Mapping Subtelomeres

In genetics, certain terms sometimes mask what scientists do not yet understand, such as "junk DNA." Similarly, the chromosomal regions just proximal to the tips--the subtelomeres--have been dubbed "buffers," ill-defined DNA sequences that somehow support the telomeres, which control the cell cycle and cellular aging. A team of researchers from the Wistar Institute in Philadelphia, the University of California, Irvine, and John Radcliffe Hospital in Oxford, UK, has used single-copy sequences in the subtelomeric regions to anchor them to and extend the draft genome sequence to the telomeres.1 And in what is becoming a repeated theme in genomics, they were surprised at what they found. Highly repetitive genome regions are notoriously difficult to assign to a particular chromosome, let alone sequence, because assembly programs can overlap them in many ways and places. So it is for the telomeres, the tips of chromosomes that in humans and other vertebrates consist of repeats of the sequence TTAGGG.2 "Using shotgun cloning, most of the human genome has been sequenced. However, this approach has missed most of the regions near telomeres, because repetitive sequences do not clone well. The subtelomeric regions will be one of the last aspects of the human genome to be completed," says Jerry Shay, a professor of cell biology at the University of Texas Southwestern Medical Center in Dallas. The exclusion of the telomeres and much of their environs from the draft sequence is largely because the bacterial artificial chromosomes (BACs) used to sequence most of the rest of the genome do not include restriction enzyme cutting sites within the TTAGGG repeats, leaving the regions right next to them something of a mystery. "The way the rest of the sequence was constructed used a BAC clone library, carrying segments of 150 to 200 kilobases of DNA. The BAC libraries were prepared using restriction enzyme digests followed by a size selection process. We expected, statistically, to see an underrepresentation of regions adjacent to the chromosome ends," explains Harold Riethman, an associate professor at the Wistar Institute. The team used larger cloning vectors, called half YACs (yeast artificial chromosomes), to overlap the subtelomeric zones with the ends of the working drafts of each chromosome.3 Then they assembled overlapping pieces cloned in smaller cosmids within the region of interest, ultimately deriving and ordering many of the subtelomeric sequences. "This technique was developed specifically to target telomeric DNA. Rather than using a BAC library, we used a yeast chromosome clone. This permits isolation of linear DNA fragments rather than circles. The key to the half YAC method is that human chromosome tips function in yeast. The idea was to chop off pieces of human chromosome ends and ligate them to a yeast sequence for chromosome replication, and allow the human telomere to function in yeast. We are able to clone the entire human chromosome end," says Riethman. Anatomy of a Subtelomere Photo: Tommy LeonardiHarold Riethman It wasn't possible to decipher all of the 46 subtelomeric regions. The five human chromosomes that are acrocentric, with one long arm and one very stunted one, yielded sequence only at the major end. Other subtelomeric regions could not be placed because the draft sequence itself in many areas consists of small, unordered pieces. Still, in several regions the draft sequence extends well into subtelomeric terrain. As with much of the rest of the human genome sequence, researchers are more in the general observation stage than the analysis stage. All of the subtelomeres have regions of 50 to 250 bases just proximal to the telomeres that seem to echo the TTAGGG theme, or a similar sequence, as if it really is a buffer of sorts, or perhaps evolution experimenting with chromosome-tip sequences. However, it quickly became clear that all subtelomeres are not the same. The number of repeats, for example, varies from chromosome to chromosome, from a mere 8 kilobases in the long arm of chromosome 7, to 300 kilobases in the short arm of chromosome 8. Of great interest to the researchers were the low-copy repeats that seem to be a defining feature of the subtelomeric regions. "Typically there are a number of interspersed repeats, multiple copies of 15 to 20 base sequences present exclusively in subtelomeric regions. The size and distribution of these duplicated segments varies from individual to individual," remarks Riethman. Duplicated stretches of DNA sequence are found in various places in the genome, and seem to be related to each other, he adds. But a subtelomere is much more than a row of repeats. When the researchers compared subtelomeric sequences to known protein-encoding genes (expressed sequence tags, or ESTs), they found 500 matches, including 62 that are clearly distal to the limits of the working draft. "Half of the predicted genes have no known function. Even if we know a gene is evolutionarily conserved, the actual function can be unknown," Riethman says. Harbor for Gene Groupings Especially intriguing is the discovery that the subtelomeres harbor many multi-gene families. These gene groupings typically include two or more genes with very similar or identical sequences that function in the same process, plus pseudogenes, which are close in sequence but do not encode protein. Like ghosts of genes past, pseudogenes are thought to represent ancient duplication and divergence events, with key mutations preventing gene expression. "Half the (subtelomeric) sequences that match sequences in the EST databases appear to be members of gene families or pseudogenes. It can be very difficult to determine which copies are pseudogenes and which are protein-encoding genes," Riethman adds. Multi-gene families may hold clues to recent evolution that the much-anticipated sequencing of the chimpanzee genome may help to clarify. "Some subtelomeric gene families and duplications have expanded subsequent to the divergence of humans from other primates. A single gene in an ape or chimp in the human genome is duplicated. In a short space of time, some event has taken place to amplify a gene family. When a gene duplication occurs, one functional copy is needed while the other acquires a new function," explains Riethman. Although we still have a lot to learn about the subtelomeric parts of the human genome, it seems clear that they have more roles than anyone anticipated. Sums up Riethman, "Are subtelomeric repeats just buffers? Are they out there for protection, to stabilize a chromosome that has lost a tip? The fact that we find so many expressed sequences within 10,000 bases of the tips argues that the subtelomeres are unlikely to be junk DNA." Ricki Lewis (rickilewis@nasw.org) is a contributing editor for The Scientist. References 1. H.C. Riethman et al., "Integration of telomere sequences with the draft human genome sequence," Nature, 409:948-51, Feb. 16, 2001. 2. R.K. Moyzis et al., "A highly conserved repetitive DNA sequence, (TTAGGG)n, present at the telomeres of human chromosomes," Proceedings of the National Academy of Sciences, 85:6622-6, 1988. 3. H.C. Riethman et al., "Cloning human telomeric DNA fragments into Saccharomyces cerevisiae using a yeast-artificial-chromosome vector," PNAS, 86:6240-4, 1989.

Mapping Subtelomeres

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