The general objective of the proposed research is to develop efficient techniques for constructing and analyzing artificial chromosomes in yeast carrying contiguous mega-base segments of exogenous (human) DNA. These methods will be directly applicable to the other projects in this program and will be made generally available to other human chromosome mapping laboratories. In addition, the phenotypic consequences of specific chromosomal aneuploidies will be studied by taking advantage of experimental approaches that are available in the yeast, S. cerevisiae. Of essential importance to this research, we have developed a series of yeast transformation vectors and strains for the propagation of large, linear, nonessential artificial chromosomes in yeast. A combination of genetic and molecular approaches will be used to accomplish the following specific experimental aims: 1) To develop efficient techniques for analyzing large DNA segments on artificial chromosomes in yeast. Improved vectors will be constructed to facilitate cloning of megabase sized exogenous DNA and methods developed for screening recombinant clones and for mapping and manipulating these cloned sequences in yeast. 2) To construct a comprehensive human library. A comprehensive library of mega-base sized overlapping clones will be constructed in yeast. Multiple overlapping clones corresponding to large contiguous segments of DNA from the Xq24, 8q24, and 9q34 regions will be isolated in conjunction with other projects in this program. The ability to clone and manipulate mega-base sized segments of DNA in a microbial host will have a profound impact on the mapping and analysis of eukaryotic genomes. Moreover, analysis of the effects of chromosomal aneuploidies in yeast may be important to a basic understanding of factors that contribute to the phenotypic abnormalities associated with aneuploidies in general.
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