Nucleotide sequence analysis of a variable region of the large subunit rRNA for identification of marine-occurring yeasts

A 230-nucleotide region of the large subunit (LSU) ribosomal RNA was examined to determine whether signature nucleotide sequences could be used for species identifications of basidiomycetous yeasts. Multiple strains of genetically defined heterothallic species ofRhodosporidium, Leucosporidium, Cystofilobasidium, andSporidiobolus demonstrated that nucleotide sequences within these species are homologous and that differences between species range from 1 to 20 or more bases. Also included in this study were several homothallic species of these teleomorphic genera and some anamorphs assigned toRhodotorula andCandida. Those results indicated close relationships among certain homothallic species, particularly in the genusMrakia, and potential relationships of homothallic and anamorphic strains to several teleomorphs. The data suggest that LSU sequences can be used for yeast identifications with the possible exception of closely related homothallic species.     

DNA quantification in nuclei of cultivated mushroom with DAPI staining

Agaricus bisporus (Lange) Imbach is actively cultivated amphithallic basidiomycete, in which various strains are primary homothallic, heterothallic or secondary homothallic. Countings of relative nuclear DNA content by means of DAPI stain and its comparison in different strains can help to understand the mushroom's life cycle features. The authors for the first time observed change of nuclear phases in basidia of A. bisporus strains with different types of life cycle and revealed that DNA content in diploid nuclei is about 1.3 times higher than in haploid ones. The method is highly sensitive and can be used for quantitative measurings of nuclear DNA even in objects with nuclei of about 1 mkm in diameter.     

Zygospore formation between homothallic and heterothallic strains of Closterium

Zygospore formation in different strains of the Closterium peracerosum-strigosum-littorale complex was examined in this unicellular isogamous charophycean alga to shed light on gametic mating strains in this taxon, which is believed to share a close phylogenetic relationship with land plants. Zygospores typically form as a result of conjugation between mating-type plus (mt⁺) and mating-type minus (mt⁻) cells during sexual reproduction in the heterothallic strain, similar to Chlamydomonas. However, within clonal cells, zygospores are formed within homothallic strains, and the majority of these zygospores originate as a result of conjugation of two recently divided sister gametangial cells derived from one vegetative cell. In this study, we analyzed conjugation of homothallic cells in the presence of phylogenetically closely related heterothallic cells to characterize the reproductive function of homothallic sister gametangial cells. The relative ratio of non-sister zygospores to sister zygospores increased in the presence of heterothallic mt⁺ cells, compared with that in the homothallic strain alone and in a coculture with mt⁻ cells. Heterothallic cells were surface labeled with calcofluor white, permitting fusions with homothallic cells to be identified and confirming the formation of hybrid zygospores between the homothallic cells and heterothallic mt⁺ cells. These results show that at least some of the homothallic gametangial cells possess heterothallic mt⁻-like characters. This finding supports speculation that division of one vegetative cell into two sister gametangial cells is a segregative process capable of producing complementary mating types.     

Sex determination in the first-described sexual fungus

The original report of sex in fungi dates 2 centuries ago to the species Syzygites megalocarpus (Mucoromycotina). The organism was subsequently used in 1904 to represent self-fertile homothallic species when the concepts of heterothallism and homothallism were developed for the fungal kingdom. In this study, two putative sex/MAT loci were identified in individual strains of S. megalocarpus, accounting for its homothallic behavior. The strains encode both of the high-mobility-group domain-containing proteins, SexM and SexP, flanked by RNA helicase and glutathione oxidoreductase genes that are found adjacent to the mating-type loci in other Mucoromycotina species. The presence of pseudogenes and the arrangement of genes suggest that the origin of homothallism in this species is from a heterothallic relative, obtained via a chromosomal rearrangement to switch two alleles into two separated loci within a single genetic background. Similar events have given rise to homothallic species from heterothallic species in ascomycete fungi, demonstrating that conserved forces shape the evolution of sex determination and speciation in highly diverged fungi.     

A new disruption vector (pDHO) to obtain heterothallic strains from both Saccharomyces cerevisiae and Saccharomyces pastorianus

Yeasts are responsible for several traits in fermented beverages, including wine and beer, and their genetic manipulation is often necessary to improve the quality of the fermentation product. Improvement of wild-type strains of Saccharomyces cerevisiae and Saccharomyces pastorianus is difficult due to their homothallic character and variable ploidy level. Homothallism is determined by the HO gene in S. cerevisiae and the Sc-HO gene in S. pastorianus. In this work, we describe the construction of an HO disruption vector (pDHO) containing an HO disruption cassette and discuss its use in generating heterothallic yeast strains from homothallic Saccharomyces species.     

Duplicated Genes Producing Transposable Controlling Elements for the Mating-Type Differentiation in SACCHAROMYCES CEREVISIAE

Mutation of the two homothallic genes, HML alpha/HMLa and HMRa/HMR alpha, in homothallic strains of Saccharomyces cerevisiae was studied. Of 11 mutants of the HML alpha gene, eight were due to a phenotypic mutation from HML alpha to HMLa, i.e., a mutation causing a change in function of the original HML allele to that of the other HML allele (functional mutation), and three were due to a defective mutation at the HML alpha gene, i.e., a mutation causing a nonfunctional allele (nonfunctional mutation). All 14 mutants of the HMRa gene, on the other hand, were due to a phenotypic mutation from HMRa to HMR alpha i.e., a functional mutation. Phenotypic reverse mutations, i.e., HMLa to HML alpha and HMR alpha to HMRa, were also observed in the cultivation of EMS (ethyl methanesulfonate) treated spores having the HO HMR alpha HMLa genotype. Mutation from heterothallic cells to homothallism was observed in a nonfunctional mutant of the HML alpha gene, by mutagenesis with EMS, but not in the functional mutants of the HML alpha and HMRa genes or in the authentic strains having the alpha HO HMR alpha HML alpha (alpha Hp) and a HO HMRa HMLa (a Hq) genotypes. These observations suggest that the functional mutation is not caused by the direct mutation from a homothallic allele to the opposite, but by replacement of a transposable genic element produced from a homothallic locus with a region of a different homothallic locus. These observations also support the controlling-element model and the cassette model, which have been proposed to explain the mating-type differentiation by the homothallic genes.     

双孢蘑菇及大肥菇的种内及种间多态性RAPD分析*

应用RAPD方法分析双孢蘑菇（Agaricusbisporus）及大肥菇（Agaricusbitorquis）的种内及种间多态性,进一步证实由于双孢蘑菇的遗传保守性,多数孢子为自体可育的异核孢子,从而导致种内相似性很高;而依赖天然杂交具四孢特性的大肥菇种内相似程度较低。研究还分析评价了蘑菇属中的这两个不同种之间的亲缘关系,为种间杂交选材提供了理论依据。     

Mating-Type Mutations in SCHIZOSACCHAROMYCES POMBE: Isolation of Mutants and Analysis of Strains with an h or h Phenotype

Mutants defective in various steps of the sexual cycle have been isolated from homothallic strains of Schizosaccharomyces pombe by Bresch, Müller and Egel (1968). These mutants include heterothallic h(+) and h(-) strains. We have isolated additional h(+) and h(- ) mutants from homothallic strains. Those mutants which are due to mutations in the mating-type region were analyzed in detail. Our results show that the mating-type gene mat2 not only has a function in copulation and meiosis, but that it also regulates the formation of the map1 gene product (map1 is a mating-type auxiliary gene). Some of the h( -) mutants have lost only one of the three functions while others are defective in at least two, and perhaps all three, functions. Further, we show that the mat1(-) allele of h(90) strains can mutate to mat1(+) but that mutations in mat2 appear to affect the mutational behavior of mat1. Finally, we describe a new inactive mating-type allele, mat2*, which is different from mat2(0) in that it can mutate to mat2(+).     

双孢蘑菇及大肥菇的种内及种间多态性RAPD分析

应用RAPD方法分析双孢蘑菇（Agaricusbisporus）及大肥菇（Agaricusbitorquis）的种内及种间多态性,进一步证实由于双孢蘑菇的遗传保守性,多数孢子为自体可育的异核孢子,从而导致种内相似性很高;而依赖天然杂交具四孢特性的大肥菇种内相似程度较低。研究还分析评价了蘑菇属中的这两个不同种之间的亲缘关系,为种间杂交选材提供了理论依据。     

DNA homologies among homothallic, pseudo-homothallic and heterothallic species of Neurospora.

Summary Highly purified DNAs from three homothallic speciesNeurospora africana, N. dodgei andN. lineolata; three reference strains representing authentic heterothallic species,N. crassa, N. intermedia andN. sitophila; and two strains of pseudo-homothallic speciesN. tetrasperma were characterized by spectrophotometry and DNA reassociation using hydroxyapatite chromatography. All of these known species are closely related on the basis of DNA characteristics such as base composition and thermal denaturation profiles of major DNA components. Minor components of ascospore DNA was, however, only 5–7% of total DNA instead of 15–20% minor component DNA shown by mycelial DNA. Species belonging to same group were not distinguishable morphologically, but all of these species were distinguishable by DNA:DNA homology studies. Greater DNA homology was noticed between DNAs of heterothallic species and DNAs of pseudohomothallic species than DNA of true homothallic species. Difference on DNA-nucleiotide sequences among homothallic species was very little. Pseudo-homothallic speciesN. tetrasperma was found to be distinctly different from homothallic species but closer to heterothallic species based on such studies.Supported in part by a contract No. E(40-1)4182 with the U.S. Energy Research and Development Administration. We are grateful to Departments of Oncology and Radiotherapy, College of Medicine, Howard University, Washington, D.C. for providing us with material assistance     

Genetic aspects of ethanol tolerance and production bySaccharomyces cerevisiae

We developed a method of hybrid selection between homothallic wild-type and heterothallic strains. The hybrids obtained were used to study the heredity of ethanol tolerance and production. Both characters segregated independently, but no ethanol-sensitive strains were able to produce high levels of ethanol. At least four genes are implicated in ethanol tolerance.     

Karyotype polymorphism correlates with intraspecific infertility in the homothallic ascomycete Sordaria macrospora

The homothallic fungus Sordaria macrospora produces perithecia with meiotically derived ascospores. In most cases, intraspecies crosses between strains from different culture collections generate fertile hybrid perithecia in the contact zone of two mycelia. However, in some of these crosses we observed a significant decrease in the fertility of the hybrid perithecia when strains of different origin were used for mating. Since we assumed that chromosome variability between the culture collection strains might contribute to this reduction in fertility, we performed pulsed‐field gel electrophoresis. In the course of our study, we were able to identify two major groups of electrophoretic karyotypes in S. macrospora culture collection strains. A quantitative analysis revealed that polymorphic karyotypes contribute to a reduction of fertility in forced crosses between strains carrying differently sized chromosomes. The observed intraspecific chromosome length polymorphism might have consequences on the speciation process of a homothallic fungus capable of sexual but not of asexual spore formation.     

Apparent bisexual behavior of yeast strains obtained from hybridization of industrial yeasts of the genusSaccharomyces with auxotrophic diploids

During a genetic study of some hybrids of brewer's and distiller's yeast strains with impaired sporulation characteristics and genetically marked auxotrophic aa and alpha alpha diploids, strains which showed positive mating reactions with both a and alpha haploid tester strains were observed. These strains proved to be homothallic and sporulated freely. The original hybrids, which appeared to be tetraploid, usually yielded sporulating single-spore clones on dissection of asci formed from them, with few or no mating strains among them. Dissection of asci from these clones yielded some single-spore clones which showed mating reactions with one or the other or both haploid tester strains, and further selection produced strains which on sporulation and dissection yielded single-spore clones which were apparently bisexual and sporulated freely. These strains proved to be homothallic, yielding single-spore clones which were all of the a mating type, and in which the mating reaction and, possibly, the action of the genes for homothallism were impaired, so that sporulating, non-mating diploids and haploids of both mating types were present in cultures originally obtained as single-spore clones.     

Induction of trehalase activity on a nitrogen-free medium: a sporulation-specific event in the fission yeast, Schizosaccharomyces pombe

Summary Kinetic experiments with synchronously sporulating cultures of a homothallic h⁹⁰ strain of Schizosaccharomyces pombe showed that trehalase activity abruptly increased in the late sporulation process, coinciding with the appearance of visible spores. Trehalase activity was absent in vegetative cells. A set of strains different in genetic constitution at the mating type loci was tested for induction of trehalase on nitrogen-free sporulation medium. The appearance of trehalase activity on the sporulation medium was observed only in sporulating cultures; cultures of homothallic strains (h⁹⁰) and diploid strains heterozygous for mating type (h⁺/h^–), and mixed cultures of heterothallic h⁺ and h^– strains. Trehalase activity was not induced in nonsporogenic strains: heterothallic haploid strains (h⁺ and h^–), diploid strains homozygous for mating type (h⁺/h⁺ and h^–/h^–) and the homothallic strain harboring the mutation in the mat2 gene, which was unable to undergo the first meiotic division. Trehalose accumulation on the sporulation medium was observed solely in the sporulating cultures. These results led us to conclude that the induction of trehalase activity as well as the accumulation of trehalose in the medium lacking nitrogen sources was a sporulation-specific event under the control of the mating type genes.     

Interconversion of Yeast Mating Types II. Restoration of Mating Ability to Sterile Mutants in Homothallic and Heterothallic Strains

The two mating types of the yeast Saccharomyces cerevisiae can be interconverted in both homothallic and heterothallic strains. Previous work indicates that all yeast cells contain the information to be both a and α and that the HO gene (in homothallic strains) promotes a change in mating type by causing a change at the mating type locus itself. In both heterothallic and homothallic strains, a defective α mating type locus can be converted to a functional a locus and subsequently to a functional α locus. In contrast, action of the HO gene does not restore mating ability to a strain defective in another gene for mating which is not at the mating type locus. These observations indicate that a yeast cell contains an additional copy (or copies) of α information, and lead to the "cassette" model for mating type interconversion. In this model, HMa and hmα loci are blocs of unexpressed α regulatory information, and HMα and hma loci are blocs of unexpressed a regulatory information. These blocs are silent because they lack an essential site for expression, and become active upon insertion of this information (or a copy of the information) into the mating type locus by action of the HO gene.     

Cell Fusion Promoting Factor Common to Homothallic and Heterothallic Mating Systems in Dictyostelium discoideum1

Cellular slime mould Dictyostelium discoideum propagates as single haploid cells and under certain environmental conditions enters into a sexual cycle called macrocyst formation. There are homothallic and heterothallic strains reported, the former being able to form macrocysts in clonal cell populations while the latter to do so only in the presence of opposite mating-type strains. Molecular basis for differential mating systems is an intersting subject totally unknown yet. In the present study, sexual cell interactions in AC4, a homothallic strain of D. discoideum, was studied in comparison with the heterothallic mating system. The conditoned medium of AC4 cells was found to promote the sexual cell fusion among themselves. In addition, it also enhanced the cell fusion between heterothallic strains. Furthermore, the conditioned medium obtained from the mated culture of heterothallic strains reported to induce the sexual cell fusion in the heterothallic strains (Saga and Yanagisawa, 1983) was found also to promote the cell fusion in AC4. These results suggest that common regulatory mechanisms operate for sexual cell fusion among different mating systems in D. discoideum.     

Induction of Heterothallic and Homothallic Zygotes in Dictyostelium discoideum by Ethylene

Dictyostelium mucoroides -7 (Dm7) and a mutant (MF1) derived from it exhibit homothallic macrocyst formation in the sexual process. As previously shown, the zygote formation during macrocyst formation is induced by a potent plant hormone, ethylene. The present work was undertaken to know if ethylene is also involved in heterothallic and homothallic macrocyst formation in D. discoideum. In heterothallic macrocyst formation between NC4 and V12M2 cells, ethionine, an analogue of methionine, inhibits macrocyst formation through arresting specifically the acquisition process of fusion competence. Such an inhibitory effect of ethionine was almost completely cancelled by co-application of ACC (1-aminocyclopropane-1-carboxylic acid), the immediate precursor of ethylene. Essentially the same effects of ethionine and ACC were also noticed on homothallic macrocyst formation in D. discoideum AC4. Thus it seems most likely that ethylene is required for the acquisition of fusion competence during macrocyst formation, and that in a variety of strains examined there is a common mechanism regulated by ethylene, beyond the difference of sexual modes.     

Evidence of Preferential Pairing of Chromosomes at Meiosis in Aneuploid Yeast

Meiotic pairing in homothallic S. cerevisiae was studied by tetrad analysis, using strains that were trisomic or tetrasomic for chromosome I. The disomic segregants of these strains produce tetrasomic spore colonies that can be distinguished by their phenotype. Results indicated the existence of preferential pairing and nonrandom assortment of chromosomes at meiosis I. The frequency of crossing over is apparently normal in at least some regions when non-preferred pairing occurs.     

Peculiarities of flor strains adapted to Sardinian sherry-like wine ageing conditions

Saccharomyces cerevisiae flor yeasts, which are subjected to stressful conditions during wine ageing, exhibit a number of characteristics which distinguish them from non-flor S. cerevisiae wine strains. In the present work, 22 flor and 14 non-flor S. cerevisiae wine strains are compared, in order to elucidate other possible peculiarities of these yeasts. The results obtained demonstrate that in contrast to the homothallic nature of the non-flor strains, 77% of the flor strains exhibit two variants of a semi-homothallic life cycle. Moreover, the flor-forming ability is shown to be inversely correlated to spore viability and the utilisation of maltose and galactose.