B Mating-Type Specificities and Pheromones in Schizophyllum

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9513513 Raper The aim of this research is to understand how a simple eukaryote distinguishes itself from many nonselves in regulating mating and development. The specific objective is to clarify the mechanism by which the multiple B mating-type genes trigger sexual development in the mushroom Schizophyllum commune. The B genes are in two linked loci, B( and B(, each of which has nine different versions, or specificities, in the world-wide population. A mating pair must be different at either B( or B( to be compatible: any of the 36 possible pairings of different specificities at either locus results in initiation of the identical developmental pathway. How does this occur? Recent analysis of one specificity at each locus, B(l and B(1, has revealed genes for pheromones and pheromone receptors similar to those found in other fungi. All previously described fungal systems, however, have only two different versions of such genes. In S. commune, there are multiple versions. A model to explain multiple B mating types in S. commune is that each specificity at each locus is defined by a unique combination of pheromone and pheromone receptor genes. These genes have coevolved so as to insure that no combination of genes contained within the B mating-type locus of a single specificity encodes a pair of molecules capable of activating sexual development in self; only molecules encoded by different specificities of that locus can interact to activate the pathway. It is proposed that the basis for multiple B mating-types in S. commune is a unique system of multiple pheromone and pheromone receptors which act as master regulators of sexual morphogenesis. The focus of this project is to test this model by defining the functional genetic components of the B(3 and B(2 loci and comparing these with the genes of the B(l and B(1 loci which are currently being studied in collaboration with two European laboratories. Genes of the wild-type B(3 and B(2 loci will also be compared to a series of existing mutated genes within these loci that cause a variety of defective phenotypes. These comparisons will begin to explain how specificity of function is determined by the genes of each locus, and why certain pairings of these genes activate development while others do not. The research is divided into three parts: 1) Determine the basis for specificity of function among wild-type alleles by comparing differences and similarities between genetic elements that define B(3 and B(2 versus B(l and B(1. 2) Gain understanding of how the B mating-type genes determine specificity for self/nonself recognition by defining a mutation within the B(2 locus that destroys this function. 3) Determine specific functions of the multiple genes within the B(2 locus by correlating a series of mutations with changes in phenotype. Also define predicted deletions within the mutants for use as recipients in transformation experiments in order to test the effect of various combinat1ons of wild-type and mutated B mating-type genes. The pattern of expression of selected B mating-type genes will be examined for evidence of regulation with respect to development. This research will be accomplished by using standard techniques of DNA cloning, sequencing, sequence analysis, nucleic acid hybridization, PCR amplification, and DNA-mediated transformation of S. commune. %%% The long-term goal of this research is an understanding of how multiple pheromones and pheromone receptors regulate mating and cell-to-cell communication during development simple multicellular eukaryote. The organism being studied is the woodrotting Basidiomycete Schlzophyllum commune. This fungus, like other members of its class, has thousands of mating types. The investigator believes that the underlying mechanisms by which a haploid individual can distinguish self from many nonselves in regulating sexual development is fundamental relevance to an understanding of multicellular development in eukaryotes generally. S.commune is an ideal model system for this study because of the relative ease with which it can be manipulated in the laboratory and the substantial background information available for molecular genetic studies. ***