Genetic Analysis of Mating Type Differentiation in PARAMECIUM TETRAURELIA. II. Role of the Micronuclei in Mating-Type Determination

The two complementary mating types, O and E, of Paramecium tetraurelia are normally inherited cytoplasmically. This property has generally been interpreted to indicate the presence of cytoplasmic factors that determine macronuclear differentiation towards O or E. In these macronuclear-cytoplasmic interactions, the micronuclei were held to be unbiased and the determination to be established in the course of macronuclear development. In order to ascertain whether the micronuclei were actually neutral, amicronucleate clones were needed and a method to produce them was developed. In crosses between amicronucleate clones and normal micronucleate clones, we have observed regular deviations from cytoplasmic inheritance: the commonest deviation is that most O amicronucleate cells become E when they receive a micronucleus from an E partner. The data can be interpreted by assuming that the micronuclei are predetermined and that the apparent "cytoplasmic" inheritance of the two mating types is due, in E cells, to E-determining factors present in the cytoplasm and in the nucleus; and, in O cells, to O-determining factors present only or mainly in the nucleus.     

Genetic analysis of mating type differentiation in Paramecium tetraurelia. III. A mutation restricted to mating type E and affecting the determination of mating type

In P. tetraurelia each cell is determined to express only one of the two complementary mating types, O and E. This determination is under cytoplasmic control and seems to be achieved only by the commitment or noncommitment to the expression of mating type E. All the previously known mutations affecting the differentiation of mating type prevent the expression of the E mating type (O-restricted mutations) without affecting the determination process. An E-restricted mutation was obtained: mtF^E. Its phenotypic properties indicate that the mutation affects the determination process itself. When an O cell becomes mtF^E/mtF^E it acquires the E mating type and an E-determining cytoplasm. We propose that this constitutive determination for the E mating type is due to the inefficiency of a factor which is normally active in an O cell. This factor would act like a repressor and stabilize the E functions under an inactive state.     

Mutational Analysis of Mating Type Inheritance in Syngen 4 of PARAMECIUM AURELIA

Six genic mutations restricting clones to mating type VII (O) were isolated in syngen 4, Paramecium aurelia. The only three extensively tested were neither allelic nor closely linked. A second type of mutation, allelic to one of the O restricted mutants, was also found. Clones homozygous for this mutant gene were selfers, producing both O and E (VIII) mating types, but only when they were progeny of mating type E parental clones. While all seven mutant genes behaved as recessives in monohybrid crosses, clones heterozygous at two different loci often demonstrated an unanticipated phenotype: selfing. The significance of the findings is discussed in relation to mating type determination and the evolution of mating type systems.     

Mating Type Determination in Stock 51, Syngen 4 of Paramecium aurelia Grown in Axenic Culture*

SYNOPSIS. The use of axenic medium permits the study of mating type determination in stock 51 (sensitive) of syngen 4 of Paramecium aurelia. A high frequency of cytoplasmically bridged pairs was correlated with a high frequency of change of mating type following conjugation in axenic medium. The direction of change was predominantly from mating type VII to mating type VIII, suggesting a dominance of type VIII cytoplasm in the clones arising from a mixed cytoplasmic ancestry. No significant effect of either lower temperature or of N_aX₃ upon the pattern of mating type determination was found. The high frequency of cytoplasmic bridges between conjugants led to the formation of many double or higher multiplex clones.     

Mating-Type Inheritance and Maturity Times in Crosses between Subspecies of TETRAHYMENA PIGMENTOSA

Subspecies 6 and 8 of T. pigmentosa (formerly syngens 6 and 8 of T. pyriformis) share a mating-type system controlled by three alleles with "peck-order" dominance at a single locus. The system is apparently closed and limited to three mating types that are homologous, but not identical, in the subspecies. These relationships are reflected in new mating-type designations.—The viability in some intersyngenic crosses is excellent, and the inheritance of major mating types in first-generation hybrids and their progeny follows the pattern of subspecies 8.—The period of immaturity is shorter than that previously reported for subspecies 8, with 50% of the subclones maturing between 46 and 100 fissions after conjugation. Maturity curves are generally sigmoid, but some are apparently biphasic. The onset of maturity in triplicate sublines from the same synclone is usually highly correlated.     

A mutation with pleiotropic effects on macronuclearly differentiated functions in Paramecium tetraurelia

The mtF^E mutation isolated in Paramecium tetraurelia affects mating type differentiation, trichocyst excretion, and viability. Its effect on mating type has already been shown to correspond to a restriction to the E mating type interpreted by an inefficiency of nuclear O-determining factors. In this paper we study the other two phenotypic characteristics whose hereditary transmission displays two unusual features. (1) In crosses between a wild-type strain and the mutant strain, the mutant characteristics do not reappear in F2 in the wild-type cytoplasmic lineage but only in F3 after the homozygous clones have undergone an additional nuclear reorganization. (2) Some F2 wild-type clones, in the mutant cytoplasmic lineage, retain some of the phenotypic characteristics of the mutant. We propose that the mtF gene product plays a role in the control of several macronuclearly differentiated functions.     

Discovery and Functional Study of a Novel Genomic Locus Homologous to Bα-Mating-Type Sublocus of Lentinula edodes

The interaction of mating pheromone and pheromone receptor from the B mating-type locus is the first step in the activation of the mushroom mating signal transduction pathway. The B mating-type locus of Lentinula edodes is composed of Bα and Bβ subloci, each of which contains genes for mating pheromone and pheromone receptor. Allelic variations in both subloci generate multiple B mating-types through which L. edodes maintains genetic diversity. In addition to the B mating-type locus, our genomic sequence analysis revealed the presence of a novel chromosomal locus 43.3 kb away from the B mating-type locus, containing genes for a pair of mating pheromones (PHBN1 and PHBN2) and a pheromone receptor (RCBN). The new locus (Bα-N) was homologous to the Bα sublocus, but unlike the multiallelic Bα sublocus, it was highly conserved across the wild and cultivated strains. The interactions of RcbN with various mating pheromones from the B and Bα-N mating-type loci were investigated using yeast model that replaced endogenous yeast mating pheromone receptor STE2 with RCBN. The yeast mating signal transduction pathway was only activated in the presence of PHBN1 or PHBN2 in the RcbN producing yeast, indicating that RcbN interacts with self-pheromones (PHBN1 and PHBN2), not with pheromones from the B mating-type locus. The biological function of the Bα-N locus was suggested to control the expression of A mating-type genes, as evidenced by the increased expression of two A-genes HD1 and HD2 upon the treatment of synthetic PHBN1 and PHBN2 peptides to the monokaryotic strain of L. edodes.     

The Search for Mating-Type-Limited Genes in the Homothallic Alga CHLAMYDOMONAS MONOICA

The non-Mendelian erythromycin resistance mutation ery-u1 shows bidirectional uniparental inheritance in crosses between homothallic ery-u1 and ery-u1⁺ strains of Chlamydomonas monoica . This inheritance pattern supports a general model for homothallism invoking intrastrain differentiation into opposite compatible mating types and, further, suggests that non-Mendelian inheritance is under mating-type (mt) control in C. monoica as in heterothallic species. However, the identification of genes expressed or required by one gametic cell type, but not the other, is essential to verify the existence of a regulatory mating-type locus in C. monoica and to understand its role in cell differentiation and sexual development. By screening for a shift from bidirectional to unidirectional transmission of the non-Mendelian ery-u1 marker, a mutant with an apparent mating-type-limited sexual cycle defect was obtained. The responsible mutation, mtl-1, causes a 1000-fold reduction in zygospore germination in populations homozygous for the mutant allele and, approximately, a 50% reduction in germination for heterozygous (mtl-1/mtl-1 ⁺) zygospores. By next screening for strains unable to yield any viable zygospores in a cross to mtl-1, a second putative mating-type-limited mutant, mtl-2, was obtained. The mtl-2 strain, although self-sterile, mates efficiently with mtl-2⁺ strains and shows a unidirectional uniparental pattern of inheritance for the ery-u1 cytoplasmic marker, similar to that observed for crosses involving mtl-1. Genetic analysis indicates that mtl-1 and mtl-2 define unique unlinked Mendelian loci and that the sexual cycle defects of reduced germination (mtl-1) or self-sterility (mtl-2) cosegregate with the effect on ery-u1 cytoplasmic gene transmission. By analogy to C. reinhardtii, the mtl-1 and mtl-2 phenotypes can be explained if the expression of these gene loci is limited to the mt⁺ gametic cell type, or if the wild-type alleles at these loci are required for the normal formation and/or functioning of mt ⁺ gametes only.     

Mating-type locus of Cryptococcus neoformans: a step in the evolution of sex chromosomes

The sexual development and virulence of the fungal pathogen Cryptococcus neoformans is controlled by a bipolar mating system determined by a single locus that exists in two alleles, α and a. The α and a mating-type alleles from two divergent varieties were cloned and sequenced. The C. neoformans mating-type locus is unique, spans >100 kb, and contains more than 20 genes. MAT-encoded products include homologs of regulators of sexual development in other fungi, pheromone and pheromone receptors, divergent components of a MAP kinase cascade, and other proteins with no obvious function in mating. The α and a alleles of the mating-type locus have extensively rearranged during evolution and strain divergence but are stable during genetic crosses and in the population. The C. neoformans mating-type locus is strikingly different from the other known fungal mating-type loci, sharing features with the self-incompatibility systems and sex chromosomes of algae, plants, and animals. Our study establishes a new paradigm for mating-type loci in fungi with implications for the evolution of cell identity and self/nonself recognition.     

Identification of the Mating-Type (MAT) Locus That Controls Sexual Reproduction of Blastomyces dermatitidis

Blastomyces dermatitidis is a dimorphic fungal pathogen that primarily causes blastomycosis in the midwestern and northern United States and Canada. While the genes controlling sexual development have been known for a long time, the genes controlling sexual reproduction of B. dermatitidis (teleomorph, Ajellomyces dermatitidis) are unknown. We identified the mating-type (MAT) locus in the B. dermatitidis genome by comparative genomic approaches. The B. dermatitidis MAT locus resembles those of other dimorphic fungi, containing either an alpha-box (MAT1-1) or an HMG domain (MAT1-2) gene linked to the APN2, SLA2, and COX13 genes. However, in some strains of B. dermatitidis, the MAT locus harbors transposable elements (TEs) that make it unusually large compared to the MAT locus of other dimorphic fungi. Based on the MAT locus sequences of B. dermatitidis, we designed specific primers for PCR determination of the mating type. Two B. dermatitidis isolates of opposite mating types were cocultured on mating medium. Immature sexual structures were observed starting at 3 weeks of coculture, with coiled-hyphae-containing cleistothecia developing over the next 3 to 6 weeks. Genetic recombination was detected in potential progeny by mating-type determination, PCR-restriction fragment length polymorphism (PCR-RFLP), and random amplification of polymorphic DNA (RAPD) analyses, suggesting that a meiotic sexual cycle might have been completed. The F1 progeny were sexually fertile when tested with strains of the opposite mating type. Our studies provide a model for the evolution of the MAT locus in the dimorphic and closely related fungi and open the door to classic genetic analysis and studies on the possible roles of mating and mating type in infection and virulence.     

Vegetative Incompatibility and the Mating-Type Locus in the Cellular Slime Mold DICTYOSTELIUM DISCOIDEUM

The genetic basis of vegetative incompatibility in the cellular slime mold, Dictyostelium discoideum, is elucidated. Vegetatively compatible haploid strains from parasexual diploids at a frequency of between 10^-6 and 10^-5, whereas "escaped" diploids are formed between vegetatively incompatible strains at a frequency of ~10^-8. There is probably only a single vegetative incompatibility site, which appears to be located at, or closely linked to, the mating-type locus. The nature of the vegetative incompatibility is deduced from parasexual diploid formation between wild isolates and tester strains of each mating type, examination of the frequency of formation of "escaped" diploids formed between vegetatively incompatible strains, and examination of the mating type and vegetative incompatibility of haploid segregants obtained from "escaped" diploids.     

A Study of Odd Mating-Type Determination Factor by Transfer of Macronuclear Karyoplasm in PARAMECIUM TETRAURELIA

The unique feature of the "B system" of mating-type determination found in Paramecium tetraurelia is the existence of a cytoplasmic difference between odd (O) and even (E) cells created and maintained by the action of their macronuclei. Thus far, the presence of a determining factor that controls the differentiation of the developing zygotic macronucleus for O mating type has not been verified. Results of crosses between cells of differing clonal age and complementary mating type suggest that, for one to two fissions after autogamy, O cells produce some factor that determines the gametic nucleus (micronucleus) as mating type O. Direct evidence for the production of O-determining factor by the young O macronucleus was obtained by transplanting young O macronuclear karyoplasm (a part of the macronucleus) into E cells: 32-35% of E exautogamous clones transformed to O; transformation of E exautogamous clones to O reached as high as 72% by transfer of young O macronuclear karyoplasm from a conjugant, 3-4 hr after mixing. This indicates that O determinants produced by the O macronucleus can also act during the sensitive period of development of the new macronucleus. These O-determining factors may be produced or activated at the sexual stage and then decrease in activity in subsequent fissions after new macronuclear reorganization.     

Alternative Phenotypic States in Genomically Identical Cells: Interstock Genetics of a Trichocyst Phenotype in PARAMECIUM TETRAURELIA

The trichocysts of most wild stocks of Paramecium tetraurelia discharge en masse in response to picric acid. In most nonresponding wild stocks, the defective phenotype is simply determined by a single recessive gene difference from the standard wild type, stock 51. However, two wild stocks, 146 and 148, which are completely homozygous at all loci, express either a nondischarge, ND, or discharge, DI, phenotype. In stock 146, both ND and DI sublines generally reproduce true to type, but observed changes are highly biased. Changes from ND to DI occur more than ten times as often as changes from DI to ND. After conjugation between ND and DI cells, genomically identical exconjugant lines from the ND parent may be either ND or DI, while those from the DI parent invariably remain DI.—Interstock crosses between stocks 146 and 51 indicate that stock 146 possesses a recessive gene, nd146, which, when homozygous in stock 51 background, produces a distinct nondischarge phenotype, KO. Crosses between stock 146 and KO phenotype nd146 homozygotes in stock 51 background demonstrate that stock 146 possesses a dominant gene, M-nd146, which modifies the defect of nd146 homozygotes, resulting in either the ND or DI phenotype. The two loci, M-nd146 and nd146, are linked and estimated to be 5.3 centiMorgans apart. Stock 148 has the same alleles as stock 146 at these loci.—Presumably M-nd146 is involved in the dual phenotypic states in stock 146, but M-nd146 nd146 homozygotes backcrossed into stock 51 are invariably discharging. The possibility that the original ND state is independent of these genes is discussed and is regarded as unlikely. The phenotypic and genetic relationship discovered in these stocks should remind population biologists that phenotypic and genotypic variability do not always have a simple relationship. The nature and frequency of epistasis in the highly inbreeding P. tetraurelia are reviewed.     

Mating Type and Sporulation in Yeast I. Mutations Which Alter Mating-Type Control over Sporulation

In Saccharomyces cerevisiae, meiosis and spore formation as well as mating are controlled by mating-type genes. Diploids heterozygous for mating type (aα) can sporulate but cannot mate; homozygous aa and αα diploids can mate, but cannot sporulate. From an αα diploid parental strain, we have isolated mutants which have gained the ability to sporulate. Those mutants which continue to mate as αα cells have been designated CSP (control of sporulation). Upon sporulation, CSP mutants yield asci containing 4α spores. The mutant gene which allows αα cells to sporulate is unlinked to the mating-type locus and also acts to permit sporulation in aa diploid cells. Segregation data from crosses between mutant αα and wild-type aa diploids and vice versa indicate (for all but one mutant) that the mutation which allows constitutive sporulation (CSP) is dominant over the wild-type allele. Some of the CSP mutants are temperature-sensitive, sporulating at 32°, but not at 23°. In addition to CSP mutants, our mutagenesis and screening procedure led to the isolation of mutants which sporulate by virtue of a change in the mating-type locus itself, resulting in loss of ability to mate.     

Regulation of Mating and Meiosis in Yeast by the Mating-Type Region

A supposed sporulation-deficient mutation of Saccharomyces cerevisiae is found to affect mating in haploids and in diploids, and to be inseparable from the mating-type locus by recombination. The mutation is regarded as a defective a allele and is designated a*. This is confirmed by its dominance relations in diploids, triploids, and tetraploids. Tetrad analysis of tetraploids and of their sporulating diploid progeny suggests the existence of an additional locus, RME, which regulates sporulation in yeast strains that can mate. Thus the recessive homozygous constitution rme/rme enables the diploids a*/α, a/a*, and α/α to go through meiosis. Haploids carrying rme show apparent premeiotic DNA replication in sporulation conditions. This new regulatory locus is linked to the centromere of the mating-type chromosome, and its two alleles, rme and RME, are found among standard laboratory strains.     

A Mendelian Mutation Affecting Mating-Type Determination Also Affects Developmental Genomic Rearrangements in Paramecium Tetraurelia

In Paramecium tetraurelia, mating type is determined during the differentiation of the somatic macronucleus from a zygotic nucleus genetically competent for both types, O and E. Determination of the developing macronucleus is controlled by the parental macronucleus through an unknown mechanism resulting in the maternal inheritance of mating types. The pleiotropic mutation mtF(E) affects macronuclear differentiation. Determination for E is constitutive in mutant homozygotes; a number of unrelated mutant characters are also acquired during development. We have examined the possibility that the mutation causes a defect in the developmental rearrangements of the germ-line genome. We show that the excision of an IES (internal eliminated sequence) interrupting the coding sequence of a surface antigen gene is impaired in the mutant, resulting in an alternative macronuclear version of the gene. Once established, the excision defect is indefinitely transmitted across sexual generations in the cytoplasmic lineage, even in a wild-type genetic context. Thus, the processes of mating-type determination and excision of this IES, in addition to their common sensitivity to the mtF(E) mutation, show a similar maternal inheritance of developmental alternatives in wild-type cells, suggesting a molecular model for mating-type determination.