DOE Human Genome Program Contractor-Grantee
5. Human Telomere Mapping and Sequencing
Robert K. Moyzis, Deborah L. Grady, Han-Chang Chi, and Harold C. Riethman
Department of Biological Chemistry, College of Medicine, University of California at Irvine, Irvine, CA 92697 and The Wistar Institute, Philadelphia, PA 19104
The Human Genome Project has undergone a dramatic shift to the goal of obtaining a "working draft" sequence of human DNA by the end of 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. Over 80% of human DNA can be rapidly sequenced 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, 43 of the 46 unique human telomeres have been obtained as half-YACs.
Using RARE (RecA-Assisted Restriction Endonuclease) cleavage, 20 of these telomere half-YAC clones (representing the telomeres of human chromosomes 1p, 1q, 2p, 2q, 4p, 6q, 7p, 7q, 8p, 8q, 9p, 12q, 13q, 14q, 17p, 17q, 18p, 18q, 19p, and 21q) have now been confirmed to represent the true telomere. An additional 11 clones (representing the telomeres of 3q, 4q, 9q, 10p, 10q, 11p, 11q, 15q, 16q, 19q, and 20p) are currently being confirmed by RARE cleavage analysis. 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). These framework telomere sequences will provide a "cap" to the worldwide genome sequencing efforts. 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 1q, 5p, 9q, 11q, 17p, and 18p have been completed. Following framework sequencing, finished sequencing will be conducted in select regions, with priority given to areas with high biological interest and/or relevant to the JGI, i.e., chromosomes 5,16, and 19. An important QC/QA aspect of our sequence analysis is the extensive confirmation of the sequence against genomic DNA by PCR-resequencing. Numerous polymorphisms in these regions, including SNPs, VNTRs, and rearrangements have been identified. Using pooled DNA PCR/sequencing, the population distribution of many of these polymorphisms can be determined rapidly.
|The online presentation of this publication is a special feature of the Human Genome Project Information Web site.|