Complete Sporulation Research Articles

Rapid Blue Light Regulation of a Trichoderma harzianum Photolyase Gene

Photolyases and blue light receptors belong to a superfamily of flavoproteins that make use of blue and UVA light either to catalyze DNA repair or to control development. We have isolated a DNA photolyase gene (phr1) from Trichoderma harzianum, a common soil fungus that is of interest as a biocontrol agent against soil-borne plant pathogens and as a model for the study of light-dependent development. The sequence of phr1 is similar to other Class I Type I eukaryotic photolyase genes. Low fluences of blue light rapidly induced phr1 expression both in vegetative mycelia, which lack photoprotective pigments, and, to a greater extent, in conidiophores. Thus, visible light induces the development of pigmented, resistant spores as well as the expression of phr1, perhaps announcing in this way the imminent exposure to the more damaging short wavelengths of sunlight. Light induction of phr1 in non-sporulating mutants shows that a complete sporulation pathway is not required for photoregulation. The light requirements for photoinduction of phr1 were not altered in dimY photoperception mutants. This suggests that photoinduction of sporulation and of photolyase expression is distinct in their photoreceptor system or in the transduction of the blue light signal.

Thin agar film for enhanced fungal growth and microscopic viewing in a new sealable fungal culture case.

This project was undertaken to find ways to enhance fungus colony maturation, to make viewing of fungal cultures easier, and to reduce disruption of the fungal structures to be observed for identification. Accordingly, a technique using a thin (0.2-mm) agar film that avoids problems inherent in traditional methods of fungal culture and identification was developed. In addition, to accommodate the 0.2-mm layer of agar film and a contiguous thicker 4-mm section of agar, a sealable fungal culture case that fits within microscope stage calipers and under the objective lenses was invented. The growth and identification of 28 organisms were evaluated in the sealable fungal culture cases and on double-pour agar plates by using potato dextrose agar in both. Compared with results obtained with the double-pour agar plates (rated as "good"), fungal growth and identification with the sealable fungal culture case were superior (rated as "excellent") (P < 0.05, chi-square test). The thin agar film limits excessive mycelial growth, while it often promotes complete sporulation or other forms of maturation of the fungal colony. More importantly, the thin agar film allows direct microscopic viewing of the developing fungal colonies. The portion of the sealable fungal culture case with the 4-mm layer of agar can be used for evaluation of colony pigment and texture. In conclusion, this new sealable fungal culture case allows direct viewing and earlier fungal species identification with greater intrinsic safety.

Molecular biology of yeast exoglucanases

Three exoglucanase (Exg) genes have been reported in Saccharomyces cerevisiae. Gene EXG1 encodes the major isoenzyme (Exgl). Differential glycosylation of the primary translation product throughout the secretory pathway results in the secretion of several glycoforms. The major glycoform (Exglb) contains two short carboxypeptidase Y-like oligosaccharides attached to both potential glycosylation sites present in the molecule. A minor glycoform (Exgla) arises from the former by elongation of the second oligosaccharide. The protein portion is processed in the secretory pathway by the Kex2 protease. Gene EXG2 encodes a 63 kDa polypeptide with 12 potential glycosylation sites. The predicted protein, Exgll, carries a signal peptide at the amino terminus and a glycosyl-phosphatidyl inositol anchoring motif at the carboxyl end. The latter appears responsible for the particulate nature of this isoenzyme, since its elimination results in the secretion of this activity into the culture medium. Gene SSG1 encodes a 52 kDa polypeptide which is specifically synthesized during sporulation of diploids. SSC1 expression is under control of both sexual (a1-α2 element) and nutritional control. Although homozygous ssg1 / ssg1 diploid strains are still able to complete sporulation, they exhibited a delay in the appearance of mature asci. Single or double disruption of EXG1 and EXG2 did not result in any relevant phenotype and the triple mutant behaved as ssg1 /ssg1. A Exgl-related enzyme is secreted by Candida albicans. All these four enzymes share 8 highly conserved regions in the same relative positions, indicating that they derive from a common ancestor. However, no clear function has so far been demonstrated for them.

SSG1, a gene encoding a sporulation-specific 1,3-beta-glucanase in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, the meiotic process is accompanied by a large increase in 1,3-beta-glucan-degradative activity. The molecular cloning of the gene (SSG1) encoding a sporulation-specific exo-1,3-beta-glucanase was achieved by screening a genomic library with a DNA probe obtained by polymerase chain reaction amplification using synthetic oligonucleotides designed according to the nucleotide sequence predicted from the amino-terminal region of the purified protein. DNA sequencing indicates that the SSG1 gene specifies a 445-amino-acid polypeptide (calculated molecular mass, 51.8 kDa) showing extensive similarity to the extracellular exo-1,3-beta-glucanases encoded by the EXG1 gene (C. R. Vazquez de Aldana, J. Correa, P. San Segundo, A. Bueno, A. R. Nebreda, E. Mendez, and F. del Rey, Gene 97:173-182, 1991). The N-terminal domain of the putative precursor is a very hydrophobic segment with structural features resembling those of signal peptides of secreted proteins. Northern (RNA) analysis reveals a unique SSG1-specific transcript, 1.7 kb long, which can be detected only in sporulating diploids (MATa/MAT alpha) but does not appear in vegetatively growing cells or in nonsporulating diploids (MAT alpha/MAT alpha) when incubated under nitrogen starvation conditions. The meiotic time course of SSG1 induction indicates that the gene is transcribed only in the late stages of the process, beginning at the time of meiosis I and reaching a maximum during spore formation. Homozygous ssg1/ssg1 mutant diploids are able to complete sporulation, although with a significant delay in the appearance of mature asci.

Cyclospora species--a new protozoan pathogen of humans.

Organisms referred to as "cyanobacterium-like bodies" have now been identified worldwide in the feces of both immunocompetent and immunocompromised patients with diarrhea. Organisms with a similar appearance have been isolated from Peruvian patients since 1985. From 1988 to 1991 we studied prospectively two cohorts of infants and young children infected with this organism. We now attempt to identify it. Fecal samples were collected weekly from the children and examined with the use of acid-fast staining and staining with a monoclonal antibody specific for cryptosporidium. Stools positive for cyanobacterium-like bodies were preserved in potassium dichromate and exposed to conditions allowing coccidian sporulation and excystation. Both unsporulated and sporulated oocysts were fixed by freeze-substitution techniques and then examined by electron microscopy. Organisms isolated from the feces of Peruvian patients and two patients from the United States were identified as belonging to the coccidian genus cyclospora, after sporulation and excystation of the oocysts according to standard techniques. Complete sporulation occurred within 5 to 13 days in oocysts maintained in potassium dichromate at 25 or 32 degrees C. Complete excystation resulted in the liberation of two sporozoites from the two sporocysts within each oocyst (cryptosporidia have four naked sporozoites within each oocyst). The presence of organelles characteristic of coccidian organisms was confirmed by electron microscopy. We have identified organisms of the genus cyclospora that are remarkably similar to cryptosporidia in their morphologic features and the diarrheal disease that they produce in humans. The complete life cycle and epidemiology of this new protozoan parasite remain to be described.

Multisensory activation of the phosphorelay initiating sporulation in Bacillus subtilis: identification and sequence of the protein kinase of the alternate pathway

The phosphorelay is the signal-transduction system recognizing and integrating environmental signals to initiate sporulation. The major signal input to the phosphorelay is an ATP-dependent kinase, KinA, responsible for phosphorylating the SpoOF protein. Mutants lacking KinA, however, still sporulate, suggesting that other kinases can fulfil its role. In order to identify these kinases, genes for kinases were isolated by hybridization using a degenerate oligonucleotide probe designed for common regions of this class of kinases. A gene for a second kinase, KinB, was isolated which gave a sporulation negative phenotype when inactivated in a kinA background. The kinB locus was sequenced and found to be a small operon consisting of the kinB gene and another gene, kapB, transcribed from a single.sigma A.-dependent promoter. Inactivation of either kinB or kapB in a kinA strain led to severe sporulation deficiency. The kinB gene coded for a 47774 M(r) protein with the carboxyl half of this protein highly homologous to the same domain of KinA. The amino-terminal domain of KinB was hydrophobic with six recognizable membrane-spanning regions. The kapB gene coded for a moderately charged, probably soluble, protein of 14,668 M(r) with no homology to any known protein. Genetic evidence suggests that KapB is required either for the function of KinB or for its expression. Although double mutants kinA kinB cannot sporulate and assume a stage 0 phenotype, the SpoA approximately P-dependent regulation of the abrB gene is normal in these strains, suggesting that low levels of SpoA approximately P accumulate even in the absence of both kinases. This accumulation is dependent on functional spo0F and spo0B genes and its source is unknown. The KinA and KinB pathways are the only pathways capable of producing sufficient Spo0A approximately P to allow initiation and completion of sporulation under laboratory conditions.

Achievement of complete Bacillus subtilis microcycle sporulation by the addition of S-adenosylmethionine and phospholipids

In an attempt to find factors that may be responsible for the initiation of sporulation, a system in which the germination and outgrowth phases were separate was applied to Bacillus subtilis. Outgrowth of the germinated spores to only the primary singlet cells was followed in chemically defined medium. Addition of specific metabolites induced the primary singlet cells to sporulate via microcycle sporulation. Experiments are described that led to complete sporulation by the addition of diaminopimelic acid, S-adenosyl- l-methionine and phosphatidylethanolamine.

Evidence for developmental commitment in the differentiating fruit body of Coprinus cinereus

Commitment of hymenial tissues of Coprinus cinereus to continued development after being explanted to agar medium was determined as a function of the stage of development reached immediately prior to explantation. Probasidia at the dikaryotic (prekaryogamy) stage at excision were arrested at that stage ; expiants made at later physiological ages did complete meiosis and/or sporulation, though at a slower rate than in vivo. Reversion to hyphal growth was observed to occur from hymenial and tramai cells but not often from probasidia. It is concluded that dikaryotic (prekaryogamy) probasidia are specified irreversibly as meiocytes and that they become determined to complete the sporulation programme during meiotic prophase I. Once initiated, maturation of basidia is an autonomous, endotrophic process. The potential of different fruit-body tissues of C. cinereus to form fruit-body primordia when explanted to agar medium was tested. Stipe bases and pseudorhizal (dark-grown) stipe bases always gave rise directly to primordia; most lamellae explanted at the dikaryotic stage showed such direct fruiting, but gills explanted after meiosis did not. This behaviour correlated with the disposition of accumulated glycogen and it is suggested that competence to fruit in these circumstances depends on the glycogen content of the tissue rather than representing commitment to a morphogenetic pathway.

Initiation of meiosis and sporulation in Saccharomyces cerevisiae does not require a decrease in cyclic AMP.

Meiosis and sporulation of Saccharomyces cerevisiae are initiated in a guanine auxotroph by guanine deprivation (E. Bautz Freese, Z. Olempska-Beer, A. Hartig, and E. Freese, Dev. Biol. 102:438-451, 1984). We used this condition to examine a hypothesis (K. Matsumoto, I. Uno, and T. Ishikawa, Cell 32:417-423, 1983) that initiation of meiosis requires a low level of cAMP. We found that, after guanine deprivation, the intracellular concentration of cAMP transiently decreased not more than 20% and not at all if the cAMP phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) was added to the medium. Under these conditions, at least 76% of the cells sporulated in the absence of IBMX, and almost 100% sporulated in its presence. The sporulating cells continually excreted cAMP and utilized the gluconeogenic carbon source. The cells failed to sporulate efficiently and to form four-spored asci if simultaneously deprived of guanine and carbon. After guanine deprivation in glucose medium, sporulation remained suppressed and intracellular cAMP was unchanged. We conclude that, under conditions of guanine starvation, cAMP deficiency is not required for initiation of meiosis and sporulation, cAMP is produced in excess and excreted to the medium, the cells sporulate better if the cAMP concentration is increased by addition of IBMX, the cells require a gluconeogenic carbon source for complete and efficient sporulation, and suppression of sporulation by glucose is not mediated by cAMP.

Initiation of meiosis and sporulation in Saccharomyces cerevisiae does not require a decrease in cyclic AMP.

Meiosis and sporulation of Saccharomyces cerevisiae are initiated in a guanine auxotroph by guanine deprivation (E. Bautz Freese, Z. Olempska-Beer, A. Hartig, and E. Freese, Dev. Biol. 102:438-451, 1984). We used this condition to examine a hypothesis (K. Matsumoto, I. Uno, and T. Ishikawa, Cell 32:417-423, 1983) that initiation of meiosis requires a low level of cAMP. We found that, after guanine deprivation, the intracellular concentration of cAMP transiently decreased not more than 20% and not at all if the cAMP phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) was added to the medium. Under these conditions, at least 76% of the cells sporulated in the absence of IBMX, and almost 100% sporulated in its presence. The sporulating cells continually excreted cAMP and utilized the gluconeogenic carbon source. The cells failed to sporulate efficiently and to form four-spored asci if simultaneously deprived of guanine and carbon. After guanine deprivation in glucose medium, sporulation remained suppressed and intracellular cAMP was unchanged. We conclude that, under conditions of guanine starvation, cAMP deficiency is not required for initiation of meiosis and sporulation, cAMP is produced in excess and excreted to the medium, the cells sporulate better if the cAMP concentration is increased by addition of IBMX, the cells require a gluconeogenic carbon source for complete and efficient sporulation, and suppression of sporulation by glucose is not mediated by cAMP.

Effects of Cortisol Implants on the PKX Myxosporean Causing Proliferative Kidney Disease in Rainbow Trout, Salmo gairdneri

The myxosporean (PKX) that causes proliferative kidney disease in salmonid fishes is found primarily in the kidney interstitium of clinically affected fish, where it invokes a severe inflammatory response. Incomplete spores are observed in the lumen of kidney tubules in recovering fish. PKX-infected rainbow trout, Salmo gairdneri, were immunosuppressed with cortisol implants to determine if a reduction in renal interstitial inflammation would enhance the development of the parasite. Immunosuppression of these fish was indicated by high plasma cortisol levels, chronic mortality due to opportunistic pathogens, and depressed leucocrits when compared to infected fish not receiving cortisol implants. Cortisol-treated PKX-infected fish had 20 times the density of interstitial PKX compared with nontreated fish, but showed less interstitial inflammation. All of the former group examined at 4 wk postimplantation exhibited intraluminal sporogonic forms of PKX, whereas no sporogonic forms were detected in the nontreated fish. Suppression of the inflammatory response clearly enhanced the parasite's ability to reach the sporogonic stage, but did not induce complete development of the spore. Possibly the kidney tubules of rainbow trout do not provide the proper physiological environment for complete sporulation of PKX and a nonsalmonid fish may be the primary host.

Description of the Goose Coccidium Eimeria stigmosa (Klimes, 1963), with Evidence of Intranuclear Development

Oocysts of Eimeria stigmosa isolated from a wild juvenile lesser snow goose (Anser caerulescens caerulescens) were used in experimental infections of laboratory-reared geese. The structure of oocysts is described; a peculiar bumpy surface and a calyx-like thickening around the micropyle are illustrated by scanning electron microscopy. Experimentally infected geese had reduced feed intake and passed blood in their feces. Six days post-inoculation (p.i.) oocysts were passed for about 24 hr. Oocyst production was not increased in dexamethasone-treated geese. Complete sporulation of oocysts occurred after about 3.5 days. Developmental stages in lesser snow geese were occasionally present throughout the intestine, but were concentrated at Meckel's diverticulum, and at foci in the ileum and colon. Meronts were found 2, 3 and 4 days p.i. and gamonts and developing oocysts were observed 4, 5 and 5.5 days p.i. All endogenous stages of E. stigmosa were present in the nucleoplasm of enterocytes, primarily at the apical half of villi. Nuclear hypertrophy occurred in infected cells and nuclei containing gamonts were enlarged up to 4 times their normal size. Mild lesions were observed in the intestine of infected geese. Patent infections were established in lesser snow, Canada (Branta canadensis) and domestic (Anser anser domesticus) geese, but oocysts were not passed by inoculated ducks.

Coccidia of Brazilian edentates: Eimeria cyclopei n.sp. from the silky anteater, Cyclopes didactylus (Linn.) and Eimeria choloepi n.sp. from the two-toed sloth, Choloepus didactylus (Linn.)

Eimeria cyclopei n.sp. is described from the silky anteater, Cyclopes didactylus, from Para State, north Brazil. Undifferentiated oocysts, passed in the faeces, complete sporulation in seven days at 26 to 28°C. Oocysts are ellipsoidal to sub-spherical, with a mean size of 28.1 × 23.6 μm: the wall is ∼1.5 to 2.0 μm thick, apparently with an outer thin, colourless membrane and two inner, thicker, striated and yellowish layers. There is no micropyle, oocyst residuum or polar body. The mean measurements of sporocysts are 19.0 × 9.0 μm, and they are slightly asymmetrical, elongate pear-shape, with a plug-shaped Steida body projecting beyond the end of the sporocyst. Sporozoites are as long as or longer than the sporocysts: The sporocyst residuum is scattered between sporozoites in younger specimens and becomes condensed into rounded mass in older ones. The endogenous stages occur in the epithelial cells of the ileum, on the lumenal side of the host-cell nucleus. Uninucleate meront, microgamont and macrogamont precursors are recognizable morphologically. Mature meronts are ∼ 20.0 × 15.7 μm some produce 12 to 20 merozoites which are ∼ 8.7 × 2.0 μm, and others 10 to 26 merozoites which are ∼ 11.4 × 2.0 to 15.0 × 3.0 μm. Mature microgamonts which are ∼27.5 × 24.1 μm, produce from 150 to 170 microgametes of ∼7.1 × 1.0 μm: microgametes have two flagella of unequal length. Mature macrogamonts are ∼ 28.4 × 24.5 μm Eimeria choloepi n.sp. is recorded from the two-toed sloth, Choloepus didactylus, from the same area of Brazil. Undifferentiated oocysts, passed in the faeces, complete sporulation in 23 days at 26 to 28°C. Oocysts with a mean size of 23.0 × 20.3 μm, have a wall ∼2.0 to 2.5 μm thick which is composed of two thick, yellowish and striated outer layers and a delicate, colourless inner one. There is no micropyle, oocyst residuum or polar granule. Mature sporocysts with a mean size of 11.3 × 7.1 μm, are ellipsoidal to egg-shaped and have a poorly developed Steida body. The sporocyst residuum is composed of a small number of large globules: The sporozoites are longer than the sporocyst and strongly recurved. The endogenous stages occur in epithelial cells of the ileum, on the lumenal side of the host-cell nucleus. Dimorphic meronts produce 8 to 18 merozoites which are either ∼13.0 × 2.0 μm or ∼13.0 × 3.0 μm. Microgamonts produce 50 to 80 microgametes of ∼8.0 × 1.0 μm. Mature macrogamonts are ∼18.3 × 17.9 μm. ac]19820212

Statistical analysis of the patterns of spore formation in Bacillus subtilis.

A description of the patterns of sporulation in chains of Bacillus subtilis cells has been obtained by fitting a statistical model to data obtained from a resuspended culture. The underlying assumptions of this model are: (a) resuspension causes an immediate response in the growing bacteria which is necessary for subsequent sporulation; (b) the probability of this response occurring is the same for all the bacteria present at the time of resuspension; (c) after resuspension some of the bacteria pass through a single round of cell division to produce a pair of sporulating bacteria, while the rest divide twice to produce four; and (d) the two sub-populations described in (c) differ in their ability to complete sporulation.

Septum formation, cell division, and sporulation in mutants of yeast deficient in proteinase B.

Mutants of Saccharomyces cerevisiae that carry lesions in the structural gene for proteinase B, prb1, have been isolated. Proteinase B was thought to be involved in activation of chitin synthase zymogen to allow septum formation during budding and to be involved in sporulation. The prb1 mutants are able to grow and divide and they form apparently normal septa that contain chitin. Most diploids homozygous for prb1 mutations are unable to complete sporulation.

Changes in regulation of ribosome synthesis during different stages of the life cycle of Saccharomyces cerevisiae

Diploid strains of Saccharomyces cerevisiae, each homozygous for one of the temperature sensitive mutations rna2, rna3, rna4, rna6 or rna8, are temperature sensitive for ribosome synthesis during vegetative growth, but are not inhibited for ribosomal synthesis at the restrictive temperature under sporulation conditions. The continued ribosome biosynthesis at the restrictive temperature (34 degrees C) during sporulation includes de novo synthesis of both ribosomal RNA and ribosomal proteins. This lack of inhibition of ribosome biosynthesis is found even when cells committed to complete sporulation are returned to vegetative growth medium. The ribosomes synthesized at 34 degrees C are apparently functional, as they are found in polyribosomes. Although the rna mutants do not regulate ribosome synthesis during sporulation, all of these diploid strains fail to complete sporulation at 34 degrees C. The cells are arrested after the second meiotic nuclear division but before ascus formation. The failure to complete sporulation at the restrictive temperature and the inhibition of ribosome biosynthesis during growth are caused by the same mutation, because revertants selected for temperature independent growth were also able to sporulate at 34 degrees C.

Sporogony of the Oocysts of Isospora canis

LEPP, D. L. & TODD, K. S., JR. 1976. Sporogony of the oocysts of Isospora canis. Trans. Amer. Micros. Soc., 95: 98-103. The sporulation process of Isospora canis oocysts is described and compared with sporogony of other Isospora species of carnivores. Oocysts had not completed sporulation after 16 days at 10 C. At 20, 30, and 35 C, sporulation was completed at 48, 16, and 16 hr, respectively. At 45 C, oocysts degenerated and did not complete sporulation.

Sporulation in D-glucosamine auxotrophs of Saccharomyces cerevisiae: meiosis with defective ascospore wall formation.

Mutants that require exogenous D-glucosamine for growth were isolated from Saccharomyces cerevisiae X2180-1A after ethyl methane sulfonate mutagenesis. Class A auxotrophs fail to grow on yeast extract-peptone-dextrose and minimal media, whereas class B auxotrophs grow on minimal medium and readily revert to grow on yeast extract-peptone-dextrose medium. Class B auxotrophs are suppressible by a class of suppressors distinct from nonsense suppressors, and their properties suggest that they are defective in a regulatory function. All 23 mutants studied were recessive and allelic, and they define a new gene designated gcn1. An analysis of a class A auxotroph revealed that it lacked L-glutamine:D-fructose 6-phosphate amidotransferase (EC 2.6.1.16) activity and indicates that GCN1 codes the amino acid sequence of this enzyme. The finding that all mutants were allelic indicates that the amidotransferase is the only enzyme responsible for D-glucosamine synthesis in S. cerevisiae. The occurrence of allelic complementation and media-conditional mutants suggests that the amidotransferase is a multimeric enzyme with an activity subject to metabolic control. Diploids homozygous for gcn1 fail to complete sporulation. They proceed through meiosis normally, as judged by the occurrence of meiotic recombination, the production of haploid nuclei, and the formation of multinucleate cells visible after Giemsa staining. However, the formation of glusulase-resistant ascospores is blocked, and deformed spores lacking the electron-dense outer layer characteristic of the normal spore wall are observed by electron microscopy. Cells that acquire the ability to synthesize D-glucosamine, because of gene conversion during meiosis, complete sporulation in a normal fashion. Thus, the GCN1 gene product appears to be synthesized late in sporulation and may prove to be a useful developmental landmark for the termination of ascospore development.

Changes of Nucleic Acids and Proteins in the Oocysts of Eimeria tenella during Sporulation

SYNOPSIS. The total content of DNA in Eimeria tenella, estimated at 5.8 × 10−12 gm/oocyst, varies little during sporulation. Its buoyant density is 1.682 gm/cm3, reflecting a G + C content of ∼41%. Thymidine is not incorporated into any TCA insoluble fraction of sporulating oocysts, but radioactivity from [3H]uridine and [3H]deoxyuridine are incorporated into RNA at a linear rate during the first 5 hr of sporulation. The labeled RNA, found mainly in the paranuclear bodies of newly formed sporozoites, contains ∼0.15 nmole [3H]uridine/106 oocysts at the completion of sporulation. One nmole of leucine is incorporated into the hot TCA insoluble fraction of 106 oocysts during the first 7 hr of sporulation after an initial lag. The incorporated amino acid is mainly in the cytoplasm of the sporozoites, and an analysis by SDS‐gel electrophoresis reveals most of the radioactivity in a narrow band with a molecular weight of ∼50,000 daltons. Incorporation of uridine and leucine, however, can be totally suppressed by respiratory inhibition. Further analysis of the proteins in the oocysts reveals that the total protein content remains relatively unchanged at 2.64 × 10−16 gm/oocyst during sporulation, but there is a shift of 13–14% of total protein from the soluble cytoplasm to the 15,000 g pellets. By polyacrylamide gel electrophoresis, a major protein band. possibly a glycoprotein, is shown in the soluble cytoplasm of unsporulated oocysts. This band disappears during sporulation.

Mating type and sporulation in yeast. II. Meiosis, recombination, and radiation sensitivity in an alpha-alpha diploid with altered sporulation control.

In wild-type S. cerevisiae, diploid cells must be heterozygous at the mating-type locus in order to sporulate. In the preceding paper, we described a number of mutants (CSP mutants), isolated from nonsporulating aa and alpha-alpha parent strains, in which sporulation appeared to be uncoupled from control by mating type. The characterization of one of these mutants (CSP1) is now extended to other processes controlled by mating type. This mutant is indistinguishable from alpha-alpha cells and unlike aalpha cells for mating factor production and response, zygote formation, intragenic mitotic recombination, and for X-ray sensitivity. The mutant apparently undergoes a full round of DNA synthesis in sporulation medium, but with delayed kinetics. Only 20% of the cells complete sporulation. Among spores in completed asci, the frequency of both intra- and intergenic recombination is the same as it is for spores produced by aalpha cells. However, experiments in which cells were shifted from sporulation medium back to minimal growth medium gave a frequency of meiotic recombination between ade2 or leu2 heteroalleles only 25% to 29% as high for CSP1 alpha-alpha diploid or CSP1 aa disomic cells as for aalpha diploid or disomic cells. Because the latter result, indicating recombination defectiveness, measured recombinant production in the entire cell population, whereas the result indicating normal recombination sampled only completed spores, we infer that all meiotic recombination events occuring in the population of CSP1 alpha-alpha cells are concentrated in those few cells which complete sporulation. This high degree of correlation between meiotic recombination and the completion of meiosis and sporulation suggests that recombination may be required for proper meiotic chromosome segregation in yeast just as it appears to be in maize and in Drosophila.