This is a first amendment of a renewal application to study the mating-type loci of Schizophyllum commune, a basidiomycete fungus. Sexual development is controlled by two linked pairs of loci mapping to two different chromosomes, Aalpha and Abeta and Balpha and Bbeta. Multiple alleles of each of the four loci are found in natural populations. Multinucleate heterokaryons which differ at Aalpha and/or Abeta and differ at Balpha and/or Bbeta are capable of producing fruiting bodies wherein meiotic spores are formed. From studies of heterokaryons which differ at only one of the A or B loci, two functionally equivalent pathways of sexual development are present; one controlled by either Aalpha or Abeta (the A pathway) and the other controlled by Balpha or Bbeta (the B pathway). Drs. Novotny and Ullrich's research centers on an understanding of the Aalpha and Abeta loci and the A pathway, which appear to be functionally distinct from the B loci. They have cloned and characterized 4 of the 9 known alleles of Aalpha. The locus encodes two genes, Y and Z, both are homeodomain proteins and the Y and Z proteins from the different alleles exhibit 49-54% and 40% identity, respectively. Transcripts of both genes are constitutively expressed in all cell types. Transformation studies using Y and Z genes from the different alleles indicates that the A pathway is activated by non-identical combinations of the two genes. They hypothesize that in non-self heterokaryons the Y protein from one allele interacts with the Z protein from the other allele and these heteromultimers act as transcriptional regulators of the developmental pathway genes. A model of this interaction is presented based on the results of their previous studies which emphasized a molecular genetic analysis of the system.
The Specific Aims of this proposal set out to explore the proposed model. Recently the Abeta6 locus was cloned. Two genes, V and W, have been identified and V protein shows no significant homology to the Aalpha Y protein except for the homeodomain region which is 41% identical. W is only partially sequenced. They plan to continue the molecular characterization of Abeta in this proposal and outline analyses similar to those carried out with the Aalpha locus. The Two-Hybrid System will be used to analyze the Y-Z protein-protein interactions using examples of both non-self and self combinations. Information from these studies will be used to guide in vitro mutagenesis studies of the interacting domains. The Two-Hybrid System will also be used to search for protein which interact with the postulated Y-Z complex. Biochemical analysis of purified epitope-tagged Y and Z proteins will be undertaken. Characteristics of the Y-Z interaction will be explored as well as other functional features of the proteins such as phosphorylation. Efforts to identify target genes controlled by the Y-Z complex will be initiated toward the end of the grant period.
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