The objective of the proposed research is to develop efficient procedures for making ordered recombinant DNA collections that represent complete fungal genomes. The clone collections to be generated will be of use for gene cloning and mapping projects and for studies of chromosomal structure, organization, and dynamics. The process of placing clones in order according to their chromosomal positions is referred to as """"""""in vitro reconstruction."""""""" Initially, the genomes of the genetic model organisms Neurospora crassa and Aspergillus nidulans will be reconstructed. After techniques have been optimized, they will be used as time permits to reconstruct the genomes medically or agriculturally important fungi, for example Candida albicans and Magnaporthe grisea. The proposed procedure will first involve construction of several cosmid clone banks in vectors with different properties. These will either be sorted into chromosome-specific subcollections by use of a colony hybridization technique or subcollections will be made directly from isolated chromosomes. Clones will be sorted into contiguous groups by using oligonucleotide probes to generate digital signatures for each clone and computer programs to determine clone relationships. Contiguous groups generated by this method will be connected by conventional chromosome walking procedures. The final contiguous groups will be oriented and aligned with the genetic maps of the organisms by hybridization with known gene probes or by DNA-mediated transformation of mutant strains.
