Viable Ascospores Research Articles

The SPS4 gene of Saccharomyces cerevisiae encodes a major sporulation-specific mRNA.

The SPS4 gene of Saccharomyces cerevisiae, a sporulation-specific gene identified previously in a differential hybridization screen of a genomic yeast DNA library, has been characterized further. The protein encoded by this gene was inferred from its nucleotide sequence to be 38,600 daltons with an isoelectric pH of 8.2. Consistent with this, two-dimensional polyacrylamide gel electrophoresis of the in vitro translation products of RNA purified by hybridization with the cloned SPS4 DNA indicated that the SPS4 gene product is a 39-kilodalton, basic protein. This protein was found to be identical in size and charge to a major, sporulation-specific protein identified in a two-dimensional polyacrylamide gel electrophoretic comparison of the in vitro translation products of total RNA from sporulating MATa/MAT alpha cells and asporogenous MAT alpha/MAT alpha cells. A MATa/MAT alpha strain homozygous for a partial deletion of the SPS4 gene appeared, however, to be unaffected in its ability to form viable ascospores.

Evidence for homothallism and vegetative compatibility in southern Diaporthe phaseolorum

Twenty-nine vegetative compatibility groups (vcgs) were identified among 297 isolates of the fungus causing soybean stem canker in the southeastern United States (southern Diaporthe phaseolorum). Of the isolates characterized, 79% were from one of the vcgs described (group 1). Barrage zones formed between isolates from different vcgs when grown on potato dextrose agar, and also formed, but not always distinctly, on cornmeal agar, Czapek-Dox agar, lima bean agar, and malt agar; barrages did not form on Sach's agar or water agar. Two types of evidence suggested that southern D. phaseolorum is homothallic. Perithecia and viable ascospores were produced by single ascospore isolates, and ascospores produced by mass isolates were always of the same compatibility group as the parental mass isolate.

Isolation and functional analysis of sporulation-induced transcribed sequences from Saccharomyces cerevisiae.

Strains of the yeast Saccharomyces cerevisiae that are heterozygous for the mating-type locus (MATa/MAT alpha) undergo meiosis and spore formation when they are starved for nitrogen and are provided with a nonfermentable carbon source such as potassium acetate. Haploids and diploids homozygous for the mating-type locus (MAT alpha/MAT alpha or MATa/MATa) are asporogenous and undergo neither meiosis nor spore formation when incubated under the same conditions. A small number of genes produce transcripts that appear to be induced specifically in sporulating cells. These transcripts either are not found or are present at much lower levels both in vegetatively growing cells and in cells from asporogenous strains that have been incubated in sporulation medium. Several genes complementary to these MATa/MAT alpha-dependent sporulation-induced transcripts were isolated from a gene-size insert yeast-lambda recombinant DNA library, by differential-plaque filter hybridization. An attempt was made to determine the function of three of these genes by mutating them in the yeast genome with in vitro mutagenesis and one-step gene replacement techniques. One gene was extensively disrupted by both a 0.3-kilobase deletion and the insertion of two large DNA sequences at different sites within the gene. Surprisingly, this compound mutation did not appear to affect meiosis or the production of viable ascospores, indicating that this gene was dispensable for differentiation. The other two genes were disrupted by simple insertion mutations at a site where it was possible that they might still possess some gene activity. These mutations also did not appear to affect sporulation. These results suggest that not all sporulation-induced genes are essential for meiosis and the production of viable ascospores under the conditions examined.

Isolation and functional analysis of sporulation-induced transcribed sequences from Saccharomyces cerevisiae.

Strains of the yeast Saccharomyces cerevisiae that are heterozygous for the mating-type locus (MATa/MAT alpha) undergo meiosis and spore formation when they are starved for nitrogen and are provided with a nonfermentable carbon source such as potassium acetate. Haploids and diploids homozygous for the mating-type locus (MAT alpha/MAT alpha or MATa/MATa) are asporogenous and undergo neither meiosis nor spore formation when incubated under the same conditions. A small number of genes produce transcripts that appear to be induced specifically in sporulating cells. These transcripts either are not found or are present at much lower levels both in vegetatively growing cells and in cells from asporogenous strains that have been incubated in sporulation medium. Several genes complementary to these MATa/MAT alpha-dependent sporulation-induced transcripts were isolated from a gene-size insert yeast-lambda recombinant DNA library, by differential-plaque filter hybridization. An attempt was made to determine the function of three of these genes by mutating them in the yeast genome with in vitro mutagenesis and one-step gene replacement techniques. One gene was extensively disrupted by both a 0.3-kilobase deletion and the insertion of two large DNA sequences at different sites within the gene. Surprisingly, this compound mutation did not appear to affect meiosis or the production of viable ascospores, indicating that this gene was dispensable for differentiation. The other two genes were disrupted by simple insertion mutations at a site where it was possible that they might still possess some gene activity. These mutations also did not appear to affect sporulation. These results suggest that not all sporulation-induced genes are essential for meiosis and the production of viable ascospores under the conditions examined.

Recessive mutations from natural populations of Neurospora crassa that are expressed in the sexual diplophase.

Wild-collected isolates of Neurospora crassa Shear and Dodge were systematically examined for recessive mutations affecting the sexual phase of the life cycle, which is essentially diploid. Seventy-four of 99 wild-collected isolates from 26 populations in the United States, India and Pakistan carried one or more recessive mutations that reduced fertility significantly when homozygous; mutations affecting spore morphology were also detected. Limited complementation tests indicate that most of the 106 recovered mutations are unique.--The recessive diplophase (= sexual phase) mutations were uncovered by crossing each wild-collected isolate to a marked two-chromosome double-reciprocal translocation strain as "balancer." Surviving progeny receive approximately 60% of their genome from the wild parent, but receive the mating-type allele from the "balancer" parent. These progeny were backcrossed to the wild parent and were also crossed with a standard laboratory strain (fl). Reduced fertility in the backcross vs. normal fertility in the cross with the laboratory standard signals the presence of a recessive mutation in the wild-collected isolate.--Most of the mutants (95 of 106) fall into two major classes: those producing barren perithecia with no or few viable ascospores (51) and those with spore maturation defects (44). Most of the recessive barrens result either from an early block in meiosis of ascus development (25) or from a late disturbance in postmeiotic ascus behavior (18).--These recessive mutations are formally equivalent to recessive lethals in higher eukaryotes and may be important in determining the breeding structure of natural Neurospora populations.

Transcriptional control of the sporulation-specific glucoamylase gene in the yeast Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, glucoamylase activity appears specifically in sporulating cells heterozygous for the mating-type locus (MAT). We identified a sporulation-specific glucoamylase gene (SGA) and show that expression of SGA is positively regulated by the mating-type genes, both MATa1 and MAT alpha 2. Northern blot analysis revealed that control of SGA is exerted at the level of RNA production. Expression of SGA or the consequent degradation of glycogen to glucose in cells is not required for meiosis or sporulation, since MATa/MAT alpha diploid cells homozygous for an insertion mutation at SGA still formed four viable ascospores.

Transcriptional Control of the Sporulation-Specific Glucoamylase Gene in the Yeast Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae, glucoamylase activity appears specifically in sporulating cells heterozygous for the mating-type locus (MAT). We identified a sporulation-specific glucoamylase gene (SGA) and show that expression of SGA is positively regulated by the mating-type genes, both MATa1 and MAT alpha 2. Northern blot analysis revealed that control of SGA is exerted at the level of RNA production. Expression of SGA or the consequent degradation of glycogen to glucose in cells is not required for meiosis or sporulation, since MATa/MAT alpha diploid cells homozygous for an insertion mutation at SGA still formed four viable ascospores.

Use of a selective medium to study the dispersal of ascospores ofSclerotinia sclerotiorum

A selective medium (potato dextrose agar with pentachloronitrobenzene and streptomycin) was used to assess dispersal of ascospores ofSclerotinia sclerotiorum in the field. This medium inhibited development ofMucor spp. andRhizopus spp. without affecting the germinability ofS. sclerotiorum ascospores or the production of sclerotia. The number of viable ascospores started increasing in December, was maximal from January to February, and decreased sharply in March. Deposition of ascospores on plates was maximal (57%) between 10:00 and 13:00 hours. Seventy-seven to 90% of the ascospores were deposited within the first 100 m from the source of inoculum and the rest were transported farther.

Isolation and partial characterization of a dominant mutant defective in the production of viable ascospores in Neurospora crassa.

A dominant mutant Asc (KH2A83), defective in ascospore formation, was obtained as a spontaneous mutation by the application of allelic complementation between inl (37401) and inl (83201(t)). The heterozygous cross of this mutant with wild type as protoperithecial parent produced 10-40% of white abortive ascospores, and that with wild type as conidial parent formed 60-90% of white abortive ascospores. The homozygous cross of Asc (KH2A83) produced 90-95% of ascospore abortion. The mutation was mapped between his-1 and al-3 at a distance of 0.8% to al-3 on LG VR. The UV sensitivity of Asc(KH2A83) was 2.8-4.0-fold higher than that of wild type, but that of Mei-2, mapped near al-3, was 2.3-fold. Whereas, unlike Mei-2, Asc(KH2A83) did not show sensitivity to methyl methanesulfonate and to histidine. Conidia of Asc(KH2A83) showed about 1% of viability in sorbose agar minimal medium, but those of Mei-2 did not show such character. Markers near Asc (KH2A83) including al-3 and inl represented low recombination recovery rates (2.6-28.9%) indicating reduced viability of ascospores which includedAsc (KH2A83). The aneuploid formation in a heterozygous cross with Asc(KH2A83) as a protoperithecial parent was estimated to be 0.9%.

Successful mating of Microsporum distortum with Nannizzia otae

Because of the close similarity between Microsporum distortum and M. canis , ten isolates of M. distortum were crossed with a pair of sexually reactive tester strains of Nannizzia otae , the teleomorph of M. canis . Six of the 10 isolates mated with the + tester strain of N. otae , producing viable ascospores. The close similarities between the anamorphic species and the successful mating between M. distortum and N. otae show that M. distortum is best recognized as a variety of M. canis rather than as a separate species.

Diploid protoplasts in Schizosaccharomyces pombe: formation, growth, and sporulation

Diploid cells of Schizosaccharomyces pombe heterozygous for the mating type region (h−/h+) were converted into protoplasts. On sporulation medium the diploid protoplasts either grew vegetatively or underwent meiosis resulting in four viable ascospores. Sporulation (without regenerating of cell wall) or growth (or conversion to cells) were found to be alternative possibilities.

Multiple mutant analysis of recombination in yeast

Summary. The RAD6, RAD50, and RAD52 loci have been identified as genes which code for functions which may act during meiotic recombination in yeast (Game et al.1980; Prakash et al. 1980). By use of the spol3-1 mutation,which allows sporulating cells to bypass the first meiotic division, the rad50-1 mutation has been directly implicated as a general meiotic Rec- mutation by examination of viable ascospores (Malone and Esposito 1981). Since the rad6-1 and rad52-1 mutations do not yield viable ascospores in the presence of spol3-1, multiple rad mutants have been constructed and analyzed. This analysis has demonstrated that in meiosis tad50-1 is epistatic to rad52-1, and rad6-1 is epistatic to rad50-1. This suggests that the order of action of these genes during meiosis is RAD6, RAD50, and then RAD52. The data for rad6-1 can be interpreted to suggest that RAD6 may not code for a recombination function,per se, although it may be required for recombination to occur. Analysis of mitotic recombination indicates that rad52-1 is epistatic to rad50-1 ; in mitosis; this is consistent with the hypothesis that the RAD50 gene codes for a recombination function required in meiosis but not in mitosis.

Development of breeding stocks of the yeast Saccharomycopsis lipolytica by methods of moderate inbreeding

Sporulation parameters of genetically labelled strains, derived from a wild strain of the alkane-utilizing yeast Saccharomycopsis lipolytica were improved by a breeding program using brother-sister crosses. Sporulation frequency, the number of four-spored asci and viability of ascospores could be significantly enhanced. To date a number of genetically well-defined strains is available that have good sporulation parameters and show a 1:1 segregation pattern of markers suitable for genetic analysis.

Monilinia Vaccinii-Corymbosi(Sclerotiniaceae): Its Biology on Blueberry and Comparison with Related Species

A six year study of Monilinia vaccinii-corymbosi in a 700 m2 site at Greenbelt, Maryland, revealed that this fungus attacks Vaccinium corymbosum and closely related species which, together, comprise the commercial blueberries of North America. Monilinia polycodii and M. taxonomic sp. 1 also grew at the site and, respectively, attacked V. stamineum and Gaylussacia baccata. Apothecia of M. vaccinii-corymbosi ejected viable ascospores for 2932 da; infection by ascospores caused leaf and shoot blight. Conidia were collected for 2547 da and they caused mummification of fruit. However the two inocula overlapped for several days when the bloom was 100%. In a growth chamber at 12 C, cups matured in 26 da beginning with the apical flattening of the stipe. Ascospores stored at 12 C, r.h. 50-65%, were viable for 23 da but no conidia germinated after 6 da. Occurrence of conidia on mummified fruits on plants of V. corymbosum and a technique to produce them in vitro are reported for the first time. Observations from inoculation of eight species of Vaccinium and one species of Gaylussacia with six species of Monilinia, and other data, revealed that the following are host specific: M. baccarum on V. myrtillus; M. ledi, anamorph on V. uliginosum, teleomorph on Ledum palustre; M. megalospora on V. uliginosum; M. oxycocci on V. macrocarpon and V. oxycoccos; M. polycodii on V. stamineum; M. urnula on V. vitis-idaea and M. taxonomic sp. 1 on G. baccata. The foregoing species, plus M. azaleae, and M. rhododendri are described, including their cultural characteristics. Useful taxonomic criteria include the extent of host specificity, the location of the ectostroma in relation to host epidermis and the parts in which it is lodged, the anatomical structure of the entostroma, and cultural characteristics. A dichotomous key is provided to delineate all Monilinia species on Monilinia vaccinii-corymbosi (Reade) Honey is an economically important fungus common on commercial blueberries in the United States and Canada. It is one of the 10 species of Monilinia Honey that attack plants of the ericaceous genera Vaccinium, Gaylussacia, Ledum, and Rhododendron. It causes symptoms rather similar to the effects of other Vaccinium-inhabiting species, and it is sometimes confused with M. urnula (Weinm.) Whet. (3, 4, 27). However, M. vacciniicorymbosi has a characteristically wide host range of related Vaccinium species and is otherwise distinct from all other Monilinia species. Monilinia has monilioid macroconidia (anamorph: Monilia), hollow-sphaeroid or (rarely) solid pseudosclerotia (a resting fungus mass enclosing substratum), and fructicolous apothecia (12, 25). The genus was segregated from Sclerotinia Fuckel, which now includes species with tuberoid, true sclerotia (a resting fungus mass) but lacking a conidial state.

Recombinationless meiosis in Saccharomyces cerevisiae.

We have utilized the single equational meiotic division conferred by the spo13-1 mutation of Saccharomyces cerevisiae (S. Klapholtz and R. E. Esposito, Genetics 96:589-611, 1980) as a technique to study the genetic control of meiotic recombination and to analyze the meiotic effects of several radiation-sensitive mutations (rad6-1, rad50-1, and rad52-1) which have been reported to reduce meiotic recombination (Game et al., Genetics 94:51-68, 1980); Prakash et al., Genetics 94:31-50, 1980). The spo13-1 mutation eliminates the meiosis I reductional segregation, but does not significantly affect other meiotic events (including recombination). Because of the unique meiosis it confers, the spo13-1 mutation provides an opportunity to recover viable meiotic products in a Rec- background. In contrast to the single rad50-1 mutant, we found that the double rad50-1 spo13-1 mutant produced viable ascospores after meiosis and sporulation. These spores were nonrecombinant: meiotic crossing-over was reduced at least 150-fold, and no increase in meiotic gene conversion was observed over mitotic background levels. The rad50-1 mutation did not, however, confer a Rec- phenotype in mitosis; rather, it increased both spontaneous crossing-over and gene conversion. The spore inviability conferred by the single rad6-1 and rad52-1 mutations was not eliminated by the presence of the spo13-1 mutation. Thus, only the rad50 gene has been unambiguously identified by analysis of viable meiotic ascospores as a component of the meiotic recombination system.

Recombinationless meiosis in Saccharomyces cerevisiae.

We have utilized the single equational meiotic division conferred by the spo13-1 mutation of Saccharomyces cerevisiae (S. Klapholtz and R. E. Esposito, Genetics 96:589-611, 1980) as a technique to study the genetic control of meiotic recombination and to analyze the meiotic effects of several radiation-sensitive mutations (rad6-1, rad50-1, and rad52-1) which have been reported to reduce meiotic recombination (Game et al., Genetics 94:51-68, 1980); Prakash et al., Genetics 94:31-50, 1980). The spo13-1 mutation eliminates the meiosis I reductional segregation, but does not significantly affect other meiotic events (including recombination). Because of the unique meiosis it confers, the spo13-1 mutation provides an opportunity to recover viable meiotic products in a Rec- background. In contrast to the single rad50-1 mutant, we found that the double rad50-1 spo13-1 mutant produced viable ascospores after meiosis and sporulation. These spores were nonrecombinant: meiotic crossing-over was reduced at least 150-fold, and no increase in meiotic gene conversion was observed over mitotic background levels. The rad50-1 mutation did not, however, confer a Rec- phenotype in mitosis; rather, it increased both spontaneous crossing-over and gene conversion. The spore inviability conferred by the single rad6-1 and rad52-1 mutations was not eliminated by the presence of the spo13-1 mutation. Thus, only the rad50 gene has been unambiguously identified by analysis of viable meiotic ascospores as a component of the meiotic recombination system.

The mutation SK(ad-3A) cancels the dominance of ad-3A+ over ad-3A in the ascus of Neurospora.

A newly induced mutant of Neurospora, when crossed with an ad-3A mutant, produces asci with four viable black and four inviable white ascospores. The survivors always contain the new mutant allele, never ad-3A. The new allele, which is called SK(ad-3A) (for spore killer of ad-3A), is located at or very near the ad-3A locus.--In crosses homozygous for ad-3A, each ascus contains only inviable white ascospores. This defect in ascospore maturation is complemented by the wild-type allele, ad-3A+ (crosses heterozygous for ad-3A and ad-3A+ produce mainly viable ascospores), but it is not complemented by the new SK(ad-3A) allele (all ad-3A ascospores from crosses heterozygous for SK(ad-3A) and ad-3A are white and inviable). In crosses homozygous for SK(ad-3A) or heterozygous for SK(ad-3A) and ad-3A+, each ascus contains only viable black ascospores. SK(ad-3A) does not require adenine for growth, and forced heterokaryons between SK(ad-3A) and ad-3A grow at wild-type rates and produce conidia of both genotypes with approximately equal frequency. Thus, the action of SK(ad-3A) is apparently restricted to ascospore formation. Possible mechanisms of the action of this new allele are discussed.

Mutagen sensitivities and mutator effects of MMS-sensitive mutants in neurospora

7 mus (mutagen-sensitive) mutants of Neurospora crassa, which are more sensitive to the toxic effects of MMS (methyl methanesulfonate) than wild-type, were investigated for cross-sensitivities to other mutagens and inhibitors. These mutants have recently been mapped in 5 new genes, mus-7 to mus-11, and mutant alleles from each gene were checked for their effects on mutation frequencies. It was found that mutants in 3 of these 5 genes showed radiation-induced mutation frequencies similat to wild-type. These included 2 alleles of the gene mus-10, which were cross-sensitive only to UV and were the only mutants that produce some viable ascospores in homozygous crosses. The mutant of the second gene, mus-8, was especially sensitive to UV and mitomycin C and produced slightly reduced frequencies of spontaneous mutation. In contrast, the mutant of the third gene, mus-7, was not UV-sensitive but showed some cross-sensitivity to X-rays; mus-7 was highly sensitive to MMS and also to histidine, which inhibits various repair-defective mutants at concentrations well below those that reduce wild-type growth. None of these mus resemble mutants previously found in Neurospora, nor do they conform clearly to mutant types identified in E. coli or yeast. On the other hand mutants in 2 further genes, mus-11, and especially 2 alleles of mus-9, are very similar to uvs-3 of Neurospora and generally resemble mutants that are considered to be defective in “error-prone” repair. They were UV- as well as X-ray-sensitive, and showed strong spontaneous mutator effects but almost no increase in recessive lethal frequencies in heterokaryons after UV-treatments.

Isolation and genetic analysis of MMS-sensitive mus mutants of neurospora.

With the aim of obtaining mutants that affect DNA repair or recombination, mutants sensitive to methylmethane sulfonate (MMS) have been isolated in the ascomycete Neurospora crassa. Seven of these mutants were backcrossed repeatedly to produce isogenic strains for measurements of relative mutagen sensitivities and for analysis of recombination frequencies. The new mus (mutagen sensitives) were compared to four previously known radiation-sensitive mutants which were shown to be cross-sensitive to MMS. Tests for allelism assigned the mus mutants to five new genes, mus-7 to mus-11, each mapping in a different linkage group. In homozygous crosses all mutants were sterile, except the two alleles of gene mus-10 which occasionally produced some viable ascospores. Complementation tests on MMS-media identified double mutant strains from many intercrosses. Such strains can be used for analysis of interactions between mutant alleles from different genes and of possible epistatic groupings for repair-deficient mutants in Neurospora. Four of these double mutant strains, all containing mus-8 and previously known mutants, were checked for survival on MMS media and their sensitivities were compared to those of their parental single mutant strains. Results indicate that mus-8 may be epistatic to uvs-2 which is deficient in excision repair, but not to mutants like uvs-3 that appear to be deficient in error-prone repair.

Emendation of the Genus Issatchenkia Kudriavzev and Comparison of Species by Deoxyribonucleic Acid Reassociation, Mating Reaction, and Ascospore Ultrastructure

The genus Issatchenkia Kudriavzev has been emended to include all nitrate-negative, multilateral budding yeast species that form unconjugated persistent asci with roughened spheroidal ascospores and have Q-7 ubiquinone in the electron transport system. Pichia kudriavzevii (Issatchenkia orientalis), Pichiaterricola, Pichia scutulata var. scutulata, and Pichia scutulata var. exigua are assigned to this genus as Issatchenkia orientalis Kudriavzev, Issatchenkia terricola (van der Walt) comb. nov., Issatchenkia scutulata var. scutulata (Phaff et al.) comb. nov., and Issatchenkia scutulata var. exigua (Phaff et al.) comb. nov., respectively. Additionally, one new species, Issatchenkia occidentalis, is described. The type strain of I. occidentalis is NRRL Y-7552 (=CBS 5459). Electron microscopy demonstrated that the ascospore walls of I. terricola have thick inner and outer layers and that the ascospores of the other species have walls with a thick inner layer and a thin, dense outer layer. With the exception of I. scutulata var. scutulata, ascospore surface ornamentation arises from the dense outer layer. Deoxyribonucleic acid reassociation studies and mating tests confirmed the recognition of four species in the genus Issatchenkia and showed Candida krusei to be the imperfect state of I. orientalis; Candida sorbosa was identified as the imperfect form of I. occidentalis. I. scutulata var. scutulata and I. scutulata var. exigua showed only 25© deoxyribonucleic acid complementarity, yet intervarietal matings formed viable ascospores. This is the lowest deoxyribonucleic acid relatedness ever shown between strains capable of genetic hybridization, and the implications of this finding are discussed.