Evolution of uni- and bifactorial sexual compatibility systems in fungi.

Mating systems, that is, whether organisms give rise to progeny by selfing, inbreeding or outcrossing, strongly affect important ecological and evolutionary processes. Large variations in mating systems exist in fungi, allowing the study of their origin and consequences. In fungi, sexual incompatibility is determined by molecular recognition mechanisms, controlled by a single mating-type locus in most unifactorial fungi. In Basidiomycete fungi, however, which include rusts, smuts and mushrooms, a system has evolved in which incompatibility is controlled by two unlinked loci. This bifactorial system probably evolved from a unifactorial system. Multiple independent transitions back to a unifactorial system occurred. It is still unclear what force drove evolution and maintenance of these contrasting inheritance patterns that determine mating compatibility. Here, we give an overview of the evolutionary factors that might have driven the evolution of bifactoriality from a unifactorial system and the transitions back to unifactoriality. Bifactoriality most likely evolved for selfing avoidance. Subsequently, multiallelism at mating-type loci evolved through negative frequency-dependent selection by increasing the chance to find a compatible mate. Unifactoriality then evolved back in some species, possibly because either selfing was favoured or for increasing the chance to find a compatible mate in species with few alleles. Owing to the existence of closely related unifactorial and bifactorial species and the increasing knowledge of the genetic systems of the different mechanisms, Basidiomycetes provide an excellent model for studying the different forces that shape breeding systems.

Sexual selection in fungi.

The significance of sexual selection, the component of natural selection associated with variation in mating success, is well established for the evolution of animals and plants, but not for the evolution of fungi. Even though fungi do not have separate sexes, most filamentous fungi mate in a hermaphroditic fashion, with distinct sex roles, that is, investment in large gametes (female role) and fertilization by other small gametes (male role). Fungi compete to fertilize, analogous to 'male-male' competition, whereas they can be selective when being fertilized, analogous to female choice. Mating types, which determine genetic compatibility among fungal gametes, are important for sexual selection in two respects. First, genes at the mating-type loci regulate different aspects of mating and thus can be subject to sexual selection. Second, for sexual selection, not only the two sexes (or sex roles) but also the mating types can form the classes, the members of which compete for access to members of the other class. This is significant if mating-type gene products are costly, thus signalling genetic quality according to Zahavi's handicap principle. We propose that sexual selection explains various fungal characteristics such as the observed high redundancy of pheromones at the B mating-type locus of Agaricomycotina, the occurrence of multiple types of spores in Ascomycotina or the strong pheromone signalling in yeasts. Furthermore, we argue that fungi are good model systems to experimentally study fundamental aspects of sexual selection, due to their fast generation times and high diversity of life cycles and mating systems.

Reconstitution of active telomerase in primary human foreskin fibroblasts: effects on proliferative characteristics and response to ionizing radiation.

PURPOSE: Telomere shortening has been proposed to trigger senescence, and since most primary cells do not express active telomerase, reactivation of telomerase activity was proposed as a safe and non-transforming way of immortalizing cells. However, to study radiation responses, it is as yet unclear whether cells immortalized by telomerase reactivation behave in a similar manner as their parental primary cells. MATERIALS AND METHODS: Primary human foreskin fibroblasts were transfected with the human catalytic subunit of telomerase, the reverse transcriptase (hTERT), and their growth characteristics and response to DNA damage were characterized. RESULTS: The sole expression of the human hTERT was sufficient to immortalize the human foreskin fibroblasts. With time in culture, the immortalized cells almost doubled their average telomeric length and the clonal population contained almost no post-mitotic fibroblasts anymore. Up to 300 population doublings, no alterations compared with the parental primary cells were seen in terms of clonogenic radiosensitivity, DNA double-strand break repair, radiation-induced increases in p53 and p21(WAF-1,CIP-1) expression, and the G1/S and G2/M cell cycle checkpoints. Moreover, mitogen-induced mitotic arrest of fibroblasts was still possible in the hTERT-immortalized clones. CONCLUSIONS: Immortalizing fibroblasts by reconstitution of active telomerase seems a good, reliable manner to generate a large source of cells with a radiation damage response similar to the primary cells.

BRCA1 and BRCA2 heterozygosity and repair of X-ray-induced DNA damage.

PURPOSE: Up to 90% of hereditary breast cancer cases are linked to germ-line mutations in one of the two copies of the BRCA1 or BRCA2 genes. Brca1 and Brca2 proteins are both involved in the cellular defence against DNA damage, although the precise function of the proteins is still not known. Some studies on a small number of samples as well as the present pilot study also suggested that BRCA1 heterozygosity may lead to impaired repair of ionizing-radiation-induced DNA double-strand breaks. The purpose of the study was to test in a larger family-matched study whether carriers of BRCA1 or BRCA2 mutations have an increased sensitivity to ionizing radiation. MATERIALS AND METHODS: In a blind study, the effect of different germ-line mutations in one allele of the BRCA1 or BRCA2 gene on the ability to repair X-ray-induced DNA breaks was investigated. Fibroblasts and lymphocytes were taken from heterozygotic individuals (BRCA1+ /- and BRCA2+ /-) with different mutations and from relatives proven to be non-carriers of the BRCA mutations. Rejoining of DNA breaks was analysed by pulsed-field gel electrophoresis (for fibroblasts) or the comet assay (for lymphocytes). RESULTS: Significant interindividual differences were found in the capacities of the fibroblasts and lymphocytes to rejoin DNA breaks induced by X-radiation. However, these differences were not related to heterozygosity in BRCA1 or BRCA2. CONCLUSIONS: Cells from carriers of mutations in one allele of the BRCA1 or BRCA2 genes have no gross defects in their ability to rejoin radiation-induced DNA breaks. Hence, these carriers may not be at risk of developing excess normal tissue reactions after radiotherapy consistent with data from recent clinical studies.

Regulation of bacterial sugar-H+ symport by phosphoenolpyruvate-dependent enzyme I/HPr-mediated phosphorylation.

The lactose-H+ symport protein (LacS) of Streptococcus thermophilus has a C-terminal hydrophilic domain that is homologous to IIA protein(s) domains of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). C-terminal truncation mutants were constructed and expressed in Escherichia coli and their properties were analyzed. Remarkably, the entire IIA domain (160 amino acids) could be deleted without significant effect on lactose-H+ symport and galactoside equilibrium exchange. Analysis of the LacS mutants in S. thermophilus cells suggested that transport is affected by PTS-mediated phosphorylation of the IIA domain. For further studies, membrane vesicles of S. thermophilus were fused with cytochrome c oxidase-containing liposomes, and, when appropriate, phosphoenolpyruvate (PEP) plus purified enzyme I and heat-stable protein HPr were incorporated into the hybrid membranes. Generation of a protonmotive force (delta p) in the hybrid membranes resulted in accumulation of lactose, whereas uptake of the PTS sugar sucrose was not observed. With PEP and the energy-coupling proteins enzyme I and HPr of the PTS on the inside, high rates of sucrose uptake were observed, whereas delta p-driven lactose uptake by wild-type LacS was inhibited. This inhibition was not observed with LacS(delta 160) and LacS(H552R), indicating that PEP-dependent enzyme I/HPr-mediated phosphorylation of the IIA domain (possibly the conserved His-552 residue) modulates lactose-H+ symport activity.

A single mutation confers vanadate resistance to the plasma membrane H+-ATPase from the yeast Schizosaccharomyces pombe.

A single-gene nuclear mutant has been selected from the yeast Schizosaccharomyces pombe for growth resistance to Dio-9, a plasma membrane H+-ATPase inhibitor. From this mutant, called pma1, an ATPase activity has been purified. It contains a Mr = 100,000 major polypeptide which is phosphorylated by [gamma-32P] ATP. Proton pumping is not impaired since the isolated mutant ATPase is able, in reconstituted proteoliposomes, to quench the fluorescence of the delta pH probe 9-amino-6-chloro-2-methoxy acridine. The isolated mutant ATPase is sensitive to Dio-9 as well as to seven other plasma membrane H+-ATPase inhibitors. The mutant H+-ATPase activity tested in vitro is, however, insensitive to vanadate. Its Km for MgATP is modified and its ATPase specific activity is decreased. The pma1 mutation decreases the rate of extracellular acidification induced by glucose when cells are incubated at pH 4.5 under nongrowing conditions. During growth, the intracellular mutant pH is more acid than the wild type one. The derepression by ammonia starvation of methionine transport is decreased in the mutant. The growth rate of pma1 mutants is reduced in minimal medium compared to rich medium, especially when combined to an auxotrophic mutation. It is concluded that the H+-ATPase activity from yeast plasma membranes controls the intracellular pH as well as the derepression of amino acid, purine, and pyrimidine uptakes. The pma1 mutation modifies several transport properties of the cells including those responsible for the uptake of Dio-9 and other inhibitors (Ulaszewski, S., Coddington, A., and Goffeau, A. (1986) Curr. Genet. 10, 359-364).

Immunological evidence for the involvement of cell wall proteins in phosphate uptake in the yeast Saccharomyces cerevisiae.

Immunological cross-reactivity between cell wall proteins obtained from two yeast genera (Candida tropicalis and Saccharomyces cerevisiae) is reported. Specific retention of two cell wall proteins from Saccharomyces cerevisiae by an immunoabsorbent column coupled with antibodies against phosphate binding protein 2(PiBP2) from Candida tropicalis allowed to generate antibodies against the proteins from S. cerevisiae. These antibodies were effective in inhibiting phosphate uptake by S. cerevisiae cells. The proteins from S. cerevisiae displayed a phosphate binding activity which was inhibited in the presence of the forementioned antibodies. These results and the observation that the amount of these proteins in the shock fluid was dependent of the growth conditions (i.e., in the presence or in the absence of phosphate) support the idea that these proteins are involved in the high affinity phosphate transport system.

Effect of ethidium bromide and DEAE-dextran on divalent cation accumulation in yeast. Evidence for an ion-selective extrusion pump for divalent cations.

The larger accumulation of Mn2+ than of Sr2+ in Saccharomyces cerevisiae is ascribed to the operation of a specific extrusion pump, presumably a Ca2+ pump, which has a higher affinity for Sr2+ than for Mn2+. The differences in accumulation levels of Mn2+ and Sr2+ attained after prolonged incubation are completely abolished in cells of which the plasmamembrane has been permeabilized with the polybase DEAE-dextran under isotonic conditions. In the permeabilized cells Sr2+ and Mn2+ accumulation levels are attained as for Mn2+ in intact cells. It is suggested that the accumulation of divalent cations into the permeabilized cells mainly represents their accumulation into the vacuoles. Also the cationic dye ethidium abolishes the differences in Mn2+ and Sr2+ accumulation. The dye increases the accumulation of Sr2+ but decreases that of Mn2+ somewhat. It cannot be distinguished yet whether its action is due to an impairment of the efflux pump or to an increase in the permeability of the plasmamembrane facilitating the divalent cations to be accumulated into the vacuoles. Ethidium does not affect the initial rates of divalent cation uptake into the vacuoles, but it effectively reduces the ultimate accumulation of the divalent cations in the DEAE-dextran permeabilized cells, possibly by competing with the divalent cations for intravacuolar binding sites. Similar results are obtained for the accumulation of Ca2+. It is concluded that the efflux pump enables the yeast cell to regulate accumulation levels of the various divalent cations to different extents.

Derepression of the high-affinity phosphate uptake in the yeast Saccharomyces cerevisiae.

Phosphate starvation derepresses a high-affinity phosphate uptake system in Saccharomyces cerevisiae strain A294, while in the same time the low-affinity phosphate uptake system disappears. The protein synthesis inhibitor cycloheximide prevents the derepression, but has no effect as soon as the high-affinity system is fully derepressed. Two other protein synthesis inhibitors, lomofungin and 8-hydroxyquinoline, were found to interfere also with the low-affinity system and with Rb+ uptake. After incubation of the yeast cells in the presence of phosphate the high-affinity system is not derepressed, but the Vmax of the low-affinity system has decreased from about 35%. Phosphate supplement after derepression causes the high-affinity system to disappear to a certain extent while in the meantime the low-affinity system reappears. The results are compared with those found in the yeast Candida tropicalis for phosphate uptake.

Uptake and accumulation of Mn2+ and Sr2+ in Saccharomyces cerevisiae.

Initial uptake of Mn2+ and Sr2+ in the yeast Saccharomyces cerevisiae was studied in order to investigate the selectivity of the divalent cation uptake system and the possible involvement of the plasma-membrane ATPase in this uptake. The initial uptake rates of the two ions were not significantly different. This ruled out a direct role of the plasma-membrane ATPase, since this ATPase is specific for Mn2+ compared to Sr2+. After 1 h uptake, Mn2+ had accumulated 10-times more than Sr2+. Influx of Mn2+ and Sr2+ remained unchanged during that time, however. The differences in accumulation level found for Mn2+ and Sr2+ could be ascribed to a greater efflux of Sr2+ as compared with Mn2+. Probably this greater efflux of Sr2+ was only apparent, since differential extraction of the yeast cells revealed that Mn2+ is more compartmentalised than Sr2+, giving rise to a lower relative cytoplasmic Mn2+ concentration.

The role of hydrogen translocating shuttles during ethanol oxidation in hepatocytes from euthyroid and hyperthyroid rats.

A quantitative study of the contribution of the malate-aspartate and glycerol-3-phosphate cycles to the translocation of reducing equivalents from cytosol to mitochondria during ethanol oxidation has been made in hepatocytes from euthyroid and hyperthyroid rats. 1. In hepatocytes from euthyroid rats both cycles have an almost equal capacity and their relative contribution to total hydrogen transport to the mitochondria depends on the conditions chosen. 2. In hepatocytes from hyperthyroid rats maximal rates of ethanol oxidation were significantly lower than in hepatocytes from euthyroid rats, even though the capacity of the glycerol-3-phosphate cycle was increased. This was due to a decreased activity of alcohol dehydrogenase.