The long term goal of these studies is to use information derived from high resolution, long-range physical maps of selected regions of the human genome to understand the molecular basis of inherited single gene and chromosomal disorders and to help understand the functional organization of the human genome. The first part of this proposal involves the testing and implementation of two new approaches to facilitate long range physical mapping. The first technique involves the insertion of retroviral vectors with extremely rare (10 base and 12 base) restriction endonuclease recognition sites into human chromosomes which will then be used to develop physical maps of 5-10Mb in one step. The second new technique to be pursued in an E. coli-based cloning system which allows the cloning of very large (>150 Kb) exogenous DNA fragments in the bacterial host. These new approaches together with existing recombinant DNA and somatic cell genetic techniques will be used to develop high resolution, long range physical maps of two specific regions of the genome. The first area is 5q21-5q23, the region surrounding the gene for familial adenomatous polyposis of the colon (APC). Mutations in this locus result in predisposition to colon cancer and the disease is inherited as an autosomal dominant trait with close to 100% penetrance. The other region to be mapped, which will also serve as the system for testing the large fragment E. coli cloning vector, is the distal portion of the short arm of chromosome 4. This region contains several regions of medical interest, including the Huntingtons disease gene and the critical region for the 4p- or Wolf-Hirschhorn syndrome, as well as several other interesting features such as a large number of VNTR sequences; at least one recombination hot spot; and a number of HTF islands. In addition to answering specific questions about these two regions of the human genome, the approaches to be developed and used in this project should aid in mapping other portions of the genome.
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