The long-term objective of this project is to achieve and verify telomeric closure of physical maps of human DNA. The cloning, mapping, and eventual sequencing of these distal chromosome regions is crucial for a complete understanding of human genetics; particular subtelomeric regions have been shown to contain important genes causing human diseases, the potential biological functions of subtelomeric DNA relative to telomere loss during aging and cancer have yet to be ascertained, and an analysis of the distribution of subtelomere-derived low-copy repeat sequences amongst individual humans and across primate species may reveal important clues with respect to human chromosome evolution and primate speciation. Analysis of a library of half-YAC clones has shown that most telomeric regions are represented in this clone collection, and it has been used successfully by ourselves and others to develop STSs and linkage markers from distal chromosome regions. However, it is shallow in coverage, some telomeres appear not to be represented, and a significant fraction of clones are mitotically unstable in the present yeast host. In addition, telomeric closure has been extremely difficult to verify at the molecular level, both because of general difficulties in the efficient construction of mid-range physical maps of human DNA and because of the abundance of low-copy repeats in subtelomeric regions, particularly adjacent to the telomere itself. We propose to construct a second-generation half-YAC library in recombination-deficient hosts in order to deepen the present clone coverage of telomeric DNA and attempt to overcome YAC stability problems in subtelomeric regions. New mapping methods, including the use of oligonucleotide-directed site-specific genomic cleavage techniques and the development of single-copy probes from subtelomeric DNA, will be refined and applied to mid-range mapping of subtelomeric DNA. Overlapping telomeric clones from both the old and the new library will be used along with these mapping techniques to establish that telomeric closure has been accomplished for individual loci, and to investigate potential large-scale telomere polymorphism described by other groups. The mapped, overlapping clones can then serve as the initial substrate for future high-resolution physical analysis and DNA sequencing of these key regions of the human genome.