A sex recognition glycoprotein is encoded by the plus mating-type gene fus1 of Chlamydomonas reinhardtii.

Sexual fusion between plus and minus gametes of the unicellular green alga Chlamydomonas reinhardtii entails adhesion between plus-specific and minus-specific "fringe" proteins displayed on the plasma membrane of gametic mating structures. We report the identification of the gene (fus1) encoding the plus fringe glycoprotein, which resides in a unique domain of the mating-type plus (mt+) locus, and which was identified by transposon insertions in three fusion-defective mutant strains. Transformation with fus1+ restores fringe and fusion competence to these mutants and to the pseudo-plus mutant imp11 mt-, defective in minus differentiation. The fus1 gene is remarkable in lacking the codon bias found in all other nuclear genes of C. reinhardtii.     

The iso1 gene of Chlamydomonas is involved in sex determination.

Sexual differentiation in the heterothallic alga Chlamydomonas reinhardtii is controlled by two mating-type loci, mt+ and mt-, which behave as a pair of alleles but contain different DNA sequences. A mutation in the mt minus-linked imp11 gene has been shown previously to convert a minus gamete into a pseudo-plus gamete that expresses all the plus gametic traits except the few encoded by the mt+ locus. Here we describe the iso1 mutation which is unlinked to the mt- locus but is expressed only in minus gametes (sex-limited expression). A population of minus gametes carrying the iso1 mutation behaves as a mixture of minus and pseudo-plus gametes: the gametes isoagglutinate but they do not fuse to form zygotes. Further analysis reveals that individual gametes express either plus or minus traits: a given cell displays one type of agglutinin (flagellar glycoprotein used for sexual adhesion) and one type of mating structure. The iso1 mutation identifies a gene unlinked to the mating-type locus that is involved in sex determination and the repression of plus-specific genes.     

Sex determination in Chlamydomonas

The sex-determination system of the unicellular green alga, Chlamydomonas reinhardtii, is governed by genes in the mating-type (MT) locus and entails additional genes located in autosomes. Gene expression is initiated by nitrogen starvation, and cells differentiate into plus or minus gametes within 6h. Reviewed is our current understanding of gametic differentiation and fertilization, initiation of zygote development, and the uniparental inheritance of organelle genomes.     

Genetic structure of the mating-type locus of Chlamydomonas reinhardtii.

Portions of the cloned mating-type (MT) loci (mt(+) and mt(-)) of Chlamydomonas reinhardtii, defined as the approximately 1-Mb domains of linkage group VI that are under recombinational suppression, were subjected to Northern analysis to elucidate their coding capacity. The four central rearranged segments of the loci were found to contain both housekeeping genes (expressed during several life-cycle stages) and mating-related genes, while the sequences unique to mt(+) or mt(-) carried genes expressed only in the gametic or zygotic phases of the life cycle. One of these genes, Mtd1, is a candidate participant in gametic cell fusion; two others, Mta1 and Ezy2, are candidate participants in the uniparental inheritance of chloroplast DNA. The identified housekeeping genes include Pdk, encoding pyruvate dehydrogenase kinase, and GdcH, encoding glycine decarboxylase complex subunit H. Unusual genetic configurations include three genes whose sequences overlap, one gene that has inserted into the coding region of another, several genes that have been inactivated by rearrangements in the region, and genes that have undergone tandem duplication. This report extends our original conclusion that the MT locus has incurred high levels of mutational change.     

Evolution of the gene encoding mitochondrial intermediate peptidase and its cosegregation with the A mating-type locus of mushroom fungi

The high level of DNA polymorphism at the mating-type loci of mushroom fungi has made the cloning of mating-type genes difficult. As an alternative to strategies that employ sequence conservation, an approach utilizing conserved gene order could facilitate the cloning of A mating-type genes from mushroom fungi. It has been shown that a gene encoding a mitochondrial intermediate peptidase (MIP) is very close ( < 1 kbp) to the A mating-type locus of two model mushroom species. In this study, the cosegregation of MIP and the A mating-type locus was studied by genotyping progeny of seven additional mushroom species using PCR and genetic crosses. No evidence of any recombination between MIP and the A mating-type locus was detected among all seven species. Phylogenetic analysis of MIP sequences from diverse mushroom species agrees with the current organismal phylogeny, suggesting the sequences are generally orthologous.     

Activation for cell fusion in Chlamydomonas: analysis of wild-type gametes and nonfusing mutants

Gametes of Chlamydomonas reinhardi become activated for cell fusion as the consequence of sexual adhesion between membranes of mating-type plus and minus flagella. By using tannic acid plus en bloc uranyl acetate staining, and by fixing at very early stages in the mating reaction, we have demonstrated the following. (a) Activation of the minus mating structure entails major modifications in the structure of the organelle, causing it to double in size and to concentrate surface coat material, termed fringe, into a central zone. (b) The unactivated plus mating structure is endowed with fringe that moves with the tip of the actin-filled fertilization tubule during activation. Pre-fusion images suggest the occurrence of a specific recognition event between the plus and minus fringes. (c) Gametes carrying the imp-1 mutation fail to form a fringe and are unable to fuse. The imp-1 mutation is linked to the mating-type plus (mt+) locus, suggesting that the gene specifying the synthesis or insertion of fringe is encoded in this sector of the genome. (d) Gametes carrying the imp-11 mutation fail to form both a normal fringe and a normal submembranous density beneath the fringe, and are also unable to fuse. The imp-11 mutation converted a wild-type minus cell into a pseudo-plus strain; a model to explain this conversion proposes that the normal imp-11 gene product represses plus-specific genes concerned with Chlamydomonas gametogenesis.     

Molecular markers for rapidly identifying candidate genes in Chlamydomonas reinhardtii. Ery1 and ery2 encode chloroplast ribosomal proteins

To take advantage of available expressed sequence tags and genomic sequence, we have developed 64 PCR-based molecular markers in Chlamydomonas reinhardtii that map to the 17 linkage groups. These markers will allow the rapid association of a candidate gene sequence with previously identified mutations. As proof of principle, we have identified the genes encoded by the ERY1 and ERY2 loci. Mendelian mutations that confer resistance to erythromycin define three unlinked nuclear loci in C. reinhardtii. Candidate genes ribosomal protein L4 (RPL4) and L22 (RPL22) are tightly linked to the ERY1 locus and ERY2 locus, respectively. Genomic DNA for RPL4 from wild type and five mutant ery1 alleles was amplified and sequenced and three different point mutations were found. Two different glycine residues (G(102) and G(112)) are replaced by aspartic acid and both are in the unstructured region of RPL4 that lines the peptide exit tunnel of the chloroplast ribosome. The other two alleles change a splice site acceptor site. Genomic DNA for RPL22 from wild type and three mutant ery2 alleles was amplified and sequenced and revealed three different point mutations. Two alleles have premature stop codons and one allele changes a splice site acceptor site.     

Mapping of the Homothallic Genes, HMα and HMa, in Saccharomyces Yeasts

Two of the three homothallic genes, HM alpha and HMa, showed direct linkage to the mating-type locus at approximately 73 and 98 strans (57 and 65 centimorgans [cM], respectively, whereas, the other, HO, showed no linkage to 25 standard markers distributed over 17 chromosomes including the mating-type locus. To determine whether the HM alpha and HMa loci located on the left or right side of the mating-type locus, equations for three factor analysis of three linked genes were derived. Tetrad data were collected and were compared with expected values by chi 2 statistics. Calculations indicated that the HM alpha gene is probably located on the right arm at 95 strans (65 cM) from the centromere and the HMa locus at approximately 90 strans (64 cM) on the left arm of chromosome III.     

The homeobox genes of <Emphasis Type="Italic">Encephalitozoon cuniculi</Emphasis> (Microsporidia) reveal a putative mating-type locus

Homeobox genes have been found in animals, fungi, and plants. Recently, the complete genomic sequence of Encephalitozoon cuniculi has become available and it was shown that this Microsporida species is related to fungi. Given this close relatedness to fungi, I have searched the genome of E. cuniculifor homeobox genes. There are 12 homeobox genes as well as one STE12 orthologue. The large number of homeobox genes when compared to the annotations, which only list one, suggests that possibly other smaller genes may have been overlooked in the published analysis and E. cuniculi may have more than 1,997 genes. Sequence and phylogenetic analyses show that the E. cuniculi homeobox genes also fall into two distinct groups, with two TALE and ten typical homeobox genes being present. Like in the mating-type locus of yeast and other fungi, one TALE and one typical homeobox are found in close proximity of each other on the chromosome, suggesting that Microsporidia also contain a mating-type locus.     

Localization of the Nic-7, Ac-29 and THI-10 Genes within the Mating-Type Locus of Chlamydomonas Reinhardtii

The tight linkage observed between the mating-type (mt) locus of Chlamydomonas reinhardtii and three auxotrophic mutations--nic-7 (nicotinamide-requiring), ac-29 (acetate-requiring), and thi-10 (thiamine-requiring)--has led to the hypothesis that recombination is suppressed in the mt region. The physical location of these three genes has been established by transformation with sets of cloned DNA from the mt region. They lie to the left and right of the highly rearranged (R) domain of the mt locus, which has been proposed to be responsible for the recombinational suppression in the region. The cloned nic-7(+) and thi-10(+) genes will be useful as selectable markers for cotransformation experiments.     

The genetic structure of the A mating-type locus of Lentinula edodes

The Shiitake mushroom, Lentinula edodes (Berk.) Pegler is a tetrapolar basidiomycete with two unlinked mating-type loci, commonly called the A and B loci. Identifying the mating-types in shiitake is important for enhancing the breeding and cultivation of this economically-important edible mushroom. Here, we identified the A mating-type locus from the first draft genome sequence of L. edodes and characterized multiple alleles from different monokaryotic strains. Two intron-length polymorphism markers were developed to facilitate rapid molecular determination of A mating-type. L. edodes sequences were compared with those of known tetrapolar and bipolar basidiomycete species. The A mating-type genes are conserved at the homeodomain region across the order Agaricales. However, we observed unique genomic organization of the locus in L. edodes which exhibits atypical gene order and multiple repetitive elements around its A locus. To our knowledge, this is the first known exception among Homobasidiomycetes, in which the mitochondrial intermediate peptidase (mip) gene is not closely linked to A locus.     

Discovery of a modified tetrapolar sexual cycle in Cryptococcus amylolentus and the evolution of MAT in the Cryptococcus species complex

Sexual reproduction in fungi is governed by a specialized genomic region called the mating-type locus (MAT). The human fungal pathogenic and basidiomycetous yeast Cryptococcus neoformans has evolved a bipolar mating system (a, α) in which the MAT locus is unusually large (>100 kb) and encodes >20 genes including homeodomain (HD) and pheromone/receptor (P/R) genes. To understand how this unique bipolar mating system evolved, we investigated MAT in the closely related species Tsuchiyaea wingfieldii and Cryptococcus amylolentus and discovered two physically unlinked loci encoding the HD and P/R genes. Interestingly, the HD (B) locus sex-specific region is restricted (～2 kb) and encodes two linked and divergently oriented homeodomain genes in contrast to the solo HD genes (SXI1α, SXI2a) of C. neoformans and Cryptococcus gattii. The P/R (A) locus contains the pheromone and pheromone receptor genes but has expanded considerably compared to other outgroup species (Cryptococcus heveanensis) and is linked to many of the genes also found in the MAT locus of the pathogenic Cryptococcus species. Our discovery of a heterothallic sexual cycle for C. amylolentus allowed us to establish the biological roles of the sex-determining regions. Matings between two strains of opposite mating-types (A1B1×A2B2) produced dikaryotic hyphae with fused clamp connections, basidia, and basidiospores. Genotyping progeny using markers linked and unlinked to MAT revealed that meiosis and uniparental mitochondrial inheritance occur during the sexual cycle of C. amylolentus. The sexual cycle is tetrapolar and produces fertile progeny of four mating-types (A1B1, A1B2, A2B1, and A2B2), but a high proportion of progeny are infertile, and fertility is biased towards one parental mating-type (A1B1). Our studies reveal insights into the plasticity and transitions in both mechanisms of sex determination (bipolar versus tetrapolar) and sexual reproduction (outcrossing versus inbreeding) with implications for similar evolutionary transitions and processes in fungi, plants, and animals.