The genus Syringospora Quinquad emend

Further and more conclusive investigations on the life-cycle ofCandida albicans by means of DNA analyses and micromanipulation are reported.Six main stages are recognizable in the life-cycle of the species under consideration. The sexually active haplophase consists of small cells which either convert to the diplophase by somatogamous autogamy or mutate to the sexually quiescent haplophase with circa 8 × 10^–15 g DNA/cell. The nascent diplophase may develop further by budding, convert into chlamydospores or give rise to asexual endospores by internal budding. After a period of dormancy, the budding, uninucleate diplophase with circa 19 × 10^–15 g DNA/cell converts when transferred toFowell's acetate agar either into uninucleate chlamydospores or, after becoming multinucleate, into gonotoconts on which the haplophase is delimited externally as buds or as conidia delimited on short sterigmata. By the cultivation of single isolated conidia it was possible to confirm that they were haploid and that heterothallism was absent. The characteristic chalmydospores formed by cultures on corn meal agar were observed to germinate on non-nutritive, 2% washed agar media. Germination occurred by bud formation which gave rise to the haplophase without the formation of a septate promycelium. Since these chlamydospores appear to function as gonotoconts, they are considered to be homologous with the teliospores of theUstilaginales.This species, together with two others also described, are consequently assigned to the redefined genusSyringospora Quinquad which is provisionally assigned to the tulasnelloid fungi.     

A new aspect of the life cycle ofCandida tropicalis

The life cycle ofCandida tropicalis has been elucidated by nuclear staining. We were able to distinguish 5 stages: (1) the sexually active unstable haplophase which either can mutate directly to (2) the sexually inactive haplophase, or is converted to (3) the diplophase by “autoploidization”. The unstable diplophase changes into the sexually inactive haplophase, which is stable, by “budding meiosis’. Chlamydospores (4), which are possibly formed by sexually inactive haploid cells, are true resting spores, and during their germination only mitosis takes place. The buds formed during germination may develop into either the sexually active, or the inactive haplophase. During budding meiosis in the diplophase, multinucleate giant cells (5) may occur. The authors do not consider the latter to be metabasidia. Instead, they might in some way be homologus with asci, and their daughter nuclei might be comparable with the nuclei of ascospore initials. These new nuclei migrate successively into new buds which process results in a short chain of haploid cells. True ascospores have not been found. From these findings it is likely thatC. tropicalis is closely related to the ascomycetes rather than to the Hemibasidiomycetes.     

The perfect and imperfect states ofSporobolomyces salmonicolor

A strain ofSporobolomyces salmonicolor from which both the haplophase and diplophase had been recovered was found to form teliospores which germinated usually by the formation of a non-septate promycelium bearing 2–4 sporidia. The strain has consequently been transferred to the new basidiomycetous yeast genusAessosporon, for which a diagnosis and type are given, and which has been assigned to the Tilletiaceae.     

Saccharomyces Marxianus Hansen

III Conclusion and Summary Zygosaccharomyces Marxianus andSaccharomyces macedoniensis belong to the same species. This species is met with in the haplophase (Z. Marxianus) as well as in the diplophase (S. macedoniensis). It was possible to bring this yeast from the haplophase into the diplophase and vice versa. By keeping this yeast during long times on maltagar it showed a tendency to change from the haplophase into the diplophase, but not into the opposite direction.It seems quite possible thatHansen, who did not describe a conjugation in this yeast, had met with the diplophase.It has been once more emphasized — at whichWinge andLaustsen and alsoLindegren andLindegren have pointed —, that the genusZygosaccharomyces is no valid genus.The yeast studied here belongs to the genusSaccharomyces and must be designated with the original name given to it byHansen:Saccharomyces Marxianus.For the sake of completeness it is mentioned here that also an imperfect stage ofS. Marxianus has been describedviz., Candida macedoniensis (A. Castellani) Berkhout (I).Saccharomyces fragrans Beijerinck has to be considered as its synonym.     

``alternative Self-Diploidization'''' or ``asd'''' Homothallism in Saccharomyces Cerevisiae: Isolation of a Mutant, Nuclear-Cytoplasmic Interaction and Endomitotic Diploidization

A mutant of Saccharomyces cerevisiae representing a novel life cycle, named "alternative self-diploidization" or "ASD" homothallism, was obtained fortuitously. In this life cycle, MAT alpha (or MATa) haplophase and MAT alpha/MAT alpha (or MATa/MATa) diplophase alternate. Germinated cells are haploid and mating. They soon become nonmating and sporogenous as they vegetatively grow. They sooner or later diploidize presumably via endomitosis. The diploid cells haploidize via normal meiosis. A single recessive nuclear mutation, named asd 1-1, is responsible for "ASD" homothallism. In the rho 0 cytoplasm, asd 1-1 cells mate even if at a low efficiency and fail to diploidize. Since pet mutations do not have such effects, we conclude that a certain mitochondrial function other than respiration is required for manifestation of "ASD" homothallism. That is, "ASD" homothallism is the result of some sort of nuclear-cytoplasmic interaction.     

On the evolution of the karyorelict ciliate life cycle: heterophasic ciliates and the origin of ciliate binary fission

Karyorelict ciliates have near diploid somatic nuclei (macronuclei) incapable of division. If selective pressure favors nuclear division, how could such macronuclei have evolved? I propose that they initially evolved in the context of a diplophase stage that consisted entirely of a non-dividing trophont that was terminated by the induction of meiosis. The diploid macronucleus then differentiated, functioned and was destroyed in the absence of cell division. Such a life cycle would necessarily be heterophasic, i.e. with alternating haploid and diploid generations. I call these ancestors heterophasic ciliates. I further propose that the ability of this diploid trophont to undergo binary fission arose de novo. Ciliate binary fission would then be a derived characteristic, which possibly evolved indepedently in more than one heterophasic ciliate lineage. A progression of steps, leading to the reduction of the haplophase and the generation of the karyorelict life cycle, is proposed. The shared possession of nuclear dimorphism with non-dividing macronuclei, conjugation, and a putative heterophasic ancestry invites further investigation of the phylogenetic relationship between heterokaryotic foraminifera and karyorelict ciliates.     

Sex and the Spread of Retrotransposon Ty3 in Experimental Populations of Saccharomyces Cerevisiae

Mobile genetic elements may be molecular parasites that reduce the fitness of individuals that bear them by causing predominantly deleterious mutations, but increase in frequency when rare because transposition increases their rates of transmission to the progeny of crosses between infected and uninfected individuals. If this is true, then the initial spread of a mobile element requires sex. We tested this prediction using the yeast retrotransposon Ty3 and a strain of Saccharomyces cerevisiae lacking Ty3. We infected replicate isogenic sexual and asexual populations with a galactose-inducible Ty3 element at an initial frequency of 1%. In two of six asexual populations, active Ty3 elements increased in frequency to 38 and 86%, due to the spread in each population of a competitively superior mutant carrying a new Ty3 insertion. Ty3 frequencies increased above 80% in all sexual populations in which transposition was induced in haplophase or in diplophase. Ty3 did not increase in frequency when active during both haplophase and diplophase, apparently because of selective sweeps during adaptation to galactose. Repressed Ty3 elements spread in sexual populations, by increasing sexual fitness. These results indicate that active Ty3 elements are more likely to become established in sexual populations than in asexual populations.     

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.     

Conditions Favoring Differentiation and Stabilization of the Life Cycle of the Yeast <Emphasis Type="Italic">Pachysolen tannophilus</Emphasis>

Conditions favoring differentiation and stabilization of the life cycle of the yeast Pachysolen tannophilus have been studied. When concentrations of the carbon source in the medium were lower than 100 g/l, it was found to be favorable to the mating of vegetative cells, both haploid and diploid. The addition of nitrogen and sulfur sources to the medium influenced the life phases of haploid cells and partially stabilized the vegetative growth of diploid cells. Enrichment of the nutrient medium with potassium, vitamins, and microelements was shown to be necessary for the formation and maturation of conjugated ascospores. Microelements, vitamins, and phosphorus in excessive amounts activated conjugation but did not provide for the distinct phases of formation of unconjugated asci and spores in the diploid cells. Possible reasons for the unstable diplophase in the yeast P. tannophilus have been discussed.__________Translated from Mikrobiologiya, Vol. 74, No. 4, 2005, pp. 483–488.Original Russian Text Copyright © 2005 by Bolotnikova, Mikhailova, Shabalina, Bodunova, Ginak.     

Spectroscopic and biochemical analysis of regions of the cell wall of the unicellular 'mannan weed', Acetabularia acetabulum

Although the Dasycladalean alga Acetabularia acetabulum has long been known to contain mannan-rich walls, it is not known to what extent wall composition varies as a function of the elaborate cellular differentiation of this cell, nor has it been determined what other polysaccharides accompany the mannans. Cell walls were prepared from rhizoids, stalks, hairs, hair scars, apical septa, gametophores and gametangia, subjected to nuclear magnetic resonance and Fourier transform infrared spectroscopy, and analyzed for monosaccharide composition and linkage, although material limitations prevented some cell regions from being analyzed by some of the methods. In diplophase, walls contain a para-crystalline mannan, with other polysaccharides accounting for 10-20% of the wall mass; in haplophase, gametangia have a cellulosic wall, with mannans and other polymers representing about a quarter of the mass. In the walls of the diplophase, the mannan appears less crystalline than typical of cellulose. The walls of both diploid and haploid phases contain little if any xyloglucan or pectic polysaccharides, but appear to contain small amounts of a homorhamnan, galactomannans and glucogalactomannans, and branched xylans. These ancillary polysaccharides are approximately as abundant in the cellulose-rich gametangia as in the mannan-rich diplophase. In the diplophase, different regions of the cell differ modestly but reproducibly in the composition of the cell wall. These results suggest unique cell wall architecture for the mannan-rich cell walls of the Dasycladales.     

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.     

Commitment to meiosis in fission yeast

Summary Mutants of Schizosaccharomyces pombe blocked during meiosis were analysed with respect to the induction of diploid mitotic division. Wild type zygotes of this yeast can form diploid colonies with a low probability (ca. 1%) when they are transferred to fresh growth medium. Mutants of three genes affecting meiosis responded to the shift by forming diploid colonies with high yield (ca. 80% of the zygotes). Hence, commitment to meiosis could not be reached by such zygotes. Zygotes of a fourth meiosis-I-deficient mutant were unable to yield diploid colonies more effectively than wild type zygotes. Morphological changes were prominent in the mutant zygotes not reaching commitment (as well as in mutant cells that could not complete conjugation), indicating that activities needed during early stages of conjugation were not turned off in these zygotes.This work was supported by NIH grant GM 13234 initially, and by DFG (SFB 46).     

Isozyme Polymorphism in Natural Populations of a Liverwort, PLAGIOCHILA ASPLENIOIDES

Assays of five Polish populations of Plagiochila asplenioides (Hepaticae, Plagiochilaceae) for peroxidase isozymes revealed three bands differing in electrophoretic mobility. The phenotypes observed appear to be governed by three loci; each locus has two alleles, one of which produces a band and the other no band, i.e., the second allele is silent, or null. Marked variability in enzyme phenotypes was found both within and between populations. The evolutionary implications of this variability are discussed in terms of the persistent haplophase and ephemeral diplophase of this organism.     

Pcp1/pericentrin controls the SPB number in fission yeast meiosis and ploidy homeostasis

During sexual reproduction, the zygote must inherit exactly one centrosome (spindle pole body [SPB] in yeasts) from the gametes, which then duplicates and assembles a bipolar spindle that supports the subsequent cell division. Here, we show that in the fission yeast Schizosaccharomyces pombe, the fusion of SPBs from the gametes is blocked in polyploid zygotes. As a result, the polyploid zygotes cannot proliferate mitotically and frequently form supernumerary SPBs during subsequent meiosis, which leads to multipolar nuclear divisions and the generation of extra spores. The blockage of SPB fusion is caused by persistent SPB localization of Pcp1, which, in normal diploid zygotic meiosis, exhibits a dynamic association with the SPB. Artificially induced constitutive localization of Pcp1 on the SPB is sufficient to cause blockage of SPB fusion and formation of extra spores in diploids. Thus, Pcp1-dependent SPB quantity control is crucial for sexual reproduction and ploidy homeostasis in fission yeast.     

Ploidy dictates repair pathway choice under DNA replication stress

This study reports an unusual ploidy-specific response to replication stress presented by a defective minichromosome maintenance (MCM) helicase allele in yeast. The corresponding mouse allele, Mcm4(Chaos3), predisposes mice to mammary gland tumors. While mcm4(Chaos3) causes replication stress in both haploid and diploid yeast, only diploid mutants exhibit G2/M delay, severe genetic instability (GIN), and reduced viability. These different outcomes are associated with distinct repair pathways adopted in haploid and diploid mutants. Haploid mutants use the Rad6-dependent pathways that resume stalled forks, whereas the diploid mutants use the Rad52- and MRX-dependent pathways that repair double strand breaks. The repair pathway choice is irreversible and not regulated by the availability of repair enzymes. This ploidy effect is independent of mating type heterozygosity and not further enhanced by increasing ploidy. In summary, a defective MCM helicase causes GIN only in particular cell types. In response to replication stress, early events associated with ploidy dictate the repair pathway choice. This study uncovers a fundamental difference between haplophase and diplophase in the maintenance of genome integrity.     

GROWTH AND MATING OF GONZUM PECTORALE (VOLVOCALES) IN DEFINED MEDIA1

Growth in defined media of 32 populations of Gonium pectorale was studied to learn more about the sexual isolation reported for this species. The 23 populations containing both mating types (+ & -) were also studied for the ability to form zygotes in defined media. A preliminary study showed that some populations grew and reproduced sexually in a defined mineral medium, whereas others appeared to require exogenous organic materials for growth and/or zygote formation. The diverse reactions exhibited by the populations indicate physiological races which may explain, in part, the occurrence of sexual isolation.     

GROWTH AND MATING OF GONIUM PECTORALE (VOLVOCALES) IN DEFINED MEDIA1

Growth in defined media of 32 populations of Gonium pectorale was studied to learn more about the sexual isolation reported for this species. The 23 populations containing both mating types (+ & -) icere also studied for the ability to form zygotes in defined media. A preliminary study showed that some populations grew and reproduced sexually in a defined mineral medium, whereas others appeared to require exogenous organic materials for growth and/or zygote formation. The diverse reactions exhibited by the populations indicate physiological races which may explain, in part, the occurrence of sexual isolation.     

Two Multiallelic Mating Compatibility Loci Separately Regulate Zygote Formation and Zygote Differentiation in the Myxomycete PHYSARUM POLYCEPHALUM

The mating of Physarum polycephalum amoebae, the ultimate consequence of which is a "plasmodium," was recently shown to be governed by two compatibility loci, matA (or mt) and matB (Dee 1978; Youngmanet al. 1979). We present evidence that matA and matB separately regulate two discrete stages of mating: in the first stage, amoebae (which are normally haploid) fuse in pairs, with a specificity determined by matB genotype, to form diploid zygotes; subsequent differentiation of the zygotes into plasmodia is regulated by matA and is unaffected by matB. Mixtures of amoebae carrying unlike matA and matB alleles formed diploids to the extent of 10 to 15% of the cells present, and the diploids differentiated into plasmodia. When only the matB alleles differed, diploid cells still formed to a comparable (5 to 10%) extent, but rather than differentiating, these diploids remained amoebae. When strains carried the same alleles of matB, formation of diploid cells was greatly reduced: in like-matB, like-matA mixtures, none of 320 cells examined was diploid; in like-matB, unlike mat-A mixtures, differentiating diploids could be detected, but at only 10(-3) to 10(-2) the frequency of unlike-matB, unlike-matA mixtures. The nondifferentiating diploid amoebae recovered from unlike-matB, like-matA mixtures were genetically stable through extensive growth, even though they grew more slowly than haploids (10-hr vs. 8-hr doubling period), and could be crossed with both haploids and diploids. The results of such higher ploidy and mixed ploidy crosses indicate that karyogamy does not invariably accompany zygote formation and differentiation.     

Conditions favoring differentiation and stabilization of the life cycle of the yeast Pachysolen tannophilus

Conditions favoring differentiation and stabilization of the life cycle of the yeast Pachysolen tannophilus have been studied. When concentrations of the carbon source in the medium were lower than 100 g/l, it was found to be favorable to the mating of vegetative cells, both haploid and diploid. The addition of nitrogen and sulfur sources to the medium influenced the life phases of haploid cells and partially stabilized the vegetative growth of diploid cells. Enrichment of the nutrient medium with potassium, vitamins, and microelements was shown to be necessary for the formation and maturation of conjugated ascospores. Microelements, vitamins, and phosphorus in excessive amounts activated conjugation but did not provide for the distinct phases of formation of unconjugated asci and spores in the diploid cells. Possible reasons for the unstable diplophase in the yeast P. tannophilus are discussed.     

Ploidy differences in Cryptococcus albidus

Presumed haploid and diploid cultures ofCryptococcus albidus were analysed for their DNA content per cell. A ratio of approximately 1:2 was obtained by relating the DNA content per cell of the two phases to ploidy. As the diplophase formed neither longitudinally septated cells nor ballistoconidia, the earlier suggestions thatCryptococcus is closely related toTremella seems less likely. On the assumption that the metabasidia ofCryptococcus are gastromycetoid, a closer relationship between this genus and the tulasnelloid fungi appears more probable.Microbiology Research Group, South African Council for Scientific and Industrial Research, Pretoria, South Africa.