A new gene affecting the efficiency of mating-type interconversions in homothallic strains of Saccharomyces cerevisiae

Homothallic strains of Saccharomyes cerevisiae are able to switch efficiently from one mating genotype to another. From a single haploid spore arise both a and mating type cells, which then self-mate to produce a colony consisting almost exclusively of nonmating a/ diploid cells. We have isolated a mutant homothallic strain that gives rise to colonies that show bisexual mating behavior. The mating reaction is always asymmetric, that is, in some colonies a mating is much stronger than mating, while others show greater than a mating.-This mating phenotype arises from the presence of three cell types in a colony: some a/ nonmating diploids and an unequal number of a and haploid cells. The predominant haploid type is that of the original cell that gives rise to the colony. This mixture of cell types arises from a very reduced efficiency of homothallic mating-type interconversions in the mutant strain.-The mutation, designated switch (swi1-1), behaves as a single genetic locus. The mutation is centromere linked, but not linked to the mating type locus or to any of the homothallism genes: HO, HMa and HM. The switch mutation does not affect the efficiency of self-mating, but rather directly affects the frequency of interconversion of mating types.     

A New Type of Mutation in SCHIZOSACCHAROMYCES POMBE: Vegetative Iodine Reaction

Colonies of Schizosaccharomyces pombe that contain ascospores (e.g., colonies of homothallic strains) turn black after treatment with iodine vapors. Heterothallic strains of S. pombe normally do not show this reaction. In experiments with the latter strains we found mutants which exhibit a positive iodine reaction though they do not contain ascospores. This phenotype is due to mutations in a new gene, vir1 (vegatative iodine reaction). The vir1 locus is not linked with the mating-type genes.--Strains of mating-type h-S are known not to give any spontaneous mating-type mutations. Mating-type mutations were also not found after treatment with nitrous acid.     

Variation of colony morphology and chromosomal rearrangement in Candida tropicalis pK233

UV irradiation treatment of the asexual yeast Candida tropicalis gave rise to morphological mutants exhibiting at least four different types of abnormal colonies on glucose-containing solid medium. These mutants were named according to their colony morphologies: 'doughnut', 'frilly', 'echinoid' and 'walnut' mutants. The doughnut mutant produced a wrinkled colony with a hollow in its central region that was rich in filamentous pseudohyphal cells. With increased incubation time, the colony gradually changed to a reticulate shape. The parent strain, which normally produced smooth colonies, gave similar colonies to those of the doughnut mutant when grown in medium containing oleic acid as carbon source. Both the frilly and the walnut mutants produced pseudohyphal cells in a similar fashion to the doughnut mutant. The echinoid mutant produced an echinulate colony morphology with aerial hyphae and contained true hyphal cells as well as pseudohyphal ones. Pulsed-field gel electrophoresis showed that the echinoid and frilly mutants had different karyotypes from that of their parent strain, suggesting the occurrence of chromosomal rearrangements associated with these morphological mutations.     

Screening for recombinant glutathione transferases active with monochlorobimane

A rapid and facile colony assay has been developed for catalytically active enzymes in combinatorial cDNA libraries of mutated glutathione transferases (GST), expressed in Escherichia coli. The basis of the method is the conjugation of glutathione (GSH) with the fluorogenic substrate monochlorobimane (MCB). This screening method makes it possible to isolate and characterize one recombinant clone that is active with MCB among thousands of inactive variants. Colonies containing GSTs that catalyze the conjugation of GSH with MCB display fluorescence under long-wavelength UV light. The fluorescence is visible instantly. One rat and 11 human GSTs representing four distinct enzyme classes were studied, and all except human GST T1-1 gave rise to fluorescent colonies. The colony assay based on MCB can consequently be broadly applied for identifying active GSTs both after subcloning of wild-type enzymes and in the screening of mutant libraries. Populations of bacteria expressing GSTs can also be analyzed by flow cytometry.     

A Technique for Predicting the Solvent-Producing Ability of Clostridium acetobutylicum

Changes in colony morphology were associated with the degeneration of solvent-producing strains of Clostridium acetobutylicum. The most efficient solvent-producing strains gave rise exclusively to colonies with dense centers containing large numbers of spores. Many outgrowths of various morphologies developed from the perimeter of such colonies after several days of incubation. The most degenerate cultures did not produce solvents and gave rise to large diffuse colonies that did not contain spores. These diffuse colonies did not produce outgrowths. Intermediate colony types were also observed. These could be derived from liquid cultures that were relatively poor solvent producers or from the outgrowths of colonies of efficient solvent-producing strains. Some of these intermediate types produced spores but did so less frequently than the high-solvent-producing strains. The spores of the intermediate types could not be distinguished from those of the most efficient solvent producers on the basis of heat sensitivity. The relationship observed between colony morphology and solvent production provides a method for predicting the solvent-producing potential of C. acetobutylicum cultures.     

Construction and Characterization of Isogenic Series of Saccharomyces cerevisiae Polyploid Strains

Tetraploid cells of Saccharomyces cerevisiae are generated spontaneously in a homothallic MATa/MATα diploid population at low frequency (approximately 10⁻⁶ per cell) through the homozygosity of mating-type alleles by mitotic recombination followed by homothallic switching of the mating-type alleles. To isolate tetraploid clones more effectively, a selection method was developed that used a dye plate containing 40 mg each of eosin Y and amaranth in synthetic nutrient agar per liter. It was possible to isolate tetraploid clones on the dye plate at a frequency of 1 to 3% among the colonies colored dark red in contrast to the light red of the original diploid colonies. Isogenic series of haploid to tetraploid clones with homozygous or heterozygous genomic configurations were easily constructed with the tetraploid strains. No significant differences in specific growth rate or fermentative rate were observed corresponding to differences in ploidy, although the haploid clones showed a higher frequency of spontaneous respiratory-deficient cells than did the others. However, a significant increment in the fermentative rate in glucose nutrient medium was observed in the hybrid strains constructed with two independent homozygous cell lines. These observations strongly suggest that the polyploid strains favored by the brewing and baking industries perform well not because of the physical increment of the cellular volume by polyploidy but because of the genetic complexity or heterosis by heterozygosity of the genome in the hybrid polyploid cells.     

Glucose and penicillin concentrations in agar medium below fungal colonies

The growth of colonies of Rhizoctonia cerealis and Penicillium chrysogenum on solid media in plate cultures was studied. When grown on defined media containing 10-50 mM-glucose, R. cerealis did not cause a significant reduction in the glucose concentration of the medium in advance of colonization, but did cause the formation of a steep glucose concentration gradient in the substrate below the colony; the medium directly below the centre of a 7 cm diameter colony of R. cerealis was exhausted of glucose even when the fungus was grown on medium containing 50 mM-glucose. Penicillin produced by colonies of P. chrysogenum accumulated in the medium in advance of fungal colonization. For a period up to about 18 d after inoculation, the concentration of penicillin in the medium throughout the plate increased with colony development and thereafter, except at the margins of the plate, decreased.     

Isolation of a homothallic mutant in <Emphasis Type="Italic">Lentinula edodes</Emphasis>

To obtain a homothallic mutant in Lentinula edodes, basidiospores derived from the common Bmut dikaryon (A1B1mut × A2B1mut) were treated with UV irradiation. Of a total of approximately 5000 monosporous cultures recovered, a single basidiospore isolate was found to produce the hyphae bearing clamp connections without mating. This mutant strain could form fruit bodies, and all its single basidiospore isolates developed into colonies with clamp connections. Such homothallic behaviors were transmitted from the mutant strain to the next generation. During the germination and following hyphal elongation in a single basidiospore of mutant strain, clamp connections were clearly detected in multicellular hyphae, which contained two nuclei in each cell. Their clamp connections were morphologically variable, viz., pseudo, abnormal, and true clamps. Amplified fragment length polymorphism (AFLP) profiles among the basidiospore isolates of mutant strain were identical, indicating that the mutant strain produced isogenic basidiospore progeny. Contribution no. 385 from the Tottori Mycological Institute     

Variation in Colony Characteristics and Symbiotic Effectiveness of Rhizobium

Seventeen cultures of Rhizobium CB756 varied in symbiotic effectiveness and contained a number of different colony types. An examination of single colony isolates from one culture of CB756 indicated that colony characteristics of most isolates were unstable and did not breed true. There was a relationship between symbiotic effectiveness and colony type of the original isolate. The 3 most ineffective sub-strains were all isolated from large, gummy colonies whereas the most effective were isolated from pinpoint, dry colonies.     

Relation between the efficiency of homothallic switching of yeast mating type genes and the distribution of cell types.

Homothallic switching of yeast mating type genes occurs as often as each cell division, so that a colony derived from a single haploid spore soon contains an equal number of MATa and MAT alpha cells. Cells of opposite mating types conjugate, and eventually the colony contains only nonmating MATa/MAT alpha diploids. Mutations that reduce the efficiency of homothallic MAT conversions yield colonies that still contain many haploid cells of the original spore mating type plus a few recently generated cells of the opposite mating type. These (a greater than alpha)- or (alpha greater than a)-mating colonies also contain some nonmating diploid cells. As an alternative to microscopic pedigree analysis to determine the frequency of mating type conversions in a variety of mutant homothallic strains, we analyzed the proportions of MATa, MAT alpha, and MATa/MAT alpha cells in a colony by examining the mating phenotypes of subclones. We developed a mathematical model that described the proportion of cell types in a slow-switching colony. This model predicted that the proportion of nonmating cells would continually increase with the size (age) of a colony derived from a single cell. This prediction was confirmed by determining the proportion of cell types in colonies of an HO swi1 strain that was grown for different numbers of cell divisions. Data from subcloning (a greater than alpha) and (alpha greater than a) colonies from a variety of slow-switching mutations and chromosomal rearrangements were used to calculate the frequency of MAT conversions in these strains.     

Effects of honey bees on colonies of Exoneura asimillima,an Australian native bee

Honey bee hives were placed, during two consecutive summers, in an experimental site which contained natural and artificially placed colonies of Exoneura asimillima, a semi-social, native bee. Two classes of colonies were studied: founders, and established colonies. Nests and contents were collected from an experimental site and three control sites following several months of exposure at the experimental site to the apiary honey bee population. Nest contents were analysed for differences among sites in colony population parameters which could have been caused by resource competition with introduced honey bees. Colony founding and overall colony survival were also considered. During the first season, the average number of large larvae plus prepupae per colony was significantly higher in the experimental site than in the control site. This difference could, however, have been the result of a two-week gap in sampling all the sites. All nest parameters showed high variability and there were no other significant differences between the two kinds of site. In the experimental site during the second season there were, relative to the control sites, significantly fewer total numbers of adult males and females in established nests and in all nests combined, significantly more immatures of all stages in founder nests and significantly lower adult male:female ratio. Although preliminary in nature, the data suggest that, in the experimental plots, E. asimillima showed: (i) increased adult emigration, (ii) increased brood rearing success, and/or (iii) relatively later colony founding, compared to the three controls. The possibility of resource competiton with honey bees causing the observed changes is discussed, along with alternative explanations.     

Growth of Streptococcal Protoplasts and L-Colonies on Membrane Filters

Membrane filters (Millipore Corp.; pore sizes 1.2 to 0.22 mum) were placed on the surface of L-phase growth medium solidified with agar. The filter and the surrounding medium were inoculated with either protoplasts or stable broth-grown L-phase variants obtained from Streptococcus faecium strain F24. The L-phase inoculum gave rise to viable L-colonies on the filters and on the medium, whereas protoplasts gave colony formation only on the medium. However, when the Millipore filters were covered by a layer of solid L-phase medium, 75 mum or greater in depth, before inoculation with protoplasts, colony formation resulted but with atypical morphology. In contrast, inoculation of protoplasts on Nuclepore and Sartorius membrane filters did give rise to L-colonies on the surface and underneath the filters after 2 days of incubation at 37 C. Submicroscopic, viable L-phase elements produced during colony formation were capable of passing through membrane filters with pore channels as small as 0.22 mum; these elements required transfer from underneath the filters to fresh agar medium in order to develop into L-phase colonies. Membrane filters were also placed on the surface of L-phase growth medium solidified with gelatin. Inoculation of the filters and surrounding medium with a lysozyme-prepared protoplast suspension gave rise to streptococci on the surface of the filters and on the medium. However, inoculation with the stable broth-grown L-phase variants gave rise to atypical colonies on the medium and only small patches of abortive growth on the filters.     

Allogeneic inhibition in a compound ascidian, Botryllus primigenus Oka. II. cellular and humoral responses in "nonfusion"reaction

Involvement of cellular and humoral responses in the “nonfusion” reaction (NFR) in a compound ascidian, Botryllus primigenus, was studied.NFR was irreversible and progressed at the same rate to completion even if one of the incompatible colonies was experimentally removed after NFR had been initiated between them. When two incompatible colonies were placed in a row with another compatible but genetically nonidentical colony between them, a rejection reaction was observed in blood vessels on the central colonies; or between the central colony and either one of the side colonies, which had fused with the central colony later than the other side colonies.It was tentatively supposed that NFR was mediated by an involvement of two types of factors. It was assumed that one was colony-specific, and the other was not necessarily colony-specific. The former was found contained in the test matrix and blood, which upon being introduced into a genetically different and incompatible colony, affected its test cells or granular amoebocytes which were then disintegrated. The latter was released from test cells upon their disintegration causing constriction of the ampullae or of the blood vessels forming an area of necrosis between reciprocally rejecting colonies.     

Susceptibility to merocyanine 540-mediated photosensitization: a differentiation marker on murine hematopoietic progenitor cells

Merocyanine 540 (MC 540) is an impermeant fluorescent dye that binds preferentially to fluidlike domains of the cell membrane. Photoexcitation of membrane-bound dye causes a breakdown of the normal permeability properties of the membrane and, eventually, cell death. We have used in vitro and in vivo clonal assays to determine the relative sensitivities of different classes of normal murine hematopoietic progenitor cells to MC 540-mediated photosensitization. Late erythroid progenitors (CFU-E) were the most sensitive cells, followed in order of decreasing sensitivity by early erythroid progenitors (BFU-E), megakaryocyte progenitors (CFU-Meg), day 7-spleen colony forming cells (day 7-CFU-S), granulocyte/macrophage progenitors (CFU-GM), and day 11-spleen colony forming cells (day 11-CFU-S). Bipotent progenitors of the granulocyte/macrophage lineage were more sensitive than unipotent macrophage progenitors but less sensitive than unipotent granulocyte progenitors. Progenitors giving rise to large granulocyte/macrophage colonies were more sensitive than progenitors giving rise to small colonies ("clusters"). We conclude that sensitivity to MC 540-mediated photosensitization is develop-mentally regulated and that differences occur even between the most closely related classes of progenitor cells. Our findings indicate the usefulness of MC 540 as a plasma membrane probe. They also support the contention that early and late-appearing spleen colonies are the progeny of two distinct classes of progenitor cells.     

The spatial scale of seed collection by harvester ants

Colonies of the seed-eating ant, Pogonomyrmex barbatus, compete with neighboring colonies for foraging areas. In a conflict over foraging area, what is at stake? This depends on how resources are distributed in time and space: if certain regions consistently provide particularly nutritious seed species, or especially abundant seeds, such regions will be of greater value to a colony. During the summer, seeds were taken from returning foragers in colonies located in 4 different vegetation types. There was no relation between the vegetation currently growing in the foraging area, and the species of seeds collected by ants. During the summer, ants collect mostly seeds produced in previous seasons and dispersed by wind and flooding. In 1991, colonies in all vegetation types collected mostly Bouteloua aristidoides; in 1992, Eriastrum diffusum and Plantago patagonica. There was no relation between colony density and numbers of seeds collected. Seed species collected by ants were compared in different colonies, and on different foraging trails within a colony. The results show that seed patches are distributed on the scale of distances between nests, not the smaller scale of different foraging trails of one colony. It appears that colonies are competing for any space in which to search for seeds, not competing for certain regions of consistently high value.     

Colony formation in agar by adult bone marrow multipotential hemopoietic cells

Erythroid colony formation in agar cultures of CBA bone marrow cells was stimulated by the addition of pokeweed mitogen-stimulated spleen conditioned medium (SCM). Optimal colony numbers were obtained when cultures contained 20% fetal calf serum and concentrated spleen conditioned medium. By 7 days of incubation, large burst or unicentric erythroid colonies occurred at a maximum frequency of 40–50 per 10⁵ bone marrow cells. In CBA mice the cells forming erythroid colonies were also present in the spleen, peripheral blood, and within individual spleen colonies. A marked strain variation was noted with CBA mice having the highest levels of erythroid colony-forming cells. In CBA mice erythroid colony-forming cells were mainly non-cycling (12.5% reduction in colony numbers after incubation with hydroxyurea or 3H-thymidine). Erythroid colony-forming cells sedimented with a peak of 4.5 mm/hr, compared with CFU-S, which sedimented at 4.25 mm/hr. The addition of erythropoietin (up to 4 units) to cultures containing SCM did not alter the number or degree of hemoglobinisation of erythroid colonies. Analysis of the total number of erythroid colony-forming cells and CFU-S in 90 individual spleen colonies gave a correlation coefficient of r = 0.93 for these two cell types. In addition to benzidine-positive erythroid cells, up to 40% of the colonies contained, in addition, varying proportions of neutrophils, macrophages, eosinophils, and megakaryocytes. Taken together with the close correlation between the numbers of CFU-S in different adult hemopoietic tissues, including individual spleen colonies, the data indicate that the erythroid colony-forming cells expressing multiple hemopoietic differentiation are members of the hemopoietic multipotential stem cell compartment.     

Possible demonstration of multipotential nature of embryonic neural retina by clonal cell culture.

The possible multipotential nature of the neural retina of early chick embryos was examined by the technique of clonal cell culture. Cultures were prepared from cells dissociated from freshly excised neural retinas of 3.5-day-old chick embryos or from cells harvested from primary highdensity cultures. The following four colony types were obtained: colonies differentiating into “lentoid bodies”; colonies with pigment cells; colonies with both “lentoid bodies” and pigment cells; and colonies comprised entirely of unidentifiable cells. Neuronal differentiation occurred frequently in the early stages of culture (up to about 10 days). In some of these neuronal colonies, “lentoid bodies” and, rarely, both “lentoid bodies” and pigment cells differentiated after a further culture period of up to 30 days. Secondary colonies established from primary colonies after 9–10 days demonstrated that these original colonies fell into four different categories: those giving rise to secondary colonies containing only “lentoid bodies,” those giving rise to pigmented colonies only, those developing both lentoid and pigmented colonies, and finally those which gave rise to secondary colonies of all three types, lentoid, pigmented, and mixed colonies. When primary pigmented colonies were recloned at about 30 days after inoculation, the differentiated pigment cells transdifferentiated into lens. Whether multispecific colonies were really of clonal origin or not is discussed. The possible presence of a multipotent progenitor cell able to give rise to multispecific clones in the neural retina of 3.5-day-old chick embryos is suggested. A sequence of differentiation starting from multipotent neural retinal cells to be terminated with lens through the differentiation of neuronal and pigment cells is hypothetically proposed.     

Pigmented variants of Tolypocladium inflatum in relation to cyclosporin A production

Summary We have isolated four distinct colony types of the fungus Tolypocladium inflatum, the producer of the immunosuppresive agent cyclosporin A: morphologically normal white, red, and orange colonies and morphologically diverse tiny brown colonies. In liquid cultures, white normal and brown colonies developed into yellow broths. The broth of the brown colony had a low final pH and low cyclosporin production, whereas orange and red colonies had dark brown and even black broths with higher final pH and high cyclosporin production. The specific production of cyclosporin A by the red colony was three times that of the white normal colonies. Offprint requests to: S. N. Agathos     

Differential proliferation of erythroid and granuloid spleen colonies following sublethal irradiation of the bone marrow donor

Spleen colonies produced by sublethally irradiated mouse bone marrow cells were compared to those produced by unirradiated marrow cells in lethally irradiated mice. Sublethally irradiated marrow cells gave rise to many fewer spleen colonies. At seven days of colony age, the ratio of erythroid colonies to granuloid colonies was lower (< 1) than for colonies formed by unirradiated marrow (2 to 3 or more). Delay of harvest of colonies to day 10 or 12 resulted in 6 to 11 fold increase in the ratio of erythroid to granuloid colonies due largely to the belated appearance of erythroid colonies.