Healing of Broken Linear Dicentric Chromosomes in Yeast

In yeast, meiotic recombination between a linear chromosome III and a haploid-viable circular chromosome will yield a dicentric, tandemly duplicated chromosome. Spores containing apparently intact dicentric chromosomes were recovered from tetrads with three viable spores. The spore containing the dicentric inherited URA3 (part of the recombinant DNA used to join regions near the ends of the chromosome into a circle) as well as HML, HMR and MAL2 (located near the two ends of a linear but deleted from the circle). The Ura⁺ Mal⁺ colonies were highly variegated, giving rise to as many as seven distinctly different stable ("healed") derivatives, some of which were Ura⁺ Mal ⁺, others Ura⁺ Mal^- and others Ura ^- Mal⁺. The colonies were also sectored for five markers (HIS4, LEU2, CRY1, MAT and THR4) initially heterozygous in the tandemly duplicated dicentric chromosome.—Southern blot and genetic analyses have demonstrated that these stable derivatives arose from mitotic break-age of the dicentric chromosome, followed by one of several different healing events. The majority of the stable derivatives contained circular or linear chromosomes apparently resulting from homologous recombination between a broken chromosome end and a homologous region on the other end of the original dicentric duplicated chromosome. A smaller proportion of events resulted in apparently uniquely healed linear chromosomes in which the broken chromosome acquired a new telomere. In two instances we recovered chromosome III partially duplicated with a novel right end. We have also found one derivative that had also experienced rearrangement of repeated DNA sequences found adjacent to yeast telomeres.     

Isolation of Auxotrophic Mutants of Diploid Industrial Yeast Strains after UV Mutagenesis

Auxotrophic mutants of the yeast Saccharomyces cerevisiae are usually isolated in haploid strains because the isolation of recessive mutations in diploids is thought to be difficult due to the presence of two sets of genes. We show here that auxotrophic mutants of diploid industrial sake yeast strains were routinely obtained by a standard mutant selection procedure following UV mutagenesis. We isolated His⁻, Met⁻, Lys⁻, Trp⁻, Leu⁻, Arg⁻, and Ura⁻ auxotrophic mutants of five sake strains, Kyokai no. 7, no. 9, no. 10, no. 701, and no. 901, by screening only 1,700 to 3,400 colonies from each treated strain. Wild-type alleles were cloned and used as markers for transformation. With HIS3 as a selectable marker, the yeast TDH3 overexpression promoter was inserted upstream of ATF1, encoding alcohol acetyltransferase, by one-step gene replacement in a his3 mutant of Kyokai no. 7. The resulting strain contained exclusively yeast DNA, making it acceptable for commercial use, and produced a larger amount of isoamyl acetate, a banana-like flavor. We argue that the generally recognized difficulty of isolating auxotrophic mutants of diploid industrial yeast strains is misleading and that genetic techniques used for haploid laboratory strains are applicable for this purpose.     

Proper Microtubule Structure Is Vital for Timely Progression through Meiosis in Fission Yeast

Cells of the fission yeast Schizosaccharomyces pombe normally reproduce by mitotic division in the haploid state. When subjected to nutrient starvation, two haploid cells fuse and undergo karyogamy, forming a diploid cell that initiates meiosis to form four haploid spores. Here, we show that deletion of the mal3 gene, which encodes a homolog of microtubule regulator EB1, produces aberrant asci carrying more than four spores. The mal3 deletion mutant cells have a disordered cytoplasmic microtubule structure during karyogamy and initiate meiosis before completion of karyogamy, resulting in twin haploid meiosis in the zygote. Treatment with anti-microtubule drugs mimics this phenotype. Mutants defective in karyogamy or mutants prone to initiate haploid meiosis exaggerate the phenotype of the mal3 deletion mutant. Our results indicate that proper microtubule structure is required for ordered progression through the meiotic cycle. Furthermore, the results of our study suggest that fission yeast do not monitor ploidy during meiosis.     

Gene Conversion and Intragenic Recombination at at the SUP6 Locus and the Surrounding Region in SACCHAROMYCES CEREVISIAE

Spontaneous secondary mutations of the ochre suppressor SUP6 were selected in a haploid strain of Saccharomyces cerevisiae . Unselected tetrads were dissected from crosses heterozygous for one of three alleles of SUP6 and for three other loci in this region which span a length of 14 map units (his2, cdc14 and met10). The study showed that all of these markers were characterized by high frequency of meiotic gene conversion and long conversion lengths which frequently extended into adjacent marked loci. Despite the high conversion frequency of SUP6 , recombination between alleles of this locus reached a maximum frequency of only 2 x 10^-3 prototrophs/spore. Although the allelic recombination frequencies were not distance dependent and consequently could not be used to order the alleles, the inequality between the two recombinant outside marker combinations among selected intragenic recombinants produced an internally consistent map of the suppressor locus. Recombination at SUP6 (whether detected as conversion in tetrads or the production of recombinants among random spores) was accompanied by significantly less than 50% outside marker recombination.     

Mating-Type Functions for Meiosis and Sporulation in Yeast Act through Cytoplasm

Given a nutritional regime marked by a low nitrogen level and the absence of fermentable carbon sources, conventional a/α diploid cells of Saccharomyces cerevisiae exhibit a complex developmental sequence that includes a round of premeiotic DNA replication, commitment to meiosis and the elaboration of mature tetrads containing viable ascospores. Ordinarily, haploid cells and diploid cells of genotype a/a and α/α fail to display these reactions under comparable conditions. Here, we describe a simple technique for sporulation of α/α and a/a cells. Cells of genotype α/α are mated to haploid a cells carrying the kar1 (karyogamy defective) mutation to yield heterokaryons containing the corresponding diploid and haploid nuclei. The kar1 strains mate normally, but nuclei in the resultant zygotes do not fuse. When heterokaryotic cells are inoculated into sporulation media, they produce asci with six spores. Four spores carry genotypes derived from the diploid nucleus and the other two possess the markers originating from the haploid nucleus, i.e., the diploid nucleus divides meiotically while the haploid nucleus apparently divides mitotically. Similarly, the a/a genome is "helped" to sporulate as a consequence of mating with α kar1 strains. The results allow us to conclude that the mating-type functions essential for meiosis and sporulation are communicated and act through the cytoplasm and that sporulation can be dissociated from typical meiosis. This procedure will facilitate the genetic analysis of strains that are otherwise unable to sporulate.     

Histone H2B subtypes are dispensable during the yeast cell cycle

Saccharomyces cerevisiae contains two histone H2B protein subtypes, H2B1 and H2B2, which differ at 4 of 130 amino acids. We describe experiments that test whether both histone H2B subtypes are required for the completion of any stage in the yeast life cycle. Frameshift mutations were introduced into cloned copies of the H2B1 and H2B2 genes. These altered genes were integrated into the yeast genome by transformation and replaced the wild-type genes through recombination. We thus obtained strains that lacked functional H2B1 or H2B2 proteins. These mutant strains survive as haploids and homozygous diploids. During vegetative growth, they divide at the same rate as wild-type cells and are able to mate, sporulate and germinate. The h2b1^? cells grew more slowly after germination than h2b2^? or wild-type spores, but otherwise the mutants were indistinguishable from each other or from wild-type cells. We also attempted to make a strain that was mutant in both genes for H2B. We examined spores derived from a diploid that is heterozygous for both histone mutations. The two genes assort independently, so we expect one in four spores to be h2b1^?h2b2^?. Of 61 spore colonies examined, none was mutant at both loci. Our results indicate that the double mutant can germinate and bud once but cannot grow further. Since the yeast life cycle can be completed in the absence of either but not both histone H2B subtypes, we conclude that neither protein has a unique essential function.     

Recessive lethality of yeast strains carrying the SUP61 suppressor results from loss of a transfer RNA with a unique decoding function

A serine-inserting ochre suppressor (SUP61) and its amber allele (SUP-RL1) in the yeast Saccharomyces cerevisiae can only be derived from or maintained in diploid strains heterozygous for the suppressor transfer RNA locus (Brandriss et al., 1975). Two models have been proposed to account for this recessive lethal phenotype. In one, lethality results from the presence of the altered gene product; excessive suppression could interfere with the proper termination of translation. In the second model, lethality is due to the loss of the wild-type function; the suppressor mutation could alter an essential gene that is present in only a single copy in the haploid genome. We have tested a set of specific genetic and biochemical predictions which uniquely distinguish these models.We first isolated several mutant strains carrying second-site mutations which lie within, or are closely linked to, the SUP61 locus. Despite the absence of any biologically detectable suppressor activity, these mutants still give rise to only two viable spores per tetrad. As in the parent, lethality is absolutely correlated with the segregation of the SUP61 allele, and thus it cannot be due solely to suppression.To demonstrate that the SUP61 mutation alters an essential function in haploid cells, a cloned copy of the wild-type gene (sup⁺) was introduced into a diploid containing SUP61 by transformation. Following sporulation, the transformant gave rise to four viable spores per tetrad. We have shown by hybridization analysis that the two spores per tetrad which have suppressor function contain the cloned sup⁺ gene and plasmid DNA integrated in tandem with the SUP61 gene.Piper (1978) has shown that the amber suppressor SUP-RL1 is derived from a tRNA_UCG^Ser gene. More recently, we and others (Etcheverry et al., 1979; Olson et al., 1981; Broach et al., 1981) have provided evidence that the gene coding for this tRNA species exists in only a single copy per haploid genome. Our ability to “cure” the recessive lethal phenotype of SUP61 now allows the conclusion that the gene altered by the suppressor mutation codes for the only isoaccepting species of tRNA^Ser which can decode UCG codons in vivo.     

Genetic Analysis of Haploids from Industrial Strains of Baker's Yeast

Strains of baker's yeast conventionally used by the baking industry in Japan were tested for the ability to sporulate and produce viable haploid spores. Three isolates which possessed the properties of baker's yeasts were obtained from single spores. Each strain was a haploid, and one of these strains, YOY34, was characterized. YOY34 fermented maltose and sucrose, but did not utilize galactose, unlike its parental strain. Genetic analysis showed that YOY34 carried two MAL genes, one functional and one cryptic; two SUC genes; and one defective gal gene. The genotype of YOY34 was identified as MATα MAL1 MAL3g SUC2 SUC4 gall. The MAL1 gene from this haploid was constitutively expressed, was dominant over other wild-type MAL tester genes, and gave a weak sucrose fermentation. YOY34 was suitable for both bakery products, like conventional baker's yeasts, and for genetic analysis, like laboratory strains.     

A method for isolating milky disease, Bacillus popilliae var. rhopaea, spores from the soil

A method based on the tyndallization procedure is described for isolation of Bacillus popilliae var. rhopaea spores from the soil. A soil suspension is diluted with a germinating medium, which promotes the germination of most spores except B. popilliae var. rhopaea, and is treated with a series of seven heat shocks (70°C for 20 min) at hourly intervals. This treatment reduced the number of contaminant spores by over 95%. The suspension is then plated out onto “J” medium which allows the germination and growth of all surviving spores including the milky disease spores. The plates are incubated anaerobically at 28°C for 7 days before the characteristic small transparent colonies of B. popilliae var. rhopaea are counted. In testing the method it was revealed that about 15% of the milky disease spores in the soil produced visible colonies, and that a spore concentration of over 1.2 × 10⁵ spores/g dry wt of soil could be quantified. This concentration of spores produces only 3% infection in Rhopaea verreauxi larvae. The method may be applicable to other varieties of B. popilliae which will grow on “J” medium.     

Flow Cytometry-based Purification of S. cerevisiae Zygotes

Zygotes are essential intermediates between haploid and diploid states in the life cycle of many organisms, including yeast (Figure 1) ¹. S. cerevisiae zygotes result from the fusion of haploid cells of distinct mating type (MATa, MATalpha) and give rise to corresponding stable diploids that successively generate as many as 20 diploid progeny as a result of their strikingly asymmetric mitotic divisions ². Zygote formation is orchestrated by a complex sequence of events: In this process, soluble mating factors bind to cognate receptors, triggering receptor-mediated signaling cascades that facilitate interruption of the cell cycle and culminate in cell-cell fusion. Zygotes may be considered a model for progenitor or stem cell function.Although much has been learned about the formation of zygotes and although zygotes have been used to investigate cell-molecular questions of general significance, almost all studies have made use of mating mixtures in which zygotes are intermixed with a majority population of haploid cells ^3-8. Many aspects of the biochemistry of zygote formation and the continuing life of the zygote therefore remain uninvestigated.Reports of purification of yeast zygotes describe protocols based on their sedimentation properties ⁹; however, this sedimentation-based procedure did not yield nearly 90% purity in our hands. Moreover, it has the disadvantage that cells are exposed to hypertonic sorbitol. We therefore have developed a versatile purification procedure. For this purpose, pairs of haploid cells expressing red or green fluorescent proteins were co-incubated to allow zygote formation, harvested at various times, and the resulting zygotes were purified using a flow cytometry-based sorting protocol. This technique provides a convenient visual assessment of purity and maturation. The average purity of the fraction is approximately 90%. According to the timing of harvest, zygotes of varying degrees of maturity can be recovered. The purified samples provide a convenient point of departure for "-omic" studies, for recovery of initial progeny, and for systematic investigation of this progenitor cell.     

Isolation of Protoplasts from Pollen Tetrads

PRODUCTION of haploid plants by anther culture is restricted to only a few taxa¹. If protoplasts could be isolated from pollen tetrads they might behave in culture similarly to somatic cell protoplasts² and serve as the starting material for the production of haploid plants for a wide range of plant species. Such isolated microspore protoplasts might also be suitable for fusion studies in relation to somatic hybridization of plants².     

Analysis of Homothallic Saccharomyces cerevisiae Strain Mating during Must Fermentation

Genetic instability and genome renewal may cause loss of heterozygosity (LOH) in homothallic wine yeasts (Saccharomyces cerevisiae), leading to the elimination of the recessive lethal or deleterious alleles that decrease yeast fitness. LOH was not detected in genetically stable wine yeasts during must fermentation. However, after sporulation, the heterozygosity of the new yeast population decreased during must fermentation. The frequency of mating between just-germinated haploid cells from different tetrads was very low, and the mating of haploid cells from the same ascus was favored because of the physical proximity. Also, mating restriction between haploid cells from the same ascus was found, leading to a very low frequency of self spore clone mating. This mating restriction slowed down the LOH process of the yeast population, maintaining the heterozygote frequency higher than would be expected assuming a fully random mating of the haploid yeasts or according to the Mortimer genome renewal proposal. The observed LOH occurs because of the linkage of the locus MAT to the chromosome III centromere, without the necessity for self spore clone mating or the high frequency of gene conversion and rapid asymmetric LOH observed in genetically unstable yeasts. This phenomenon is enough in itself to explain the high level of homozygosis found in natural populations of wine yeasts. The LOH process for centromere-linked markers would be slower than that for the nonlinked markers, because the linkage decreases the frequency of newly originated heterozygous yeasts after each round of sporulation and mating. This phenomenon is interesting in yeast evolution and may cause important sudden phenotype changes in genetically stable wine yeasts.     

Ordered Linear Tetrads Are Produced by the Sporulation of Newly Formed Zygotes of Saccharomyces cerevisiae

Diploid Saccharomyces cerevisiae strains normally sporulate to produce tetrahedral unordered asci containing four spores (tetrads). We report that when newly formed zygotes are subjected to the same sporulation conditions, they form predominantly linear ordered tetrads. We show that the two spores from each end of such a linear tetrad invariably contain nonsister centromeres. Spore viability, recombination and independence of centromere segregation appear unaffected.     

Mutants of the Killer Plasmid of SACCHAROMYCES CEREVISIAE Dependent on Chromosomal Diploidy for Expression and Maintenance

Mutants of the killer plasmid of Saccharomyecs cerevisiae have been isolated that depend upon chromosomal diploidy for the expression of plasmid functions and for replication or maintenance of the plasmid itself. These mutants are not defective in any chromosomal gene needed for expression or replication of the killer plasmid.—Haploids carrying these mutant plasmids (called d for diploid-dependent) are either unable to kill or unable to resist being killed or both and show frequent loss of the plasmid. The wild-type phenotype (K⁺R⁺) is restored by mating the d plasmid-carrying strain with either (a) a wild-type sensitive strain which apparently has no killer plasmid; (b) a strain which has been cured of the killer plasmid by growth at elevated temperature; (c) a strain which has been cured of the plasmid by growth in the presence of cycloheximide; (d) a strain which has lost the plasmid because it carries a mutation in a chromosomal mak gene; or (e) a strain of the opposite mating type which carries the same d plasmid and has the same defective phenotype, indicating that the restoration of the normal phenotype is not due to recombination between plasmid genomes or complementation of plasmid or chromosomal genes.—Sporulation of the phenotypically K⁺R⁺ diploids formed in matings between d and wild-type nonkiller strains yields tetrads, all four of whose haploid spores are defective for killing or resistance or maintenance of the plasmid or a combination of these. Every defective phenotype may be found among the segregants of a single diploid clone carrying a d plasmid. These defective segregants resume the normal killer phenotype in the diploids formed when a second round of mating is performed, and the segregants from a second round of meiosis and sporulation are again defective.     

Applied Usage of Yeast Spores as Chitosan Beads

In this study, we present a nonhazardous biological method of producing chitosan beads using the budding yeast Saccharomyces cerevisiae. Yeast cells cultured under conditions of nutritional starvation cease vegetative growth and instead form spores. The spore wall has a multilaminar structure with the chitosan layer as the second outermost layer. Thus, removal of the outermost dityrosine layer by disruption of the DIT1 gene, which is required for dityrosine synthesis, leads to exposure of the chitosan layer at the spore surface. In this way, spores can be made to resemble chitosan beads. Chitosan has adsorptive features and can be used to remove heavy metals and negatively charged molecules from solution. Consistent with this practical application, we find that spores are capable of adsorbing heavy metals such as Cu²⁺, Cr³⁺, and Cd²⁺, and removal of the dityrosine layer further improves the adsorption. Removal of the chitosan layer decreases the adsorption, indicating that chitosan works as an adsorbent in the spores. Besides heavy metals, spores can also adsorb a negatively charged cholesterol derivative, taurocholic acid. Furthermore, chitosan is amenable to chemical modifications, and, consistent with this property, dit1Δ spores can serve as a carrier for immobilization of enzymes. Given that yeast spores are a natural product, our results demonstrate that they, and especially dit1Δ mutants, can be used as chitosan beads and used for multiple purposes.     

Morphological variants of Moniliophthora roreri on artificial media and the biotroph/necrotroph shift

Moniliophthora roreri (Mr) causes frosty pod rot of Theobroma cacao in a hemibiotrophic association. The Mr biotroph-like phase has not been studied in culture. Mr spores (isolates Co12, Co52, and B3) were germinated on high (V8) and low (BPMM) nutrients with different media hardness (0.5% to 3% agarose). Germination was high on V8 media. Hardness affected germination on BPMM. Most colonies on V8 were slow-growing, failing to sporulate. Colony morphology depended on the isolate. On BPMM, exaggerated mycelia formed of limited length with enlarged cells. On agarose, rapidly expanding sporulating necrotrophic colonies formed rarely. Co12 and B3 spores were germinated on V8 and BPMM with low melting point (LMP) agarose. Slow-growing colonies of B3 on BPMM were unstable on LMP agarose, often forming slow-growing/rapidly expanding hybrids. Slow-growing colonies are hypothesized to represent the biotrophic phase. One nucleus was common in Mr cells, other than spores. Binucleate cells were occasionally observed in aged cells of slow-growing mycelia. Co52 cells often had more than two nuclei per cell after germination. Mr mycelia cells typically carry a single nucleus, being considered haploid. Biotroph- and necrotroph-like mycelia displayed differential gene expression but results were inconsistent with published in vivo results and require further study.     

Mal3, the Schizosaccharomyces pombe homolog of EB1, is required for karyogamy and for promoting oscillatory nuclear movement during meiosis

Two successive rounds of chromosome segregation following a single round of DNA replication enable the production of haploid gametes during meiosis. In the fission yeast Schizosaccharomyces pombe, karyogamy is the process where the nuclei from 2 haploid cells fuse to create a diploid nucleus, which then undergoes meiosis to produce 4 haploid spores. By screening a collection of S. pombe deletion strains, we found that the deletion of 2 genes, mal3 and mto1, leads to the production of asci containing up to 8 spores. Here, we show that Mal3, the fission yeast member of the EB1 family of conserved microtubule plus-end tracking proteins, is required for karyogamy, oscillatory nuclear movement, and proper segregation of chromosomes during meiosis. In the absence of Mal3, meiosis frequently initiates before the completion of karyogamy, thus producing up to 8 nuclei in a single ascus. Our results provide new evidence that fission yeast can initiate meiosis prior to completing karyogamy.     

The Green Monster Process for the Generation of Yeast Strains Carrying Multiple Gene Deletions

Phenotypes for a gene deletion are often revealed only when the mutation is tested in a particular genetic background or environmental condition^1,2. There are examples where many genes need to be deleted to unmask hidden gene functions^3,4. Despite the potential for important discoveries, genetic interactions involving three or more genes are largely unexplored. Exhaustive searches of multi-mutant interactions would be impractical due to the sheer number of possible combinations of deletions. However, studies of selected sets of genes, such as sets of paralogs with a greater a priori chance of sharing a common function, would be informative.In the yeast Saccharomyces cerevisiae, gene knockout is accomplished by replacing a gene with a selectable marker via homologous recombination. Because the number of markers is limited, methods have been developed for removing and reusing the same marker^5,6,7,8,9,10. However, sequentially engineering multiple mutations using these methods is time-consuming because the time required scales linearly with the number of deletions to be generated.Here we describe the Green Monster method for routinely engineering multiple deletions in yeast¹¹. In this method, a green fluorescent protein (GFP) reporter integrated into deletions is used to quantitatively label strains according to the number of deletions contained in each strain (Figure 1). Repeated rounds of assortment of GFP-marked deletions via yeast mating and meiosis coupled with flow-cytometric enrichment of strains carrying more of these deletions lead to the accumulation of deletions in strains (Figure 2). Performing multiple processes in parallel, with each process incorporating one or more deletions per round, reduces the time required for strain construction.The first step is to prepare haploid single-mutants termed ''ProMonsters,'' each of which carries a GFP reporter in a deleted locus and one of the ''toolkit'' loci—either Green Monster GMToolkit-a or GMToolkit-α at the can1Δ locus (Figure 3). Using strains from the yeast deletion collection¹², GFP-marked deletions can be conveniently generated by replacing the common KanMX4 cassette existing in these strains with a universal GFP-URA3 fragment. Each GMToolkit contains: either the a- or α-mating-type-specific haploid selection marker¹ and exactly one of the two markers that, when both GMToolkits are present, collectively allow for selection of diploids.The second step is to carry out the sexual cycling through which deletion loci can be combined within a single cell by the random assortment and/or meiotic recombination that accompanies each cycle of mating and sporulation.