The centromeric regions of human chromosomes are dominated by a class of tandemly related DNA, alpha satellite. The alpha satellite DNA family consists of an extensive group of related, highly divergent repeats, based on a fundamental monomer repeat length of approximately 171 basepairs. Long tandem arrays of alpha satellite, estimated to be hundreds of thousands to millions of basepairs in length, are located at the centromeric region of each chromosome and are characterized by a multimeric higher-order repeat unit whose organization and sequence is specific for a particular chromosome. In total, the alpha satellite family comprises at least several percent of all human DNA and serves as a molecular model for an even larger proportion of the genome which consist of other families of tandemly related satellite DNA. Because conventional strategies for mapping human chromosomes cannot easily accommodate long megabase stretches of repetitive DNA, a novel and directed approach is proposed for the efficient, high-resolution physical and genetic mapping of the centromeric region of each human chromosome. The proposed studies are based on two well-established basic properties of centromeric DNA in the human genome: the previously documented chromosome-specificity of the alpha satellite DNA family as well as the high degree of Mendelianly inherited DNA polymorphism associated with this DNA. Specifically, it is proposed: (i) to isolate representative DNA probes for the alpha satellite subsets of all 24 types of human chromosome: (ii) to describe high frequency DNA polymorphisms for each of the alpha satellite probes for the development of a series of centromere-based genetic linkage maps of human chromosomes, as part of the international collaboration of the Centre d'Etude de Polymorphisme Humain; (iii) to develop long-range restriction maps of each centromeric region, using alpha satellite probes and pulsed-field gel electrophoresis and taking advantage of rare polymorphic restriction sites within each tandem array to construct high-density maps; (iv) to isolate, characterize, and sequence junction fragments at the edges of each alpha satellite array on both the proximal long and short arms of each chromosome, using chromosome """"""""hopping"""""""" techniques and cloning the ends of large alpha satellite-containing restriction fragments; and (v) to expand by chromosome walking and/or hopping these junction clones to extend the physical and genetic maps of each centromere approximately 100- 200 kb on each side of the alpha satellite array.
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