Research Project
6358177
DESCRIPTION (provided by applicant): The long-term objective of the proposed research is to gain an understanding of the genetic elements controlling mushroom development, which are largely uncharacterized to date. In addition to their value as an agricultural crop, mushrooms are the source of many medicinally active compounds that inhibit tumors and microbial growth. The gene FRT1 of the model basidiomycete fungus Schizophyllum commune is able to induce the production of mushrooms (fruiting) when integrated into certain haploid homokaryotic transformation recipients. This inappropriate induction of fruiting is dependent upon the FRT1 transgene being of a different type or "idiomorph" than the resident version of this gene, suggesting a self/non-self interaction. Only one version of FRT1 has been examined to date. Specific aim 1 will focus on the isolation and characterization of an alternate version of FRT1. A delta-frtl null strain will be transformed by DNA from a "non-self" genomic library, looking for complementation. Comparative sequence analysis of two versions of FRTJ will guide mutagenesis experiments, exploring structure-function relationships and idiomorph specificity. It is hypothesized that fruiting in transgenic strains occurs in part because of Frtlp heterodimer formation, releasing an activator protein that would otherwise interact with monomeric Frtlp. Specific aim 2 will explore this possibility, using a directed yeast two-hybrid approach. Potential sites for interaction will be mapped by deletion analysis and site-directed mutagenesis. Specificity regions will be defined by testing of chimeric FRTJ constructs in S. commune. The goal of specific aim 3 is to isolate and characterize other genes likely to be critical to fruiting. First, FRT1 will be used as bait in two-hybrid screens of commune cDNA libraries to isolate genes encoding proteins interacting with Frtlp. Second, a delta-frtl null strain will be subjected to REMI (Restriction Enzyme Mediated Integration) mutagenesis to generate tagged mutants that suppress the null phenotype. Rescued flanking DNA will be used as hybridization probes to isolate wild-type genomic clones of the tagged genes.
