The telomeres and adjacent regions were not fully included in the working draft sequence announced last summer because they have unique properties that make them particularly difficult to isolate and analyze.

In addition, the Wistar researchers found that the adjacent, or subtelomeric regions, appear to display more variation from individual to individual than other parts of the genome.

They also reported that the subtelomeric regions are gene-rich, suggesting that these areas may serve essential functions and are not simply buffers of nonfunctional "junk DNA" next to the telomere, as some have thought.

Their study was published in the February 15 Nature, a special issue focusing on the Human Genome Project in which the publicly-funded International Human Genome Sequencing Consortium's working draft sequence is published.

"Telomeres and subtelomeric regions make up less than 1 percent of the human genome, but they're quite important," says Harold C. Riethman, Ph.D., lead author on the study and associate professor at The Wistar Institute. "When the cellular machinery that maintains telomeres becomes damaged, a cell fails to divide properly, which is one of the hallmarks of cancer."

In addition to cell division, telomeric DNA mediates many important biological activities, such as cellular aging, movement and localization of chromosomes within the nucleus, and transcriptional regulation of subtelomeric genes.

To analyze the telomeric regions, the researchers developed a specialized yeast cloning vehicle -- called half-YAC, or half-yeast artificial chromosome -- to produce copies of human DNA regions linked to telomeres.

Cloning, or making copies of, DNA is an important step in genomic sequencing. The working draft sequence was produced using a bacterial cloning vehicle that fails to include substantial portions of telomeric DNA, but Riethman's clones include most of these regions.

While the primary aim of Riethman's study was to integrate the telomeres with the working draft sequence, analysis of the subtelomeric regions following sequencing revealed several interesting features.

The researchers found that some subtelomeric regions have very large and complex sets of DNA repeat sequences, whereas others have almost no repeated DNA segments. For at least 18 of the 46 total telomeres, the precise combination and organization of these repeated DNA segments at a single telomere can vary significantly from person to person.

"This pattern of large-scale variation is not common in other parts of the genome," Riethman says. "It suggests that telomere regions are one of the most malleable regions of the human genome and may be evolving more rapidly than other regions, although this is still highly speculative." He intends to explore the significance of this variation near the telomeres in future studies.

In addition, the scientists found that many subtelomeric regions contain DNA sequences corresponding to both known and unknown expressed genes. Human subtelomeric sequences have been proposed by some to serve solely as a buffer between the very ends of the chromosomes and the vital internal chromosomal sequences.

"The presence of many gene sequences indicates that the subtelomeric regions may serve essential functions and are not simply dispensable junk DNA," Riethman says.

In their study, the scientists succeeded in connecting 34 of 46 telomeric regions to the working draft human genome sequence. Twelve telomere ends still remain to be connected.

Seven of these 12 remaining telomeres could not be connected either because the working draft sequence does not yet extend into these regions or because the telomere clones contained repeated DNA segments that make it difficult to determine where the clones overlap with the working draft sequence.

The other five telomeres not yet connected to the working draft sequence were unstable in both yeast and bacteria, preventing the scientists from cloning and analyzing them.

The other co-authors at The Wistar Institute are Zhaoying Xiang, Ph.D., Sheila Paul, Eleanor Morse, and Xue-Lan Hu. Co-authors at other institutions are Jonathan Flint, of the John Radcliffe Hospital in Oxford, United Kingdom, and Hans H.-C. Chi, Deborah L. Grady, and Robert K. Moyzis of the University of California at Irvine.

Funding for the work was provided by the National Institutes of Health and the Department of Energy.

The Wistar Institute is an independent nonprofit biomedical research institution dedicated to discovering the basic mechanisms underlying major diseases, including cancer and AIDS, and to developing fundamentally new strategies to prevent or treat them.

The Institute is a National Cancer Institute-designated Cancer Center -- one of the nation's first, funded continuously since 1968, and one of only 10 focused on basic research. Founded in 1892, Wistar was the first institution of its kind devoted to medical research and training in the nation.

[Contact: Marion Wyce]

13-Feb-2001