Carlene A. Raper

Pheromones and Pheromone Receptors as Mating-Type Determinants in Basidiomycetes

Sexual variability within the fungi is extreme. For example, some fungi have only two mating types, while others have thousands. Is there a common genetic basis for these different mating systems? If so, how do the genes function to allow recognition of only a few, as contrasted to many thousands of potential mates?

Pulsed-field gel electrophoretic analysis of Schizophyllum commune chromosomal DNA

SummarySix chromosomal DNA bands of Schizophyllum commune have been resolved using transverse alternating field electrophoresis. The estimated sizes of the chromosomal DNAs ranged from 5.1 to 1.2 megabase pairs (Mb), the total genome size being approximately 35–36 Mb. Chromosomal length polymorphisms were found between the two S. commune isolates examined. The DNA bands corresponding to the two chromosomes containing the A and B mating-type loci were identified in hybridization experiments using probes specific to their respective linkage groups. The utility of eluted chromosomal DNAs as hybridization probes to select clones from genomic libraries, and the use of these clones in transformation experiments to identify genes of interest, are discussed.

B-MATING-TYPE GENES INFLUENCE SURVIVAL OF NUCLEI SEPARATED FROM HETEROKARYONS OF SCHIZOPHYLLUM

Abstract Separation of single haploid nuclei from dikaryons of Schizophyllum commune , via protoplast formation and regeneration, has revealed significant asymmetric ratios of the two component nuclear types despite the strict 1:1 ratio that prevails in these highly structured heterokaryons. Skewed ratios of one type of nucleus to the other were previously shown to be characteristic of hemicompatible heterokaryons in which the A -mating-type alleles were identical but the B -mating-type alleles were different. This study has determined a genetic basis for this phenomenon by demonstrating that alleles of the B -mating-type genes influence nuclear survival. Specificity of alleles at bothBα andBβ loci has been shown to correlate with percentage survival of the component nuclear types recovered from both kinds of heterokaryons, suggesting differential effects of these series of multiple alleles on nuclear function in monokaryons isolated from heterokaryons. The alleles can be put in an hierarchical order with respect to this function. Mutation to loss or impairment ofB function results in a shift in hierarchical order relative to the progenitor allele. Evidence is given for an effect on nuclear division as a basis for the differential recovery of nuclei carrying different B -gene alleles. A scheme with some analogy to the system for establishing mitotic synchrony in cells of opposite mating type in Saccharomyces cerevisae is suggested as a possible basis for the observed effects of the B -mating-type genes in basidiomycetes.

The mushroom-inducing gene Frt1 of Schizophyllum commune encodes a putative nucleotide-binding protein

Fruiting bodies (mushrooms) can be induced to form in unmated, normally non-fruiting strains of the basidiomycete fungus Schizophyllum commune by the ectopic genomic integration of a cloned gene called Frt1. Thus, the normal requirement of mating for mushroom formation is bypassed. Sequence analysis of genomic and cDNA clones revealed that the Frt1 gene encodes a predicted polypeptide of 192 amino acids, interrupted by three short introns. The FRT1 protein is predicted to be of Mr 21625 and does not have significant overall similarity to any known proteins. Analysis of the predicted amino acid sequence revealed the presence of a P-loop motif, a conserved sequence found in nucleotide-binding proteins. A potential site for Mg2+ binding is predicted to reside next to the P-loop at Thr24. The possible functional significance of these and other residues within FRT1 was examined using site-directed mutagenesis, followed by transformation of these mutant alleles of Frt1 back into S. commune. Mutation of the middle glycine of the P-loop completely abolished the fruit-inducing activity of cloned Frt1. Substitution of an alanine residue for Thr24 also resulted in mutant clones with no fruit-inducing activity. The possibility of an interaction between two closely spaced threonine residues within FRT1 was suggested by transformation experiments ultilizing mutant Frt1 alleles with specific combinations of mutations at these sites. Taken together, the results of our mutagenesis experiments suggest the possibility that activity of the predicted FRT1 protein could be altered by nucleotide binding and coordination of Mg2+. Northern blot hybridization experiments indicate that Frt1 activity is probably not controlled at the transcriptional level.Fruiting bodies (mushrooms) can be induced to form in unmated, normally non-fruiting strains of the basidiomycete fungus Schizophyllum commune by the ectopic genomic integration of a cloned gene called Frt1. Thus, the normal requirement of mating for mushroom formation is bypassed. Sequence analysis of genomic and cDNA clones revealed that the Frt1 gene encodes a predicted polypeptide of 192 amino acids, interrupted by three short introns. The FRT1 protein is predicted to be of Mr 21625 and does not have significant overall similarity to any known proteins. Analysis of the predicted amino acid sequence revealed the presence of a P-loop motif, a conserved sequence found in nucleotide-binding proteins. A potential site for Mg2+ binding is predicted to reside next to the P-loop at Thr24. The possible functional significance of these and other residues within FRT1 was examined using site-directed mutagenesis, followed by transformation of these mutant alleles of Frt1 back into S. commune. Mutation of the middle glycine of the P-loop completely abolished the fruit-inducing activity of cloned Frt1. Substitution of an alanine residue for Thr24 also resulted in mutant clones with no fruit-inducing activity. The possibility of an interaction between two closely spaced threonine residues within FRT1 was suggested by transformation experiments ultilizing mutant Frt1 alleles with specific combinations of mutations at these sites. Taken together, the results of our mutagenesis experiments suggest the possibility that activity of the predicted FRT1 protein could be altered by nucleotide binding and coordination of Mg2+. Northern blot hybridization experiments indicate that Frt1 activity is probably not controlled at the transcriptional level.

Identification of a polyadenylated transcript correlated with development in the sexual cycle of Schizophyllum

Abstract A polyadenylated transcript of about 580 nucleotides has been determined to occur preferentially in the dikaryon of Schizophyllum commune. It was apparent in blots of RNA from the vegetative dikaryon (sexually differentiated cells which are normally capable of developing into fruiting bodies) and not apparent in blots of RNA from the two monokaryotic homokaryons (sexually nondifferentiated cells) that were mated to obtain the dikaryon. It was detected by probing gel blots of poly(A)+ RNA from the dikaryon and from each of the two component unmated monokaryons simultaneously with a subset of cDNAs specific to the dikaryon. The “dikaryon-specific” cDNAs were obtained by isolating poly(A)+ RNA from the dikaryon prior to fruiting, preparing 32P-labeled cDNA from this RNA, and selecting those cDNA sequences that did not hybridize to the poly(A)+ RNA of the two component monokaryons. A probe consisting of dikaryotic cDNA that did hybridize to the poly(A)+ RNA of the monokaryons served as the control. A relatively small degree of gene regulation at the transcriptional level appears to be involved in differentiation of the dikaryon from the component monokaryons. A series of successive hybridizations (“cascade hybridization”) of 32P-labeled cDNA from the dikaryon with poly(A)+ RNA from the monokaryons revealed only 2% of the starting material as noncomplementary to the RNA of the monokaryons. This is in marked contrast to the high degree of transcriptional regulation observed for developmental processes in other fungi such as Aspergillus nidulans.

Why Study Schizophyllum

The process of mating, fertilization, fruiting, meiosis and spore formation is regulated by two kinds of genetic factors residing at the A and B mating-type loci, earlier called incompatibility factors A and B. Over the eight decades since Knieps discovery, revelations about the genetic, biochemical and molecular underpinnings of this bizarre system have made an exciting story (see list of selected references, below). While other interesting aspects of Schizophyllum have been explored, notably the hydrophobins of Wessels and associates (reviewed in W essels, 2000), a principal focus over the years has been on mating compatibility and sexual development. Although Schizophyllum commune s main role in nature is to recycle carbon by breaking down celluose and xylans in fallen wood (Clarke and Yaguchi, 1986; Bray and Clarke, 1995), it has been documented occasionally as a pathogen in fruit orchards (Latham, 1970; Oprea, et al, 1995) and also in immunologically compromised humans (Buzina et al, 2001).