IDENTIFYING YAC CONTIGS BY RECOMBINATION

The specific aim of this proposal is to develop a method for using genetic recombination to identify overlapping fragments cloned in YAC vectors. A yeast artificial chromosome (YAC) vector will be designed that can be used to construct libraries that will complement previously existing YAC libraries (made, for example, in YAC4). The second library using this new vector will be considered in a yeast strain of opposite mating type to the first. This will permit the formation of hybrid diploids between the two libraries. The new vector will be designed to permit the selection and counterselection of reciprocally recombined YAC chromosomes present only in those diploids that contain homologous overlapping sequences. The test for overlapping sequence homology will be genetic recombination. Genetic markers in the two vectors will be arranged so that homologous recombination between the two will result in one stable selected chromosome and one unstable counter-selected one. Using this strategy: (1) libraries of Herpesvirus (HV) DNA will be constructed in the new vector as well as YAC4, (2) the minimal length necessary for a recombination signal using spontaneous mitotic recombination, induced mitotic recombination and meiotic recombination will be determined, (3) libraries of Schizosaccharomyces pombe will be constructed in the two complementing vectors and methods will be developed for multiplexing to order both libraries, and (4) the largest YAC possible in yeast will be determined. If successful, this technology will be eventually applied to organisms with larger genomes such as Drosophila, plants and mammals. The overall goal of this project is to facilitate the ordering of cloned genomic fragments for the eventual complete analysis of any large genome(including physical mapping and sequencing).