequency of genetically altered cells in the population.     

Effects of Polyenes, Detergents, and Other Potential Membrane Perturbants on an Osmotolerant Yeast, Saccharomyces rouxii

The osmotolerance of Saccharomyces rouxii 48-28 was confirmed with both NaCl- and KCl-fortified growth media, with more tolerance being exhibited for the potassium salt. Washed and buffered cells from unfortified medium were challenged with a variety of compounds (and also with physical treatments) that potentially would elicit membrane perturbations. The efficacy of these brief treatments was judged primarily by monitoring subsequent viability. Change in the degree of expression of beta-fructofuranosidase (EC 3.2.1.26), which is cryptic in young cells of S. rouxii, was a second criterion. There was a linear correlation between cell death and enzyme expression for treatments with polyenes, detergents, some organic solvents which did not denature the enzyme, and various freeze-thaw regimens in graded amounts of glycerol. The species is relatively insensitive to polyene antimycotics, the order of decreasing effect being filipin, nystatin, and amphotericin B. S. rouxii was found to be less sensitive to osmotic shock than is Saccharomyces cerevisiae, but in neither species is beta-fructofuranosidase released to the medium. The sensitivity of S. rouxii to ionic detergents, but not to nonionic detergents, was rationalized as being due to cell wall discrimination against larger micelles for the nonionic examples. This was confirmed by showing that protoplasts were sensitive to both classes. In cultures older than 5 days the normal agreement between colony-forming units and methylene blue exclusion (another test of viability) no longer held. Delayed fermentation of sucrose by S. rouxii, which is a diagnostic feature of the species, is explained by death of some cells, expression of their beta-fructofuranosidase, and utilization of the monosaccharides by the surviving cells.     

Physiological changes induced by salt stress in a salt-tolerant soy-yeast, Saccharomyces rouxii

Abstract In Streptococcus cremoris SK11, different permutations of a total of 8 plasmids were observed within and between cultures of various origins. All showed similar growth rates in milk. Those variants which carried a 34-MDa plasmid, pSK112, were resistant to bacteriophage øSK11G, whereas those from which the plasmid was absent or had been cured were sensitive to this phage. Plasmid pSK112 was shown to confer resistance by reduced phage adsorption. These observations have important potential for the development of phage-resistant dairy cultures.     

Extensive Recombination of a Yeast Diploid Hybrid through Meiotic Reversion

In somatic cells, recombination between the homologous chromosomes followed by equational segregation leads to loss of heterozygosity events (LOH), allowing the expression of recessive alleles and the production of novel allele combinations that are potentially beneficial upon Darwinian selection. However, inter-homolog recombination in somatic cells is rare, thus reducing potential genetic variation. Here, we explored the property of S. cerevisiae to enter the meiotic developmental program, induce meiotic Spo11-dependent double-strand breaks genome-wide and return to mitotic growth, a process known as Return To Growth (RTG). Whole genome sequencing of 36 RTG strains derived from the hybrid S288c/SK1 diploid strain demonstrates that the RTGs are bona fide diploids with mosaic recombined genome, derived from either parental origin. Individual RTG genome-wide genotypes are comprised of 5 to 87 homozygous regions due to the loss of heterozygous (LOH) events of various lengths, varying between a few nucleotides up to several hundred kilobases. Furthermore, we show that reiteration of the RTG process shows incremental increases of homozygosity. Phenotype/genotype analysis of the RTG strains for the auxotrophic and arsenate resistance traits validates the potential of this procedure of genome diversification to rapidly map complex traits loci (QTLs) in diploid strains without undergoing sexual reproduction.     

Characterization of Chromosome Stability in Diploid,Polyploid and Hybrid Yeast Cells

Chromosome instability is a key component of cancer progression and many heritable diseases. Understanding why some chromosomes are more unstable than others could provide insight into understanding genome integrity. Here we systematically investigate the spontaneous chromosome loss for all sixteen chromosomes in Saccharomyces cerevisiae in order to elucidate the mechanisms underlying chromosome instability. We observed that the stability of different chromosomes varied more than 100-fold. Consistent with previous studies on artificial chromosomes, chromosome loss frequency was negatively correlated to chromosome length in S. cerevisiae diploids, triploids and S. cerevisiae-S. bayanus hybrids. Chromosome III, an equivalent of sex chromosomes in budding yeast, was found to be the most unstable chromosome among all cases examined. Moreover, similar instability was observed in chromosome III of S. bayanus, a species that diverged from S. cerevisiae about 20 million years ago, suggesting that the instability is caused by a conserved mechanism. Chromosome III was found to have a highly relaxed spindle checkpoint response in the genome. Using a plasmid stability assay, we found that differences in the centromeric sequence may explain certain aspects of chromosome instability. Our results reveal that even under normal conditions, individual chromosomes in a genome are subject to different levels of pressure in chromosome loss (or gain).     

Saccharomyces cerevisiae forms actin ring structures in sporulation, similarly to Zygosaccharomyces rouxii

Yeast filamentous actin (F-actin) exists mainly as patches and cables. Previously, we investigated the behavior of F-actin during sporulation of Zygosaccharomyces rouxii and found a novel actin ring localized around the spore periphery in zygotic asci at a late stage of sporulation. To clarify whether the actin rings are also formed in sporulation in the model yeast Saccharomyces cerevisiae, we observed the distribution of F-actin in sporulating S. cerevisiae by rhodamine-phalloidin staining and confocal laser scanning microscopy. Ringlike actin structures were detected at the peripheral regions of S. cerevisiae spores in globose asci. When asci of S. cerevisiae were induced to become zygotic, actin rings were more obvious than those in globose asci. These results indicate that S. cerevisiae forms characteristic actin ring structures at a late stage of sporulation, similarly to Z. rouxii.     

Association of Chromosome Loss with Centromere-Adjacent Mitotic Recombination in a Yeast Disomic Haploid

Experiments designed to characterize the association between disomic chromosome loss and centromere-adjacent mitotic recombination were performed. Mitotic gene convertants were selected at two heteroallelic sites on the left arm of disomic chromosome III and tested for coincident chromosome loss. The principal results are: (1) Disomic chromosome loss is markedly enhanced (nearly 40-fold) over basal levels among mitotic gene convertants selected to arise close to the centromere; no such enhancement is observed among convertants selected to arise relatively far from the centromere. (2) Chromosome loss is primarily associated with proximal allele conversion at the centromere-adjacent site, and many of these convertants are reciprocally recombined in the adjacent proximal interval. (3) Partial aneuploid exceptions provisionally identified as carrying left arm telocentrics have been found. A testable model is proposed suggesting that centromere involvement in genetic recombination may precipitate segregational disfunction leading to mitotic chromosome loss.     

Periplasmic Structure in Saccharomyces rouxii (Boutroux), an Osmophil

Electron micrographs of ultrathin sections of S. rouxii displayed electrondense, membrane-circumscribed structures between the protoplasmic membrane and the cell wall. These periplasmic bodies were numerous in cells from a 3-day culture and absent or rare in older cells. Periplasmic bodies were fewer and smaller (flattened) in specimens grown in a medium fortified with 10% sucrose; they were not detected in cells grown in 20% sucrose. A brief treatment with ethyl acetate caused the periplasmic bodies of young cells to become electron light. Periplasmic bodies were most prevalent in the regions of the bud scars and were often accommodated within large invaginations in the protoplasmic membrane. In general, conditions which favor the prevalence and electron density of periplasmic bodies are those which also mask the activity of β-fructofuranosidase in this species.     

Effect of Ploidy and Mutagens on Bromodeoxyuridine Resistance in Haploid and Diploid Frog Cells

THE frog embryo cell line ICR 2A is the first established haploid vertebrate cell line¹. In haploid cells recessive mutations should be detectable at a frequency 10⁶ to 10⁹ times greater than expected in diploid cells; mutagen treatment should increase the yield further. These predictions are useful to test whether variants arising in culture are the result of gene mutation. To apply this test to frog cells, mutations for thymidine kinase were sought. Such mutants were first obtained by exposing mouse L cells to the thymidine analogue 5-bromodeoxyuridine (BUdR); a loss of thymidine kinase activity prevented the lethal incorporation of BUdR into DNA². The new phenotype was considered to be the result of gene mutation because of its heritability and eventually because of data from Luria-Delbrück fluctuation analyses³ (a test of the spontaneity or non-inducibility of a process, not its cause). The question of origin was further complicated by a number of factors: (1) the necessity of a long, repeated, exposure to BUdR²; (2) the high mutation rate (up to 10^?3) compared with bacterial mutants (10^?910^?6)^4,5; and (3) the presence of resistant clones with intermediate enzyme levels^4,5.     

In Vitro Induction of Haploid, Diploid, and Triploid Androgenic Embryoids and Plantlets in Datura metal L.

Pollen plantlets of Datura metel L. have been successfully rearedin vitro through anther culture on a nutrient medium supplementedwith coconut milk (15 per cent v/v) The early segmentation patternof the proembryos and their subsequent differentiation intoheart, torpedo, and cotyledonary stages were traced. More than100 plantlets were grown to maturity on soil. They grew normallyand produced flowers. Root-tip and leaf-squash preparationsshowed variable numbers of chromosomes in their cells, and thisindicated the occurrence of ploidy plants among the population.Frequency determinations showed the predominant types to bediploid (70 per cent), triploid (24 per cent), and haploid (6per cent). Differences in the morphology of mature plants inregard to floral size, meiotic irregularities in PMCs, formationof giant pollen grains, differential fruitset, somatic mosaicismin respect of chromosome number, and aneuploidy of cells, wererecorded. The origin of diploid and triploid plants is discussed.     

Karyotype Variability in Yeast Caused by Nonallelic Recombination in Haploid Meiosis

Chromosomes of altered size were found in the meiotic products of a haploid Saccharomyces cerevisiae strain by pulsed field gel electrophoretic separation of whole chromosomes. About 7% of haploid meioses produced chromosomes that differed by >/=10 kb from their wild-type counterparts. Chromosomes most often became enlarged or shortened due to recombination events between sister chromatids at nonallelic sequences. By this mechanism chromosome III acquired tandem arrays of up to eight extra copies of the ~100 kb MAT-HMR segment during repeated rounds of haploid meioses. Enlarged chromosomes III were unstable and changed their size during meiosis more often than remaining unchanged. Altered chromosomes appeared also as the products of intrachromatid recombination and of reciprocal translocations caused by ectopic recombination between nonhomologous chromosomes. In diploid meiosis, chromosomes of altered size occurred at least 10 times less frequently, whereas in mitotic cultures cells with altered karyotypes were virtually absent. The results show that various forms of ectopic recombination are promoted by the absence of allelic homologies.     

Albinism in Plants: A Major Bottleneck in Wide Hybridization,Androgenesis and Doubled Haploid Culture

Albinism is a common problem encountered in interspecific crosses and tissue culture experiments including anther culture and generation of doubled haploids. It is characterized by partial or complete loss of chlorophyll pigments and incomplete differentiation of chloroplast membranes. This in turn impairs photosynthesis and the plants eventually die at a young stage without reaching maturity. Environmental conditions such as light, temperature, media composition and culture conditions play some role in determining the frequency of albino plant formation. Genetic factors are even more important, and are major determinants in albinism. Genetic studies in different crops show that it is a recessive trait governed by many loci. Both the nuclear and chloroplast genomes affect albinism and incompatibilities between the two are a probable cause of many pigment defects in hybrid progenies. Such incompatibility has been reported in a large number of angiosperms. The mechanisms behind these incompatibilities are poorly understood. Studies of plastid DNA inheritance together with observations using electron microscopy have established that the transmission of plastids can be maternal, paternal or biparental, even within the same genus, especially following wide crosses; contrary to the widespread belief that plastids are almost always transmitted from the maternal parent. Albinism has been overcome in some crop species through somatic hybridization and development of cybrids (cytoplasmic hybrids). However, the strict requirement of efficient protoplast regeneration is a major limitation of these techniques. This review focuses on albinism following interspecific crosses or development of doubled haploids facilitated by tissue culture experiments, underlying mechanisms, and the possibilities for dealing with this important biotechnological limitation.