Screening and organizing a large collection of clones for those that contain specific sequences is central to many applications in molecular biology. Standard hybridization methods for E. coli, phage, and yeast are of wide spread utility but are not ideally suited for all applications. Although colony hybridization techniques have proven effective as a screening method for identifying YAC clones containing single-copy genes, methods to facilitate the systematic and rapid identification of overlapping contiguous clones containing human genomic sequences are required. To this end we will develop new yeast artificial chromosome cloning vectors that will allow the direct visual identification of contiguous sequences by a replica plating assay. The system involves the preparation of two YAC libraries in yeast strains of opposite mating types and fusion of these cells into diploids that contain contiguous YACs that can undergo intermolecular homologous meiotic recombination. To reduce the load of the YAC containing transformants that would be required for the mating screen, we will prepare the chromosome 11 specific library from FACS-sorted DNA preparations. In addition to developing a recombination method for identifying contiguous sequences, we propose to develop an E. coli-yeast transposon mutagenesis system to allow the manipulation of cloned mammalian DNA segments through DNA-mediated transformation. This approach will allow the rapid targeting of sequences such as cDNAs onto existing YACs for such purposes as mapping exons, determining gene orientation within a YAC, identifying homologues, identifying structural features like 5' ends, and the construction of restriction maps of large DNA segments.
