|Genome Sequencing Section
DOE Human Genome Program Contractor-Grantee Workshop
5. Human Telomere Mapping and Sequencing
Han-Chang Chi1, Deborah
L. Grady1, Harold C. Riethman2, and Robert K. Moyzis1
The Human Genome Project has undergone a dramatic shift this year to the goal of obtaining a 'framework' sequence of human DNA in just a few years. 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. A significant fraction (20%) of human DNA contains a high percentage of repetitive sequences, is unstable in most cloning vectors, and exhibits extensive polymorphisms both between individuals and populations. Producing quality maps and sequence in such regions, which faithfully represent human genomic DNA, will be a continuing challenge. One such region is represented by human telomeres. Following the discovery and cloning of the human telomere repeat (TTAGGG)n by our laboratory ten years ago, numerous investigations have implicated this sequence or genes near telomeres as likely targets for alterations during cellular aging and cancer progression.
Nearly all human telomeres have now been cloned as yeast artificial chromosomes by functional complementation. During the last year, our laboratory finished the 0.23Mb 7q telomere sequence (GenBank accession AF027390), the first RARE (RecA-Assisted Restriction Endonulease) cleavage confirmed telomere region to be sequenced directly up to the terminal (TTAGGG)n repeat. Nine overlapping cosmids and two PCR products obtained from the 7q telomere YAC clone HTY146 (yRM2000) were sequenced using a Sample Sequencing (SASE)-parallel primer walking strategy. In total, 18% of this telomeric sequence required extensive PCR and non-standard sequencing methods to finish. Confirmation of the sequence against human genomic DNA was conducted by PCR-sequencing, using primer sets picked every 20kb. The submitted sequence is a faithful representation of human DNA, containing less than one error in 10,000 bases. Computer and experimental analysis uncovered numerous open reading frames, expressed sequence tags (ESTs), and potential exons dispersed along the entire 226 kb region, as well as 6 single nucleotide polymorphisms (SNPs), 19 variable number of tandem repeats (VNTRs) and 20 microsatellite repeats. The first and second exons for the human vasoactive intestinal peptide receptor 2 (VIPR2) gene were localized approximately 191 kb internal to the (TTAGGG)n terminal repeat. This neuropeptide system is involved in a diverse set of physiological functions including smooth muscle relaxation, electrolyte secretion, and vasodilation. Primer pairs picked to amplify the regions of 7q containing VNTRs uncovered extensive polymorphisms in the limited numbers of individuals examined to date. We are nearing completion of mapping and sequencing two additional telomeres, 9q and 11q, chosen because these regions contain a limited amount of subtelomeric repeats. In addition, SASE analysis is being initiated on 14 additional telomeres that have been confirmed by RARE cleavage (1q, 2p, 2q, 6q, 7p, 8p, 8q, 12q, 13q, 14q, 17p, 18p, 18q, and 21q) in order to prioritize our next targets for finished genomic sequencing.
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