The relationship of flagellar length to sexual signalling in Chlamydomonas

The flagella of Chlamydomonas reinhardi are required for the initiation of mating between opposite mating type gametes. It has been suggested that flagellar length is a crucial factor in a cell's ability to transmit and receive the sexual signals necessary for fusion. Mating type + (mt⁺) cells of gam-5, a mutant which is characterized by variable length, paralyzed flagella, were mated with wild-type, mt⁻ cells. Activation of the mating structures of the gam-5 gametes, and therefore successful signalling, was demonstrated for cells with flagella as short as 1.5 μm (less than 1/6 normal length). Because this mutant displays aberrant axonemal structures, and because various mutants with other defects in axonemal structure are also able to mate, it seems likely that the flagellar membrane may provide the main conduit for gametic sexual signals.     

–SH and S–S involvement in Chlamydomonas mating

Reagents that block or cross-link sulfhydryl (–SH) groups and those that reduce disulfide (S–S) bonds have been tested for their effects on mating in Chlamydomonas reinhardii. Wild-type (wt) gametes of mating type + (mt⁺) and mt^?, and a fusion-defective mt^? mutant, gam-11, were studied. Differential sensitivities of mt⁺ vs mt^? and of wt mt^? vs gam-11 mt^? were analyzed. Concentrations of reagents that did not disrupt flagellar agglutination, the first stage of the mating reaction, were generally used. Pretreatment of mt⁺ gametes with the membrane permeable –SH reducing agent dithiothreitol (DTT) inhibits flagellar sexual signaling at concentrations that do not inhibit any part of the mating reaction of mt^? gametes. Wt mt^? is more sensitive than wt mt⁺ to inhibition by low concentrations of p-chloromercuribenzoate sulfonate (pCMBS), an organic mercurial. The membrane-impermeable reducing agent, reduced glutathione (GSH), also preferentially inhibits wt mt^?. Gam-11 mt^?, a fusion-defective mutant, which has been used to study the sensitivity of the adhesion of the plasma membrane-associated mating structures, is less sensitive to GSH and pCMBS inhibition that is wt mt^?. DDT and pCMBS cause an increase in mating structure adhesion in pretreated gam-11. The differential inhibition of pair and group formation during gam-11 × wt mt⁺ matings has suggested a possible mechanism for mating structure adhesion.     

Replication of bacteriophage lambda DNA dependent on the function of host and viral genes. I. Interaction of red, gam and rec

We have studied the role of the red and gam genes in lambda replication, after infection of wild type and two recombination deficient hosts. Our results show that the rate of phage DNA replication is abnormally low in the absence of red function, in rec⁺ as well as rec^? (A^? and A^?B^?) bacteria. It appears that the virus general recombination proteins play some role in lambda replication that cannot be assumed by the general recombination proteins of its bacterial host. The red^? defect in replication results in a decrease in the total amount of intracellular phage DNA. This DNA, nevertheless, seems normal in structure and is matured and packaged with good efficiency.In rec⁺ and recA^? hosts infected with gam^? mutants, the rate of lambda replication is also low, but in this case, abnormal DNA structures are produced at late times. The gam mutation seems to alter the program of replication such that circular molecules are produced not only at early times, but continuously, throughout the lytic cycle. This, and other facts, suggest that the gam protein is required for the transition from “early” to “late” replication. This requirement for gam function is not observed in recA^?B^? hosts, in which gam mutants replicate at a normal rate and produce DNA indistinguishable from that made by wild type phage. Thus, the gam requirement seems to involve an interaction of this phage protein with the product of the host's recB gene. Other evidence for such interaction comes from our finding that, in vivo, the gam protein does inhibit presumed action of the host's BC nuclease.In the gam^? mutant infections, which are blocked in late replication, absence of a general recombination system seems to create a severe defect in maturation of intracellular phage DNA. This defect, unlike the one affecting λ replication rate, can be alleviated by either the red or rec functions and is correlated with the inability of the mutant phages to make DNA concatemers. Since other late functions (i.e. late messenger RNA production) appear to be normal, we conclude that concatemer formation, via replication or recombination, is an essential step in phage development.     

Mutants of the fission yeast Schizosaccharomyces pombe which alter the shift between cell proliferation and sporulation

Summary Mutants of S. pombe have been isolated which undergo conjugation and sporulation in rich medium, conditions which are normally inhibitory for these processes. Two of these mutants are also able to sporulate from the haploid state in the absence of heterozygosity at the mating type locus. These recessive mutants define a single nuclear gene called ran1 which is unlinked to mating type. It is proposed that the ran1 gene codes for an inhibitor in the control of the initiation of conjugation and sporulation. In wild type cells the inhibitory effect is released by nutritional starvation and heterozygosity at the mating type locus. This allows the cells to proceed to sporulation. The ran1 mutants are unusual in that they attempt to undergo a reductional meiotic division from the haploid state. They are also genetically unstable and generate extragenic suppressors at high frequency.     

Mutants inducible for sexual agglutinability in Saccharomyces cerevisiae

Summary We have isolated the mutants, T55s-41(a) and T562s-161 () which have no sexual agglutinability when cultured at 28°C, but become sexually agglutinable by the action of the sex pheromone produced by respective opposite mating type. The sex-specific glycoproteins responsible for sexual agglutination were detected in the mutants treated with the opposite mating type pheromone, but not in those treated with the same mating type pheromone.The induction of sexual agglutinability by the pheromone required both nitrogen and carbon sources and was inhibited by cycloheximide. The induction by the pheromone of sexual agglutinability was much more sensitive to osmotic shock and Triton X-100 in T55s-41 than in H1-0, an inducible a strain found in our stock cultures. When cultured at 22°C both T55s-41 and T562s-161 produced respective agglutination substances without the sex pheromones.H1-0 carried more than one genes responsible for the inducibility (inducible genes). The inducible genes carried by T55s-41 and T562s-161 were recessive, possibly linked to none of the mating type locus, thr4 and his 4, and shown to be identical. The inducible gene in H22, an inducible a strain found in our stock cultures and at least one of the inducible genes in H1-0 were linked to the mating type locus. All the inducible genes observed so far were not specific to the mating type in their action.     

Complementation betweenPhycomyces mutants of mating type (+) with abnormal phototropism

Summary Twelve mutants ofPhycomyces blakesleeanus with defects in sporangiophore phototropism (genotypemad) were obtained from a wild type of the (+) mating type by mutagenesis with nitrosoguanidine. These mutants were tested for genetic complementation against standard (+)mad mutants derived from sexual crosses between the isogenic (+) strain and established (-)mad mutants (Ootaki et al., 1974; Eslava et al., 1976). Heterokaryons for complementation tests were obtained by grafting stage I sporangiophores. The (+) mutants were also investigated for their sensory responses such as photoinduction of sporangiophores and avoidance. The mutants were grouped into two classes, based on the phenotypic classification scheme of Bergman et al. (1973). There were eleven class 1.2 mutants and one class 2 mutant. Complementation tests revealed that all eleven class 1.2 mutants carry the genemadC and the class 2 mutant carriesmadD. There was no evidence that any were double mutants. These results are consistent with the phenotypic classification and with the complementation results of themad mutants of the (-) mating type.     

Isolation and characterization of spontaneous srl-recA deletion mutants in Escherichia coli K-12

Summary A method was developed for the isolation of spontaneous mutants of Escherichia coli K-12 with deletions extending from the srl operon to the adjacent recA gene. The srl-recA deletion mutants were extremely sensitive to DNA-damaging agents; unable to support growth of the feckless red gam mutant bio11; and recombination-deficient in transduction and in conjugation. They therefore resembled recA point mutants such as recA13. The existence of these recA deletion mutants shows that the recA gene is not essential for viability.     

New tools for mitochondrial genetics of Chlamydomonas reinhardtii: manganese mutagenesis and cytoduction

Summary A novel and efficient genetic procedure is described for generating mitochondrial mutants of the green alga Chlamydomonas reinhardtii. The development of a mutagenesis procedure using manganese cations and the application of cytoduction techniques resulted in a combined approach for the generation and analysis of mitochondrial mutants. Although mitochondrial mutations are inherited in sexual crosses from the minus mating type parent, the cytoduction technique can be used to transfer mitochondrial mutations into recipient strains with different genetic backgrounds, irrespective of their mating type. Cytoduction allows the transfer of mitochondrial markers from diploid to haploid cells also, which is of great benefit since diploid cells do not germinate in C. reinhardtii. We report here the isolation and characterisation of eight mutants, which are resistant to the antibiotics myxothiazol and mucidin. The mutants all have point mutations in the mitochondrial gene for apocytochrome b. Using in vitro-amplified cytb gene fragments as probes for direct DNA sequencing, three different types of single base pair substitutions were revealed in all mutants tested. In particular, amino acid substitutions in the mutant apocytochrome b polypeptide have been identified at residues 129, 132 and 137, which have been implicated in forming part of an antibiotic-binding niche. The amino acid substitution at position 132 has not been so far described for mutant apocytochrome b in any other organism, prokaryotic or eukaryotic. The genetic approach presented here confirms C. reinhardtii as a model system that is unique among plant cells.     

Mating type‐dependent partner sensing as mediated by VEL1 in Trichoderma reesei

Sexual development in the filamentous model ascomycete Trichoderma reesei (syn. Hypocrea jecorina) was described only a few years ago. In this study, we show a novel role for VELVET in fungi, which links light response, development and secondary metabolism. Vel1 is required for mating in darkness, normal growth and conidiation. In light, vel1 was dispensable for male fertility but essential for female fertility in both mating types. VEL1 impacted regulation of the pheromone system (hpr1, hpr2, hpp1, ppg1) in a mating type‐dependent manner and depending on the mating partner of a given strain. These partner effects only occurred for hpp1 and hpr2, the pheromone precursor and receptor genes associated with the MAT1‐2 mating type and for the mating type gene mat1‐2‐1. Analysis of secondary metabolite patterns secreted by wild type and mutants under asexual and sexual conditions revealed that even in the wild type, the patterns change upon encounter of a mating partner, with again distinct differences for wild type and vel1 mutants. Hence, T. reesei applies a language of pheromones and secondary metabolites to communicate with mating partners and that this communication is at least in part mediated by VEL1.     

Deletion of the mating-type sequences in Podospora anserina abolishes mating without affecting vegetative functions and sexual differentiation

The mating-type locus of Podospora anserina controls fusion of sexual cells as well as subsequent stages of development of the fruiting bodies. The two alleles at the locus are defined by specific DNA regions comprising 3.8 kb for mat+ and 4.7 kb for mat–, which have identical flanking sequences. Here we present the characterization of several mutants that have lost mat+-specific sequences. One mutant was obtained fortuitously and the other two were constructed by gene replacement. The mutants are deficient in mating with strains of either mat genotype but are still able to differentiate sexual reproductive structures. The loss of the mating type does not lead to any discernible phenotype during vegetative growth: in particular it does not change the life span of the strain. The mutants can recover mating ability if they are transformed with DNA containing the complete mat+ or mat– information. The transformants behave in crosses as do the reference mat+ or mat– strains, thus indicating that the transgenic mat+ and mat– are fully functional even when they have integrated at ectopic sites.     

Mutation in the <Emphasis Type="Italic">cspH-cspG</Emphasis> gene cluster enhances expression of heat-shock proteins and SOS repair system of <Emphasis Type="Italic">Escherichia coli</Emphasis>

The recessive radioresistance allele gam12 cloned in plasmid pBC4042-gam12 slightly increases the radiation resistance of Escherichia coli wild-type cells. Meanwhile, irradiation by γ-rays induces transition of gam ^r 12 mutation to the homozygous state and causes a 3.37-fold increase in radiation resistance of these cells. The mutation gam ^r 12 was located at 22.68 min of the chromosomal map in the region of cspH-cspG gene cluster of cold-shock proteins. Sequence analysis of gam12 allele revealed the nucleotide sequence of cold-shock gene cspG and insertions in the C-terminal part of the gene. Translation of mutant cspG gene can lead to synthesis of a truncated product that represents the N-terminal protein fragment with motifs governing binding with DNA and RNA. Analysis of the Escherichia coli genome revealed motifs recognized by proteins of the cspA family in genes of cold shock, heat shock, SOS regulon, and other systems. These data suggest the possibility of involvement of mutant RNA-chaperones of type CspA′ and CspG′ in the expression of key genes in systems of SOS repair and recombination or auxiliary stress systems, including heat-shock proteins, in radiation resistant mutants of E. coli.     

A Schizosaccharomyces pombe gene, ksg1, that shows structural homology to the human phosphoinositide-dependent protein kinase PDK1, is essential for growth, mating and sporulation

Fission yeast (Schizosaccharomyces pombe) requires inositol for growth, mating and sporulation. To define putative genes that are involved in the processing and transduction of the inositol signal, mutants that are temperature sensitive for growth and sporulation were selected on a medium containing non-limiting amounts of inositol. Two such mutants (ksg1-208 and ksg1-358) were analyzed, which are impaired in mating and sporulation at 30° C and undergo growth arrest in the G2 phase of the cell cycle at 35° C. The ksg1 gene was isolated by functional complementation. It maps on the left arm of chromosome II and encodes a putative 592-amino acid protein which exhibits good structural homology to a human 3-phosphoinositide-dependent protein kinase (PDK1) and its rat and Drosophila homologues. The two mutants have the same substitution at amino acid position 159: a glycine residue is replaced by glutamic acid. Deletion of the gene is lethal for haploid cells. We propose that ksg1 is involved in one or several phosphoinositide signalling processes that are responsible for control of the life cycle. Received: 24 September 1998 / Accepted: 8 November 1998     

RARE MALE MATING ADVANTAGE: A REVIEW

1. The phenomenon of frequency-dependent selection with an advantage for the rare type over the common type is intriguing because it implies balancing selection. Thus the high level of genetic variability as found in natural populations can be explained without the necessity of considerable genetic load. Rare male mating advantage is here defined as frequency-dependent male sexual fitness with the rare type of male favoured. Such a rare male effect has been found to be very widespread in insects, at least under laboratory conditions, but there are several problems associated with this phenomenon which will be discussed in this review. 2. To determine whether male mating success is frequency-dependent, the quantity to be considered, most appropriately, is male sexual fitness of the one type relative to the other type (K_M). Other approaches are discussed and it is shown that they confound differential mating success with frequency dependence of mating success. Moreover, it is shown that Levene's indices, previously designed as a measure of differential mating success, confound mating success with assortment, making these indices less useful. 3. The theoretical relationship between frequency dependence of male mating success and total sexual fitness is more complicated than would be expected beforehand. Some examples are given to clarify this issue. 4. Statistical tests to determine frequency dependence of male mating success have often been carried out in the past by determining the significance of deviations from random mating for each male type frequency separately. This procedure must be considered incorrect, because a change in male mating success over frequencies has to be tested. A correct way to do this is by testing all frequencies together in one single statistic, with only a moderate assumption about the type of frequency dependence. When mating success depends on frequency in a more irregular way, alternative tests are available, in which mating success at one frequency can be tested against any other frequency. 5. The rare male effect has been studied most thoroughly in Drosophila, and has been demonstrated for many Drosophila species. The effect has been demonstrated for some other insects as well, and also for vertebrates. The rare male effect has been found for types of males differing in specific genotype (visible mutants, karyotypes), genetic background and geographic origin. A rare male effect has also been demonstrated for non-genetic properties such as temperature of rearing. Though much less common than rare male mating advantage, there are some examples of rare female mating advantage. The expression of the rare male effect may be affected by several factors, such as age of the females, temperature or experimental approach. 6. Only a few studies on rare type mating advantage in Nature have been carried out, but some positive evidence is available. 7. It is pointed out that mating success will be frequency-dependent if both types of males differ pronouncedly in mating behaviour, but the nature of this frequency dependence may vary. If one type of male is more vigorous than the other type, a one-sided rare male effect can be explained on the basis of male–male competition. Quite often, though, a two-sided rare male effect has been found for males not greatly differing in mating behaviour or mating success. Three types of explanations are considered: (a) The females prefer to choose the rare type of male; (b) The rare type of male compensates for its rarity by becoming more sexually active; (c) No individual behaviour is altered. 8. As to the question whether the rare male effect could be an artefact of the experimental setup, the following points are relevant; (a) Biases as a result of alternate marking resulting in rare male mating advantage are theoretically possible, but are shown to be unimportant in explaining the rare male effect for Drosophila. (b) An effect analogous to the effect of alternate marking due to finite sample size is conceivable, but probably less important than suggested by Bryant et al. (1980). (c) When males are not sampled randomly with respect to place in the vial, bias may be introduced, resulting in a spurious rare male effect. (d) We feel that the standpoint of Bryant et al. (1980) that there is not yet any good evidence for the existence of a rare male effect is not sustained. Positive proof in favour of the existence of the rare male effect is that the effect can be modified by changing only one factor, such as odour. The artifacts just mentioned cannot explain why a rare male effect should be present in the one but absent in the other situation.     

Effect of ethylmethanesulfonate on Chlamydomonas reinhardi

Summary Ethylmethane sulfonate (EMS) is known to cause a considerably high mutation rate in higher plants. In our experiments with Chlamydomonas reinhardi however, the mutagenic effect was unexpectedly low, whereas the toxic effect was quite remarkable. It is supposed that the reason for the low rate of mutants is the high toxicity, since non-toxic EMS concentrations induce no mutants. The toxic effect on Chlamydomonas cells is caused not only by the products of hydrolysis of the EMS, but also by the EMS itself. The damaged cells begin to bleach, furthermore they are not able to deliver their daughter cells. To a certain degree both effects are reversible. Finally it was found that the sensivity to EMS was higher in cells of the mating type — than it was in those of the+mating type.     

Mutants with decreased differentiation to plasmodia in Physarum polycephalum

Summary Mutant (APT) amoebae that display reduced ability to form plasmodia asexually were isolated by the use of an enrichment procedure. The results of reconstruction experiments show that the procedure enriches only for mutants blocked early in the pathway from amoeba to plasmodium. Mutants were isolated from four parents, two of which produce plasmodia asexually because they carry the allele mth of the mating type locus, and two because they carry gad (greater asexual differentiation) mutations. The APT mutants varied widely in the frequency of residual plasmodium formation, which occurred, in some cases, by reversion. The mutants, called apt (amoeba to plasmodium transition), were recessive in diploids and linked to the mating type (mt) locus. Mutants derived from the gad parents, unlike the parents themselves, crossed readily with heterothallic amoebae. Progeny analysis from such crosses indicates that both gad mutations are linked to mt. The mutants derived from one of the mth parents fell into two groups on the basis of their ability to cross with the mutants derived from the mt2 gad-8 parent. The result suggests that the mth-derived mutants represent two or more complementation groups. Mutants derived from the mt2 gad-8 parent cross with mt2 amoebae and hence display an altered mating specificity.     

Lambda red-mediated synthesis of plasmid linear multimers in Escherichia coli K12

Summary Expression of the red ⁺ and gam ⁺ genes of bacteriophage in plasmids cloned in Escherichia coli wild-type cells leads to plasmid linear multimer (PLM) formation. In mutants that lack exonuclease I (sbcB sbcC), either of these functions mediates PLM formation. In order to determine whether PLM formation in sbcB sbcC mutants occurs by conservative (break-join) recombination of circular plasmids or by de novo DNA synthesis, thyA sbcB sbcC mutants were transferred from thymine- to 5-bromo-2-deoxyuridine (BUDR)-supplemented medium, concurrently with induction of red ⁺ or gam ⁺ expression, and the density distribution of plasmid molecular species was analyzed. After a period of less than one generation in the BUDR-supplemented medium, most PLM were of heavy/heavy density. Circular plasmids, as well as chromosomal DNA, were of light/light or light/heavy density. These results indicate that Red or Gam activities mediate de novo synthesis of PLM in sbcB sbcC mutants. Examination of plasmid DNA preparations from sbcB sbcC mutants expressing gam ⁺ or red ⁺ reveals the presence of two molecular species that may represent intermediates in the PLM biosynthesis pathway: single-branched circles (-structures) and PLM with single-stranded DNA tails. While Gam-mediated PLM synthesis in sbcB mutants depends on the activity of the RecF pathway genes, Red-mediated PLM synthesis, like Red-mediated recombination, is independent of recA and recF activities. One of the red ⁺ products, protein, suppresses RecA deficiency in plasmid recombination and PLM synthesis in RecBCD^– Exol^– cells. The dependence of PLM synthesis on the RecE, RecF or Red recombination pathways and the dependence of plasmid recombination by these pathways on activities that are required for plasmid replication support the proposal that PLM synthesis and recombination by these pathways are mutually dependent. We propose the hypothesis that DNA double-stranded ends, which are produced in the process of PLM synthesis, are involved in plasmid recombination by the RecE, RecF and Red pathways. Conversely, recombination-dependent priming of DNA synthesis at 3 singles-tranded DNA ends is hypothesized to initiate PLM synthesis on circular plasmid DNA templates.Abbreviations PLM plasmid linear multimers - BUDR 5-bromo-2-deoxyuridine - bp base pair     

Microarray and real-time PCR analyses reveal mating type-dependent gene expression in a homothallic fungus

Sordaria macrospora is a homothallic ascomycete which is able to form fertile fruiting bodies without a mating partner. To analyze the molecular basis of homothallism and the role of mating products during fruiting body development, we have deleted the mating type gene Smta-1 encoding a high-mobility group domain (HMG) protein. The ΔSmta-1 deletion strain is morphologically wild type during vegetative growth, but it is unable to produce perithecia or ascospores. To identify genes expressed under control of Smta-1, we performed a cross-species microarray analysis using Neurospora crassa cDNA microarrays hybridized with S. macrospora targets. We identified 107 genes that are more than twofold up- or down-regulated in the mutant. Functional classification revealed that 81 genes have homologues with known or putative functions. Comparison of array data from ΔSmta-1 with those from three phenotypically similar mutants revealed that only a limited set of ten genes is deregulated in all mutants. Remarkably, the ppg2 gene encoding a putative lipopeptide pheromone is 500-fold down-regulated in the ΔSmta-1 mutant while in all other sterile mutants this gene is up-regulated. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Fusion-defective mutants ofChlamydomonas eugametos

Summary Mutant strains of the unicellular green algaChlamydomonas eugametos are described which are defective in sexual fusion. All mutants are mating type plus (mt⁺). They are unable to fuse because none of them is capable of protruding a mating structure through the cell wall, neither during sexual agglutination nor after adding dibutyryl-cAMP or compounds that raise the intracellular calcium level, treatments that are effective in wild type cells. Evidence is presented that these mutants lack the lytic enzyme activity which is normally involved in the local hydrolysis of the cell wall to allow the protrusion of the mating structure. Furthermore, a simple light microscopic method is presented to determine the presence of activated mating structures.     

Binding of calmodulin to Nuf1p is required for karyogamy in<Emphasis Type="Italic"> Saccharomyces cerevisiae</Emphasis>

The role of calmodulin (CaM) during mating in Saccharomyces cerevisiae was examined by using a set of Phe-to-Ala substitutions. We identified ten CaM mutants that exhibited significantly reduced mating efficiencies when crossed to a strain of the opposite mating type harboring the same CaM mutation. Most of the mating-defective CaM mutants were bilateral, i.e., they also exhibited mating defects, albeit minor ones, when crossed to the wild type. When strains carrying different bilateral CaM mutations were mated, the mating efficiencies recovered dramatically. We termed this phenomenon "intragenic mating complementation", and classified the mating-defective CaM mutations into two intragenic mating complementation groups. Two mutant alleles belonging to different groups showed minor defects in cell adhesion and cell fusion, but exhibited severe defects in karyogamy. CaM is known to bind to the essential spindle pole body component Nuf1p. This binding appears to be important for karyogamy because the nuf1 ^C911R mutation, which impairs CaM-Nuf1p binding, resulted in a severe defect in karyogamy. Indeed, the two mating-defective CaM mutations were found to compromise formation of the CaM/Nuf1p complex, and the mating defects of these two CaM mutants were suppressible by a dominant, CaM-independent, mutation in NUF1. Taken together, these results suggest that loss of CaM binding to Nuf1p causes a defect in karyogamy, thereby inhibiting productive mating.Communicated by C. P. Hollenberg     

Direct mating between diploid sake strains of Saccharomyces cerevisiae

Various auxotrophic mutants of diploid heterothallic Japanese sake strains of Saccharomyces cerevisiae were utilized for selecting mating-competent diploid isolates. The auxotrophic mutants were exposed to ultraviolet (UV) irradiation and crossed with laboratory haploid tester strains carrying complementary auxotrophic markers. Zygotes were then selected on minimal medium. Sake strains exhibiting a MATa or MATα mating type were easily obtained at high frequency without prior sporulation, suggesting that the UV irradiation induced homozygosity at the MAT locus. Flow cytometric analysis of a hybrid showed a twofold higher DNA content than the sake diploid parent, consistent with tetraploidy. By crossing strains of opposite mating type in all possible combinations, a number of hybrids were constructed. Hybrids formed in crosses between traditional sake strains and between a natural nonhaploid isolate and traditional sake strains displayed equivalent fermentation ability without any apparent defects and produced comparable or improved sake. Isolation of mating-competent auxotrophic mutants directly from industrial yeast strains allows crossbreeding to construct polyploids suitable for industrial use without dependence on sporulation.