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.     

A Saccharomyces Cerevisiae Rad52 Allele Expressing a C-Terminal Truncation Protein: Activities and Intragenic Complementation of Missense Mutations

A nonsense allele of the yeast RAD52 gene, rad52-327, which expresses the N-terminal 65% of the protein was compared to two missense alleles, rad52-1 and rad52-2, and to a deletion allele. While the rad52-1 and the deletion mutants have severe defects in DNA repair, recombination and sporulation, the rad52-327 and rad52-2 mutants retain either partial or complete capabilities in repair and recombination. These two mutants behave similarly in most tests of repair and recombination during mitotic growth. One difference between these two alleles is that a homozygous rad52-2 diploid fails to sporulate, whereas the homozygous rad52-327 diploid sporulates weakly. The low level of sporulation by the rad52-327 diploid is accompanied by a low percentage of spore viability. Among these viable spores the frequency of crossing over for markers along chromosome VII is the same as that found in wild-type spores. rad52-327 complements rad52-2 for repair and sporulation. Weaker intragenic complementation occurs between rad52-327 and rad52-1.     

A mutation in SPC42, which encodes a component of the spindle pole body, results in production of two-spored asci in Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae, SPC42 is an essential gene, which encodes one of the major components of the spindle pole body (SPB). We report on a mutation in the SPC42 gene (spc42-102) that results in a sporulation-specific defect. Mitotic growth of haploid and diploid spc42-102 strains is normal and both exhibit the same growth rates as the isogenic wild-type strains. Many diploid spc42-102/spc42-102 cells undergo normal meiotic nuclear divisions, producing four haploid nuclei. However, a significant fraction of meiotic spc42-102/spc42-102 cells contain two immature SPBs and aberrant nuclei that are not surrounded by a prospore membrane. Some 40% of the resultant asci contain only two spores, while wild-type diploid cells almost always produce four-spored asci. Segregation of auxotrophic markers that are tightly linked to the centromere reveals that two-spore asci formed from spc42-102/spc42-102 diploid cells exclusively contain nonsister haploid spores. Western analysis and measurements of the fluorescent signal from an Spc42p-GFP (green fluorescent protein) fusion reveal that the mutant strain fails to accumulate Spc42p at meiosis. Thus, our results suggest that insufficiency of Spc42p during meiosis results in a pair of immature nonsister SPBs that are not enclosed by prospore membrane.     

Sporulation in the budding yeast Saccharomyces cerevisiae

In response to nitrogen starvation in the presence of a poor carbon source, diploid cells of the yeast Saccharomyces cerevisiae undergo meiosis and package the haploid nuclei produced in meiosis into spores. The formation of spores requires an unusual cell division event in which daughter cells are formed within the cytoplasm of the mother cell. This process involves the de novo generation of two different cellular structures: novel membrane compartments within the cell cytoplasm that give rise to the spore plasma membrane and an extensive spore wall that protects the spore from environmental insults. This article summarizes what is known about the molecular mechanisms controlling spore assembly with particular attention to how constitutive cellular functions are modified to create novel behaviors during this developmental process. Key regulatory points on the sporulation pathway are also discussed as well as the possible role of sporulation in the natural ecology of S. cerevisiae.     

Chromosomal loci of genes controlling site-specific restriction endonucleases of Bacillus subtilis

Summary In Saccharomyces cerevisiae, diploid strains which are respiratory deficient (e.g., rho^–) or are homozygous for the mating-type locus (i.e., either a/a or /) are unable to sporulate. In order to induce sporulation in these nonsporulating strains, the technique of protoplast fusion mediated by polyethylene glycol was adopted. In this study, the products of protoplast fusion were induced to sporulate without reversion to normal cells.Protoplasts from a respiratory-deficient diploid strain were mixed with those from a respiratory-competent haploid one carrying mitochondrial drug resistance markers, treated with 30% polyethylene glycol-4000 and 25 mM CaCl₂, and incubated in 0.1 M potassium acetate containing 0.8 M sorbitol as an osmotic stabilizer. After two days' incubation, asci with three to eight spores were formed at a frequency of 1×10^–3 to 2×10^–4. Sporulation was also observed in products of fusion between an a/a diploid and haploid strains and between an / diploid and a haploid strains. The analysis of the genotypes of spores revealed that when fusion products were cultured under conditions for sporulation, karyogamy did not take place, diploid nuclei underwent meiosis, and both diploid and haploid nuclei were able to develop into spores.     

Life Cycle of Culicosporella lunata (Hazard & Savage, 1970) Weiser, 1977 (Microspora) as Revealed in the Light Microscope with a Redescription of the Genus and Species1

Light microscopy studies of Culicosporella lunata (Hazard & Savage), a parasite of the mosquito Culex pilosus (Dyar & Knab), revealed two sporogonial sequences. One sequence begins with diplokaryotic meronts that undergo repeated nuclear divisions to produce sporogonial plasmodia with nuclei in diplokaryotic arrangement. These plasmodia form rosette-like clusters of sporoblasts during incomplete cytokinesis and, eventually, binucleate spores. These spores initiate infections in healthy larvae when they ingest spores. The second sequence begins with diplokaryotic meronts that undergo karyogamy and meiosis to form Thelohania-like sporonts and haploid spores. Anomalies are often observed in these sporonts which result in aberrant spores, usually fewer than eight, in an accessory (pansporoblastic) membrane. Normal haploid spores are morphologically similar to those of species of Amblyospora. The genus and the type species are redefined based on new information presented here and it and the type species are placed in the family Amblyosporidae.     

Fine structure of plasmodial nuclei in the slime moldPhysarum polycephalum I. Comparison of diploid and haploid vegetative mitosis

Summary The same basic ultrastructural features of interphase and mitotic nuclei were found for both the haploid Colonia and the diploid wild type strains of the myxomycete,Physarum polycephalum. Differences in nuclear size and chromocenter numbers were observed, but the nucleolar cycle and the intranuclear and acentriolar type of mitosis characteristic of the plasmodial stage of the diploid is present in haploid plasmodia, ruling out any relation between ploidy level and type of mitotic figure.     

NUCLEAR DNA CONTENT OF MYXAMOEBAE AND PLASMODIA IN SIX NON-HETEROTHALLIC ISOLATES OF A MYXOMYCETE,DIDYMIUM IRIDIS

The nuclear DNA content of six non-heterothallic isolates of the myxomycete Didymium iridis was measured by combining the Feulgen reaction with absorption microspectrophotometry. This allowed us to distinguish between homothallic (sexual) and apogamic (non-sexual) isolates. Four of the isolates studied, Panamanian 4 and 5, California 1, and Missouri 1 are homothallic. Moreover, the average DNA content of the myxamoebal and plasmodial nuclei (0.32 and 0.61 respectively) does not differ significantly from the calculated haploid and diploid values for heterothallic isolates of D. iridis (0.34 and 0.63). Hence, it is concluded that in each of these isolates the myxamoebae are haploid and the plasmodia diploid. In two of the isolates investigated, Georgia 1 and Hawaii 1, the DNA content of the myxamoebal and plasmodial nuclei did not differ significantly. Therefore, in both of these isolates the plasmodia appear to develop apogamically. In addition the mean DNA values recorded for the Ha-1 isolate suggest that it is aneuploid.     

Peptidyl-tRNA hydrolase in Bacillus subtilis,encoded by spoVC,is essential to vegetative growth,whereas the homologous enzyme in Saccharomyces cerevisiae is dispensable

Peptidyl-tRNA hydrolase in Escherichia coli, encoded by pth, is essential for recycling tRNA molecules sequestered as peptidyl-tRNA as a result of pre-mature dissociation from the ribosome during translation. Genes homologous to pth are present in other bacteria, yeast and man, but not in archaea. The homologous gene in Bacillus subtilis, spoVC, was first identified as a gene involved in sporulation. A second copy of spoVC, under the control of the xyl promoter, was integrated into B. subtilis at the amy locus. In this background, interruption of the original gene was possible provided that expression of the copy at the amy locus was induced. When spoVC was interrupted, both vegetative growth and sporulation were dependent on xylose, showing that SpoVC is essential. The role of SpoVC in sporulation is discussed and appears to be consistent with previous hypotheses that a relaxation of translational accuracy may occur during sporulation. The homologous gene in Saccharomyces cerevisiae, yHR189W, has been interrupted in both haploid and diploid strains. The mutant haploid strains remain viable, as do the yHR189W mutant spores obtained by tetrad dis-section, with either glucose or glycerol as carbon source, showing that the yHR189W gene product is dispensable for cell growth and for mitochondrial respiration.     

GROWTH AND DEVELOPMENT OF THE MYXOMYCETE PERICHAENA VERMICULARIS. II. CHROMOSOME NUMBERS AND NUCLEAR CYCLES

Chromosome counts of dividing nuclei of the myxomycete Perichaena vermicularis indicate a number of 25 ± 2 in the amoebae and 50 ± 4 in the Plasmodia, confirming earlier reports that amoebae are haploid and plasmodia diploid. Chromosome numbers obtained from nuclei during sporangial development indicate a fluctuation in the location of meiosis influenced by environmental conditions. The implications of these observations are discussed in reference to past conflicting evidence of the location of meiosis.     

Isolation and properties of merodiploid strains with F-factor including the pts-region of the Escherichia coli K-12 chromosome]

A technique of hybridization of haploid methanol-utilizing yeast Pichia pinus MH4 is worked out using UV- and N-nitrosoguanidine-induced auxotrophic mutants. Vegetative diploid cultures are isolated. Tetrad analysis and random spore analysis have revealed a meiotic nature of spores, recombination of genetic material in the process of sporulation and the chromosomal nature of some mutations. A possibility to construct a genetic map of the yeast Pichia pinus MH4 is demonstrated on the basis of tetrad analysis. Three linkage groups are revealed. The life cycle in a homothalic haploid yeast, Pichia pinus, was demonstrated. They are capable to form zygotes and meiotic spores under conditions preventing vegetative growth.     

淡黄绒泡菌和全白绒泡菌孢囊形成过程显微观察的染色差异

为了研究黏菌孢囊形成过程中显微结构的变化,文中探讨了番红-固绿和铁帆-苏木精染色条件下淡黄绒泡菌和全白绒泡菌孢囊不同发育阶段显微结构的差异显示效果。结果表明：在幼孢囊中原质团有种水平的割裂,大割裂和微割裂,这些割裂和孢丝及孢子的形成有关;全白绒泡菌囊轴表现了和孢囊柄不一致的状态和染色结果;番红-固绿染色下,淡黄绒泡菌在孢囊形成前期原质团被染成淡红色,可以分辨出大量游离存在的细胞核;孢囊壁及囊轴被染成绿色,孢子灰绿色;全白绒泡菌原质团被染成绿色,初期可见较厚孢囊壁,囊轴绿色。铁帆-苏木精染色下,淡黄绒泡菌和全白绒泡菌原质团均被染成灰色,囊壁不明显,成熟孢子发生皱缩。     

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.     

Twenty-Six Chromosomal Genes Needed to Maintain the Killer Double-Stranded RNA Plasmid of SACCHAROMYCES CEREVISIAE

The double-stranded RNA killer plasmid gives yeast strains carrying it both the ability to secret a protein toxin and immunity to that toxin. This report describes a new series of mutants in chromsomal genes needed for killer plasmid maintenance (mak genes). These mutants comprise 12 complementation groups. There are a total of at least 26 mak genes. Each mak gene product is needed for plasmid maintenance in diploids as well as in haploids. None of these mak mutations prevent the killer plasmid from entering the mak- spores in the process of meiotic sporulation. Complementation between mak mutants can be performed by mating meitoic spores from a makx/+ plasmid-carrying diploid with a maky haploid. If x = y, about half the diploid clones formed lose the killer plasmid. If x not equal to y, complementation occurs, and all of the diploid clones are killers.     

Inheritance of the 2μm DNA Plasmid from Saccharomyces

A variety of Saccharomyces strains were examined for the presence of 2micro DNA and, if present, for the pattern of fragments produced by its digestion with site-specific (restriction) endonucleases. Two strains were found that did not contain detectable levels of 2micro DNA, and two strains contained 2micro DNA molecules having only one EcoRI restriction endonuclease recognition site rather than the usual two.-A haploid containing 2micro DNA with one EcoRI restriction site was mated with a haploid containing 2micro DNA with two EcoRI restriction sites and the resulting diploid maintained both types during vegetative growth. Sporulation of the diploid produced four spores, and the clones from these spores contained both types.-A haploid lacking 2micro DNA was mated with a haploid containing 2micro DNA and the resulting diploid contained 2micro DNA. The four clones derived from the haploid spores after sporulation of this diploid all contained 2micro DNA. A rho(-) strain without 2micro DNA was mated to a rho(+) strain with 2micro DNA, and heteroplasmons were selected that had received the nucleus from the strain without 2micro DNA and the mitochondria from the strain with 2micro DNA. Twelve of twenty-four such clones contained 2micro DNA.-I conclude that: (1) the different types of 2micro DNA identified in these strains do not restrict one another, (2) the different types are inherited extrachromosomally, (3) lack of 2micro DNA in two strains is not due to the absence of genes needed for maintenance and (4) the approximately 100 copies of 2micro DNA contained within a single cell are probably clustered within one or a few cytoplasmic organelles.     

Spore number control and breeding in Saccharomyces cerevisiae: a key role for a self-organizing system

Spindle pole bodies (SPBs) provide a structural basis for genome inheritance and spore formation during meiosis in yeast. Upon carbon source limitation during sporulation, the number of haploid spores formed per cell is reduced. We show that precise spore number control (SNC) fulfills two functions. SNC maximizes the production of spores (1-4) that are formed by a single cell. This is regulated by the concentration of three structural meiotic SPB components, which is dependent on available amounts of carbon source. Using experiments and computer simulation, we show that the molecular mechanism relies on a self-organizing system, which is able to generate particular patterns (different numbers of spores) in dependency on one single stimulus (gradually increasing amounts of SPB constituents). We also show that SNC enhances intratetrad mating, whereby maximal amounts of germinated spores are able to return to a diploid lifestyle without intermediary mitotic division. This is beneficial for the immediate fitness of the population of postmeiotic cells.     

Alternative modes of organellar segregation during sporulation in Saccharomyces cerevisiae

Formation of ascospores in the yeast Saccharomyces cerevisiae is driven by an unusual cell division in which daughter nuclei are encapsulated within de novo-formed plasma membranes, termed prospore membranes. Generation of viable spores requires that cytoplasmic organelles also be captured along with nuclei. In mitotic cells segregation of mitochondria into the bud requires a polarized actin cytoskeleton. In contrast, genes involved in actin-mediated transport are not essential for sporulation. Instead, efficient segregation of mitochondria into spores requires Ady3p, a component of a protein coat found at the leading edge of the prospore membrane. Other organelles whose mitotic segregation is promoted by actin, such as the vacuole and the cortical endoplasmic reticulum, are not actively segregated during sporulation but are regenerated within spores. These results reveal that organellar segregation into spores is achieved by mechanisms distinct from those in mitotic cells.     

Corn Smut Dikaryon in Culture

A TYPICAL smut life cycle has three phases—diploid, haploid and dikaryon¹. Diploid spores (teliospores) formed in the host tissue are a resting phase. They undergo meiosis at germination to form haploid vegetative cells which are usually yeast-like. The dikaryon is the pathogenic phase and is made up of cells with two haploid nuclei. It is initiated by the fusion of two compatible non-pathogenic haploid cells and the formation of an infection hypha by the fusion product.     

Nuclear migration in Saccharomyces cerevisiae is controlled by the highly repetitive 313 kDa NUM1 protein.

We have isolated a novel gene (NUM1) with unusual internal periodicity. The NUM1 gene encodes a 313 kDa protein with a potential Ca2+ binding site and a central domain containing 12 almost identical tandem repeats of a 64 amino acid polypeptide. num1-disrupted strains grow normally, but contain many budded cells with two nuclei in the mother cell instead of a single nucleus at the bud neck, while all unbudded cells are uninucleate. This indicates that most G2 nuclei divide in the mother before migrating to the neck, followed by the migration of one of the two daughter nuclei into the bud. Furthermore, haploid num1 strains tend to diploidize during mitosis, and homozygous num1 diploid or tetraploid cells sporulate to form many budded asci with up to eight haploid or diploid spores, respectively, indicating that meiosis starts before nuclear redistribution and cytokinesis. Our data suggest that the NUM1 protein is involved in the interaction of the G2 nucleus with the bud neck.     

A novel role of Dma1 in regulating forespore membrane assembly and sporulation in fission yeast

In fission yeast Schizosaccharomyces pombe, a diploid mother cell differentiates into an ascus containing four haploid ascospores following meiotic nuclear divisions, through a process called sporulation. Several meiosis-specific proteins of fission yeast have been identified to play essential roles in meiotic progression and sporulation. We report here an unexpected function of mitotic spindle checkpoint protein Dma1 in proper spore formation. Consistent with its function in sporulation, expression of dma1(+) is up-regulated during meiosis I and II. We showed that Dma1 localizes to the SPB during meiosis and the maintenance of this localization at meiosis II depends on septation initiation network (SIN) scaffold proteins Sid4 and Cdc11. Cells lacking Dma1 display defects associated with sporulation but not nuclear division, leading frequently to formation of asci with fewer spores. Our genetic analyses support the notion that Dma1 functions in parallel with the meiosis-specific Sid2-related protein kinase Slk1/Mug27 and the SIN signaling during sporulation, possibly through regulating proper forespore membrane assembly. Our studies therefore revealed a novel function of Dma1 in regulating sporulation in fission yeast.