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Exceptional Chromosome Regions Workshop I |
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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