Exceptional Chromosome Regions Workshop I



Human Telomere Mapping and Sequencing

R.K.Moyzis, H. Chi, D.L. Grady, and H. Riethman
Dept of Biol Chem, College of Med, Univ of CA, Irvine, CA 92697, and The Wistar Institute, Philadelphia, PA 19104

The Human Genome Project has accomplished the goal of obtaining a "working draft" sequence of human DNA this year. Such a framework sequence will catalyze gene discovery and functional analysis, and allow finished sequencing to be focused on regions of the highest biomedical priority. Such finished sequence can be obtained in the next few years by highly automated, high throughput sequencing centers. However, a significant fraction of the human genome will not be sequenced and/or assembled to completion by such approaches, as demonstrated by the recent sequence of human chromosome 22 (Dunham et. al., Nature 402, 489-495, 1999). These are regions that contain 1) a high percentage of repetitive DNA sequences; 2) internal tandem duplications, including multigene families; and/or 3) are unstable in all current sequencing vectors. Producing quality DNA sequence of these regions, which faithfully represents genomic DNA, will be a continuing challenge.

Telomeres, the ends of the linear DNA molecules in human chromosomes, exhibit both high levels of repetitive DNA composition and cloning instability. In addition, extensive heterogeneity exists in these regions between various individuals. Half-YAC clones are uniquely suited as starting material for the sequence analysis of human telomeric regions. The inability to clone the extreme end of human chromosomes in bacterial vectors, including BACs, is well known. Due to the lack of appropriate restriction sites in the terminal (TTAGGG)n regions, as well as the necessary size selection involved in BAC library construction, the most terminal BAC clones will be 20-200Kb from the true DNA ends. By functional complementation in yeast, however, the true human telomeric end can be cloned. To date, 44 of the 46 unique human telomeres have been obtained as half-YACs.

Using RARE (RecA-Assisted Restriction Endonuclease) cleavage, 22 of these telomere half-YAC clones (representing the telomeres of human chromosomes 1p,1q,2p,2q,4p,6q,7p,7q,8p,8q,9p,11p,12q,13q,14q,16p,17p,17q,18p,18q,19p,and 21q) have now been confirmed to represent the true telomere. Further, the majority of these YAC clones have been integrated with the BAC contigs being used for large-scale sequencing (either by hybridization of DNA probes with these BAC libraries or by DNA sequence matches). Given the new goals of the Human Genome Project, we have initiated framework sequencing on these clones, as well as the most terminal BACs identified from our chromosome 5 mapping project (Peterson et.al., Genome Res 9, 1250-1267, 1999). A combination of cosmid and plasmid end sequence analysis, combined with extensive restriction enzyme mapping of the original YAC, results in highly ordered framework sequences. To date, framework sequence of 17 half-YAC clones has been completed (1q,2p,2q,7p,7q,8p,9q,10p,10q,11p,11q,13q,15q,16q,17p,18p,and 18q), as well as the most distal BAC localized to 5p. An important QC/QA aspect of our sequence analysis is the extensive confirmation of the sequence against genomic DNA by PCR-resequencing, and somatic cell hybrid mapping. This analysis has uncovered extensive polymorphism in these regions, including SNPs, VNTRs, and widespread genomic rearrangements and duplications. Despite this complicated repetitive genomic organization, many confirmed and putative protein coding regions are found as well.

Many of the techniques used successfully to map and sequence human telomeric regions (especially RARE cleavage) should be applicable to other human ECRs, including centromeric regions. Given the high level of polymorphism uncovered, however, it is proposed that extensive human population sampling and primate DNA characterization be incorporated into such studies.

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