Unravelling genetic secrets

ELIZABETH BLACKBURN AT her laboratory in San Francisco. She said at a conference in 1980 that telomeres were made up of short repeated pieces of nucleotides, the building blocks of DNA.-PAUL SAKUMA/AP

EACH of our body cells contains genetic information packed in a compact structure called a chromosome. A long piece of the thread-like DNA molecule that carries our genes is wound up and bunched together in this structure. Every time a cell divides to form two new cells, the chromosomes need to be completely and correctly replicated so that each of the progeny cells receives an exact copy of the genetic blueprint from the parent cell. One of the long-standing fundamental questions in biology was how the chromosome replicated completely every time, including its ends, which should be susceptible to wear and tear during multiple cell divisions.

In fact, soon after the DNA structure was unravelled by Francis Crick, James Watson and Maurice Wilkins, James Watson and Alexei Olovnikov independently posed the end replication problem. They argued on theoretical grounds that there should be an incomplete replication in the new DNA molecule as copying of the very end of one of its strands cannot be complete. This would, over time, result in chromosome shortening and reduced viability. There also seemed to be some supportive experimental evidence in the observation that human somatic cells in culture had a very limited lifespan. Clearly, the molecular machinery in the human system had to have an in-built corrective mechanism to prevent this rapid degradation.

This years Nobel Prize in Physiology or Medicine goes to three American scientists who showed that the solution to this paradox lay in the ends of the chromosomes, called telomeres, which are continually replenished and maintained by an enzyme called telomerase. Elizabeth Blackburn of the University of California, San Francisco, Carol Greider, Elizabeth Blackburns former student who is now at the Johns Hopkins University School of Medicine in Maryland, and Jack Szostak of the Harvard Medical School share this years coveted prize equally.

Way back in the 1930s, Nobel award-winning biologists Herman Muller and Barbara McClintock recognised the importance of chromosome ends in ensuring chromosomal stability. They had observed that broken chromosomes were unstable and susceptible to rearrangements and fusion. But what these chromosome ends were and how the stability was achieved remained a mystery until Elizabeth Blackburn arrived on the scene in the 1970s to work in the laboratory of Joseph Gall at Yale University after her PhD at Cambridge under Fred Sanger. Elizabeth Blackburn likens telomeres to shoe-lace ends, called aglets, which prevent shoelaces from getting frayed or worn out.

Using her experience with the early DNA sequencing techniques that she had learnt at Cambridge, she was able to elucidate the DNA sequence at the ends of the chromosome in a pond-dwelling unicellular organism called Tetrahymena thermophila. This was before [automatic] DNA sequencing was working at all. There were only ways of patching together information. I realised that the ends are something that I could do. And nobody knew what the ends of chromosomes were like, Elizabeth Blackburn said in an interview to Frontline (April 24). Gall, whom Elizabeth Blackburn joined, had earlier discovered that Tetrahymena had small linear chromosomes and that in the nucleus of its cells were very, very tiny chromosomes.

According to Elizabeth Blackburn, there were 10,000 to 20,000 copies in the nucleus, and they were all very small, which meant lots of ends. By growing plenty of them just in big flasks, one could get enough material to do direct molecular studies. Normally chromosomes are so long that very few ends of DNA are left. Because methods of sensitivity then [were such that] you still needed enough purified material to work with, Elizabeth Blackburn explained how she came to choose Tetrahymena as the organism to work with to study telomeres.

At a conference in 1980, Elizabeth Blackburn presented the results of her work that telomeres were made up of short repeated pieces of nucleotides, the building blocks of DNA. This caught the attention of Szostak. He had found that plasmids, which are linear DNA molecules usually found in bacteria that are external to the chromosomes and that can replicate independent of the chromosomal DNA, were extremely unstable and prone to recombination and rearrangement when introduced into yeast cells. This observation was similar to what Muller and Barbara McClintock had found with broken chromosomes.

This brought Elizabeth Blackburn and Szostak together and they decided to do an experiment that crossed the kingdom or species boundaries. They combined the sequences from the Tetrahymena telomeres to the plasmids and put them into the yeast cells. They found that the Tetrahymena telomere seemed to protect the plasmid from degradation. These striking results were published in 1982. As telomere DNA from one organism protected chromosomes in an entirely different one, this demonstrated a fundamental biological mechanism across species that is evolutionarily conserved. This was also confirmed by the fact that the telomere DNA from different species of plants and animals, from amoeba to humans, contained the characteristic sequences that Elizabeth Blackburn had identified from Tetrahymena.

Soon afterwards, Elizabeth Blackburn and Carol Greider started investigating how the telomeres were synthesised using biochemical techniques. A natural way to approach the problem was to see whether any unknown enzyme was involved in the process. On Christmas day in 1984, Carol Greider found signs of enzyme activity in a cell extract. I was just excited about what I was doing at the time I had actually done the experiment several days before but then it takes a few days for the autoradiograph to develop. So I went in on Christmas day just to see what was there. I wouldnt necessarily saythat one shouldnt take breaks and take Christmas off. But if you are excited about something then go ahead and do it, remarked Carol Greider in her recent post-award interview to the Nobel website.

It was Elizabeth Blackburn and Carol Greider who named the enzyme telomerase. They also showed that it was a reverse transcriptase containing an RNA (ribonucleic acid) component and a protein component. The RNA contains the characteristic nucleotide sequence required to build the nucleotide repeats of telomere and serves as a template for replenishing the telomere with the protein component being responsible for the enzyme activity or the synthesis. They also showed that if you blocked this characteristic RNA sequence, the replenishing activity was severely compromised.

Telomerase thus extends telomere DNA, which in turn provides the requisite platform to enable the DNA polymerase to copy the entire length of the chromosome without leaving out its very ends. Before the two discovered the role of an enzyme in telomere maintenance, there was little understanding of how chromosomal stability was achieved. What we showed was what [the] telomere DNA structure looked like. And data were perfectly clear and people accepted that. [But] the idea that there would be telomerase [was] something that hadnt been seen before, Elizabeth Blackburn pointed out in her Frontline interview.

Then, internally, she said, you have to do a lot of convincing yourself that the data are not an artefact. We pretty much could answer in our minds that this wasnt just an artefact where, say, some random DNA polymerase was copying and contaminating fragments of DNA that came through from the cell extracts. So we had really ruled out why this might have been not a new enzyme but just some variant enzyme reaction carried out by enzymes that people already knew about. Though a scientist who had expressed views earlier about how telomeres might be replicated based on paper models had expressed reservations about our work, I didnt take it as a serious critique. But we were thinking: Were we being fooled? For about a year, we were just going back and forth trying to think of experiments that could undercut [the conclusion] but it was really something new.

Around the same time, Szostak isolated mutant yeast whose cells had gradually shortening telomeres. Such cells grew poorly and eventually stopped dividing. Elizabeth Blackburn and her co-workers engineered mutations of the enzyme and observed similar effects in Tetrahymena. In both cases, premature cellular ageing, or senescence, was observed, whereas functional telomeres were seen to prevent chromosomal damage and delay senescence. Carol Greiders group showed that senescence in human cells was also delayed with a supply of telomerase. These experiments from different perspectives considered together have enabled a complete and unambiguous understanding of the functional roles of telomeres and telomerase.

Further research has demonstrated that the DNA sequence in the telomere also attracts proteins that form a kind of sheath around the fragile ends of the telomere. In her Frontline interview, Elizabeth Blackburn explained the operative protective mechanism and the critical length of the telomere that needs to be maintained in human cells thus: The way proteins get attracted to the telomerated DNA to form a sheath is very dynamic; the proteins come on and off a little bit like a swarm of bees. If you looked at a swarm of bees, there is a shape; there is a protection there. But if you looked closely enough, you get to see that the individual bees that its made up of may be changing all the time, flitting in and out. You see an overall shape of protection, but its changing every minute, probably every second.

[Similarly here,] it must be something to do with the rates at which proteins come on and off. When its only a hundred nucleotides, the rates at having the sheath being formed as opposed to being unformed seem to be unsatisfactory. We dont know why from a physical point of view, we just have to infer. So in humans we get a rough idea; we know that telomeres cant really fall below that length for cells being able to keep renewing themselves, keep multiplying, which is what cells have to do to keep the immune system being built up, the skin and all parts of the body that have to continue to maintain different shapes throughout life.

These discoveries have not only added a new dimension to our understanding of cellular processes, but they also have significant medical implications, particularly in ageing, cancer, stem cell maintenance and genetic disorders. An important implication clearly is that telomere shortening could be the reason for ageing, not only in individual cells but also in the organism as a whole. But ongoing research has shown that the ageing process is much more complicated and depends on many different factors, telomere shortening being just one of them. Understanding the process of ageing is an area of intense current research.

Unlike normal cells, which do not divide frequently, cancer cells seem to divide and proliferate infinitely, which means that their telomeres are preserved indefinitely. How do they avoid senescence? Clearly, understanding the role of telomerase activity would be crucial to unravelling this process. One clear observation is the increased telomerase activity in cancer cells in 80-90 per cent of tumours; a great deal more than what is commensurate with their telomere lengths, which are not particularly long, unlike what is seen in normal cells where it is much more closely regulated and reasonable in amount.

Now its not a 100 per cent, Elizabeth Blackburn has pointed out (see Frontline, May 8). Some cancer cells get by with low telomerase. You know the exceptions and those would be instructive to study, but if you just look at the generality of human cancer cells its almost like a defining feature. So we have to take notice of what cancers are telling you; weve got to learn something from this. And certainly the telomeres are maintained but what is revealing are these other things.

Recent research has also shown that telomerase is not there just to accumulate DNA for the telomeres; they seem to have other functions as well. These other functions we dont understand at all but we see them when we perturb just telomerase in a very targeted way, Elizabeth Blackburn has said. According to her, these other functions that telomerase has seem to push cancer cells towards having properties that make them more malignant. Notwithstanding the above unknowns, the study of telomerase activity in cancer cells has increased the understanding of basic cancer biology.

These findings have also led to an idea that perhaps cancer could be treated by eradicating telomerase, but this approach could lead to collateral damage to other normal cells. Another approach is to use the technique of RNA interference to bring down telomerase levels, but not entirely eradicate, in cancerous cells and make them less malignant. Several studies and approaches are under way in this field, including clinical trials of vaccines against cells with elevated telomerase activity.

Similarly, some genetic diseases are now known to be caused by telomerase defects, including certain forms of congenital aplastic anaemia. In this, insufficient cell divisions in the stem cells of the bone marrow lead to severe anaemia. Certain skin and lung genetic disorders are also known to be caused by telomerase defects. Ongoing work towards understanding the molecular mechanisms around the compromised telomere and telomerase functions in genetic diseases provides possibilities of better diagnosis and development of therapies for genetic diseases.

The field that Elizabeth Blackburn, Carol Greider and Szostak have opened up is brimming with intense research activity today. As Carol Greider said in her Nobel interview, there are still many unanswered questions. Some of the detailed biochemistry, she said, isnt yet entirely worked out, about how the enzyme actually uses a small template reiteratively and probably, more importantly, exactly how its regulated in elongating the telomere. Very clearly the establishment of length equilibrium in cells so that telomeres can maintain the length is very critical. And both the level of telomerase as well as its regulation by other proteins and modifications clearly play a role in establishing that equilibrium.