The ability to isolate and maintain large fragments of DNA as artificial chromosomes in yeast (YACs) has ushered in a new era in the analysis of large genomes. Advances in this technology will be critical to the accomplishment of the goals put forth for the Human Genome Initiative. Improvements in the areas of generation and analysis of the libraries of human DNA will greatly facilitate progress towards meeting the goal of a contiguous ordered array of clones representing the entire human genome, the starting point for sequencing the genome. This proposal takes advantage of several useful features of yeast biology to advance these goals, in particular the extremely efficient pathway for homologous recombination. We propose to develop a set of YAC vectors that will allow several different types of genetic manipulation of YACs including chromosome walking using a genetic strategy. Walking will be accomplished by mating of a given YAC clone in yeast to a library of YACs in yeast made with suitable genetically marked vectors. Colonies containing pairs of YACs will be tested for the ability to undergo homologous recombination, allowing determination of those YACs containing overlapping stretches of genomic DNA. A novel in vivo method for the directed cloning of particular defined segments of genomic DNA will be explored. This strategy called """"""""DNA capture"""""""" utilizes the ability of yeast to gap repair DNA between two distantly located but genetically linked probes using total uncut genomic DNA as its repair template. The probes, attached to YAC vectors, are introduced into the yeast cells along with uncut chromosomal DNA via transformation, followed by selection for stable inheritance of the YAC markers. This strategy will also be used to explore the non-enzymatic production of genomic YAC libraries by using repetitive DNA sequences instead of unique sequences as probes for the DNA capture. A strategy for the genetic identification of YACs homologous to unique non-repetitive probes win be developed. This method will utilize homologous recombination enhanced by induced double-stranded breaks in DNA to activate expression of a defective gene on one arm of a YAC clone. A variation of this technique, """""""" cDNA capture"""""""", can also be employed to identify all cDNAs in a library that are homologous to a particular YAC, a need that will arise later in the development of the Human Genome Initiative. The use of cre-lox-mediated site-specific recombination in yeast will be investigated for two purposes. First, efficient removal of CENs and selectable markers from YACs will be explored to facilitate the genetic selections for homologous recombination designed earlier in this proposal. Ibis will be entail developing a negative selection against lox sites. Secondly, circularization of YACs via site-specific recombination will be examined as a tool to alter the topological state of YACs to facilitate purification of YAC DNA away from yeast chromosomal DNA. Circularization of YACs will also be used to generate molecules that can successfully replicate in E. coli, a second method facilitating the isolation of YAC DNA.
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