Here is a very valuable resource that I think may turn out to be a front page link for this forum. It is the Encyclopedia of Life Sciences. I would love to see a formalized relationship developed between what we are doing and what they are at some point. hint hint....
[roll]
This is the link to their homepage
http://www.els.net/e...essionid=publicAlso here are the links to the two articles that I posted on the topic of Aging that refer to Telomers
http://www.nature.co...7/990527-1.htmlhttp://www.nature.co...8/990318-8.htmlLL
http://www.els.net/e...277135c8d3a4f7fTelomeres in Cell Function: Cancer and Ageing --------------------------------------------------------------------------------
Accepted for publication:March 2001
Mary-Lou Pardue
Gregory DeBaryshe Massachusetts Institute of Technology, Cambridge, Massachusetts, USA
Massachusetts Institute of Technology, Cambridge, Massachusetts, USA
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Keywords: cancer and ageing chromosomes DNA replication telomeres telomerase
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Telomeres are dynamic structures that make up the ends of chromosomes. They have important roles in assuring that genetic material is divided equally when cells multiply, and it is thought that they may be involved in regulating cell division in human ageing and cancer.
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Introduction
The ends of chromosomes in the nuclei of all eukaryotic organisms are specialized structures called telomeres. Telomeres are composed of deoxyribonucleic acid (DNA) (with associated proteins). In every organism studied, telomeric DNA consists of long head-to-tail arrays of repeated DNA sequences; the exact nucleotide sequence of the repeat depends on the species of the organism carrying the chromosomes. The cell can compensate for DNA loss from chromosome ends by adding more copies of the repeats, although some cells appear to lose the ability to make such additions. Telomere-associated proteins are less well understood. We do not know how many such proteins there are, nor do we know what most of them do; they present some of the most important unsolved questions about telomeres. See also:Chromosomes: higher order organization;Telomeres
In addition to compensating for loss of terminal DNA, telomeres have several important jobs that we know about and probably others that we do not yet know. They do at least four jobs in all organisms:
1. As mentioned above, telomeres provide a mechanism to compensate for underreplication of the ends of linear DNA molecules.
2. They keep true chromosome ends from fusing with other chromosome ends or with broken chromosomes to make chimaeric chromosomes. Chimaeric chromosomes can wreak havoc at cell division.
3. They distinguish true chromosome ends from breaks in DNA. Unrepaired chromosome breaks will activate a checkpoint in the cell cycle, which causes that cell to stop dividing until the break can be rejoined, either correctly or incorrectly.
4. In some cells, telomeres control the positions of chromosomes within the nucleus.
Recently, telomeres have become of significant medical interest because there is evidence to associate them with both ageing and cancer in humans, although possibly not in other organisms. The evidence comes from several findings: See also:Ageing;Cancer cytogenetics
•Extreme shortening of telomeres causes cells to become senescent and lose the ability to divide.
•Telomeres in older people are shorter than telomeres in younger people, suggesting chromosomes gradually lose DNA from their ends.
•Early studies of telomerase, the enzyme that adds the DNA repeats to chromosome ends, failed to detect telomerase activity in normal human somatic, i.e. nongermline, cells but did find activity in tumour tissue. This suggested that telomerase activity ends sometime during embryonic development and, if restored in later life, could lead to uncontrolled cell growth and cancer.
These and other findings supported a model of telomerase as a kind of clock controlling life span. Because chromosome ends are not fully replicated by DNA polymerase, they shorten at each cell division unless active telomerase compensates for this loss (see below, Problem of replication). The model proposed that eventually this shortening would cause loss of important genes and cells would die. Reactivation of the inactive telomerase in somatic cells would stop loss from the chromosome end, producing immortality, a characteristic of cancerous cells. See also:Ageing genes: gerontogenes;Cancer
As we have learned more about telomeres, it has become clear that this simple model is wrong in several respects. Nevertheless, in humans, both ageing and cancer appear to be associated with changes in telomeres; a major goal of those who study telomeres is to understand why these changes occur and what they really mean. Telomeres in all organisms share some fundamental jobs and characteristics. They also have some important species-specific differences. These differences are important because species-specific differences have historically provided the major clues for unravelling these kinds of genetic mysteries; in fact, differences are so useful that geneticists have almost accepted as dogma the exhortation ‘cherish your exceptions’. See also:Chromosomes and cancer;Chromosome structure
Importance of Ends in DNA Replication and Chromosome Segregation
Eukaryotic organisms have much larger genomes than do bacteria and viruses. These large genomes present significant problems for complete replication of DNA and for its accurate distribution to daughter cells. Eukaryotes have evolved several strategies to cope with these problems. Their DNA is divided into multiple chromosomes, apparently to keep the molecules at a manageable size; nonetheless, most of these chromosomes are much larger than the single chromosome that contains the entire genome of the typical bacterium. Bacterial chromosomes are circular DNA molecules, whereas chromosomes of eukaryotes are linear DNA molecules. It is experimentally possible to form a circular chromosome in eukaryotes by forcing recombination between the ends of a linear chromosome; however, these circles have a strong tendency to be lost at cell division or to break open. It has been suggested that such large circles may become tangled during replication or cell division, whereas the smaller bacterial chromosomes can avoid these problems. See also:Evolution of genome organization;Eukaryotic chromosomes;Bacterial chromosome
Problem of replication
Dividing a genome into multiple linear molecules may make the DNA easier to handle, but it presents other problems. The first of these is the problem of completely replicating the ends of a linear DNA molecule during cell division (Figures 1 and 2).
Figure 1
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Building blocks of chromosomal DNA. DNA, deoxyribonucleic acid, is a molecule whose building blocks are four nucleotides. The DNA nucleotides (mononucleotides) are, themselves, formed by the chemical linking of one of four ‘bases’ (cytosine, thymine, adenine or guanine) to a modified 5-carbon sugar, b-D-2 deoxyribose (indicated by aqua pentagons with the carbon positions numbered in red). The sugar is modified by the replacement of the hydroxyl (OH-) group on the so-called 5' carbon (shown at the upper left end of the sugar molecule) by a phosphate (PO42-) group. These mononucleotides are linked to form unidirectional, polarized molecules of single-stranded DNA. This linkage takes place when the OH- on the 3' carbon of the sugar moiety is replaced by a phosphodiester bond to the 5' carbon on the next sugar of the linear molecule, as shown in the dinucleotide. The polymer grows by repeated linking of monomers to the 3'-OH on the chain of mononucleotides.
Figure 2
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DNA polymerase cannot completely replicate the end of linear DNA. This diagram illustrates the replication of the end of linear DNA in the absence of a special mechanism for end replication. The final stages of replication of one end of the chromosome are shown. Replication has initiated at an internal origin, and, for simplicity, is drawn as proceeding only to the right (dotted lines on the left indicate that most of the chromosome is not shown). The replication machinery moves toward the end of the chromosome but polymerization proceeds 5'®3' along each daughter strand. Base pairing makes paired strands antiparallel. Thus, one strand (the leading strand) can be synthesized continuously, 5'®3' while the opposite strand (the lagging strand) must be synthesized backwards in short fragments as the machinery moves along the chromosome. Parental DNA strands are blue, new DNA is aqua. DNA polymerase can initiate polymerization only if provided with a 3'-OH primer. The 3'-OH is supplied by short (usually 8–12 base pairs) RNA fragments (pink). When synthesis is completed, the RNA is removed, leaving gaps, which are filled by DNA polymerase, primed by the 3'-OH of the DNA upstream of the gap. Because the final gap on the lagging strand has no upstream 3'-OH, it cannot be filled and it will lack the last few nucleotides at its 5' end. Similarly, at the other end of the chromosome (not shown), the other daughter strand will also lack a few nucleotides at its 5' end.
DNA replication starts at many different sites (origins of replication) along each chromosome and is carried out by an enzyme called DNA polymerase. DNA polymerase will initiate DNA replication only after a primer molecule, a short piece of ribonucleic acid (RNA), is transcribed at an origin of replication. This RNA provides an ‘upstream’ hydroxyl (OH-) group to which DNA polymerase attaches the first nucleotide of the new DNA. (‘Upstream’ to ‘downstream’ defines the direction in which successive nucleotides are added by the polymerase. When DNA synthesis is complete, the RNA is degraded, leaving a short gap in the new strand at each origin of replication. Except for the gaps at the extreme ends of the chromosome, gaps are flanked on both ends by newly replicated DNA. The DNA on the upstream side of the gap provides a hydroxyl to prime synthesis to fill the gap. At the upstream end of each strand of a linear chromosome, removal of the terminal primer leaves a gap with no upstream attachment point, thus DNA polymerase cannot fill this final gap. See also:DNA replication;Eukaryotic replication origins and initiation of DNA replication;Eukaryotic DNA polymerases;Eukaryotic replication fork
After the chromosome is replicated, each of the two daughter DNA molecules will therefore have one strand that is a few nucleotides shorter than its mother strand. When the shorter strand is copied in the next round of replication, both strands of the double-stranded granddaughter will have lost those terminal nucleotides. Thus, without some compensating mechanism, replication would lead to slow but continual shortening of chromosomes. Organisms with linear chromosomes must have special mechanisms to prevent this progressive loss at each round of replication. Many bacteria avoid the problem of a gap at the end because, in their circular chromosome, every gap has upstream DNA to prime synthesis. See also:Bacterial replication fork: synthesis of lagging strand
Telomeres solve the problem of underreplication of chromosome ends
Eukaryotes are not the only organisms that have the problem of completely replicating linear DNA. Viruses, which also use linear DNA to carry their genetic information, have a number of seemingly simple ways to avoid leaving a gap when the last RNA primer is removed. For instance, adenoviruses use a protein covalently bound to the 5' nucleotide (Figure 1) of their DNA molecule to prime synthesis; parvoviruses have short palindromic sequences at the ends of their DNA. These palindromic sequences can fold back on themselves to prime DNA synthesis. These neat, tidy solutions are a contrast to the telomeres on eukaryotic chromosomes, which are much more dynamic and seem to require more of the cell’s resources. See also:Viral replication
Telomerase and reverse transcription
In summary, in eukaryotes, chromosomal telomeres are composed of long head-to-tail arrays of species-specific DNA repeats. In almost all cases, these repeats are produced by an enzyme called telomerase. The process is illustrated in Figures 3 and 4. See also:Chromosomes: noncoding DNA (including satellite DNA);Short DNA sequence repeats
Figure 3
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Telomerase compensates for DNA loss by underreplication. This diagram illustrates how telomerase compensates for loss of terminal nucleotides on linear DNA. The enzyme extends the parental strand by reverse transcription of sequence from an RNA template that is part of the enzyme. Each end has many repeats of this short sequence. These repeats can be replicated by DNA polymerase to make double-stranded DNA. Although this replication will result in loss of terminal sequence, as in Figure 1, the loss can be replaced by reverse transcription of telomerase RNA. Parental DNA is in dark blue, with telomerase repeats in lighter blue dashes. The new DNA strand is aqua and RNA primers are pink.
Figure 4
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Reverse transcription by telomerase and retrotransposons (relatives of retroviruses). This diagram compares the action of telomerase with that of the reverse transcriptases of retrotransposons. The catalytic subunit of telomerase and an unknown number of accessory proteins (all protein components are represented by the yellow area) associates with the chromosome end and base pair the 3' end of the template segment of its RNA component (red) with the last few terminal nucleotides of the last repeat on the chromosome. (The template segment is a stretch in the enzyme’s RNA that contains 1.2–1.9 copies of the sequence forming the organism’s telomeres. This segment is depicted by the letters in the RNA template. The figure shows the template from Tetrohymena, the first telomerase discovered.) Primed by the 3'-OH on the end of the upper DNA strand, the enzyme copies one complete repeat from its template and then moves to realign with the new end of the strand and repeat its action. Most retrotransposable elements (retroviruses and retrotransposons) insert themselves into the interior of the chromosome. However, in fruitflies (Drosophila), two different retrotransposons can be reverse transcribed directly on to the ends of the chromosomes, where they form a unique telomere structure. The reverse transcriptases of retrotransposable elements differ from telomerase in several ways, including: (1) they reverse transcribe the entire length of their RNA template (usually several thousands of nucleotides) rather than a short defined segment (usually 5–10 nucleotides) within their RNA component; and (2) they do not use base pairing to align their RNA template with the chromosome. The catalytic subunit of reverse transcriptase and possible associated proteins are yellow. The RNA template is red and only the 3' end is shown. A, C, G, and T represent the four free dNTP (deoxynucleotide triphosphate) derivatives of the bases adenine, cytosine, thymine or guanine.
Telomerase carries an RNA molecule that serves as a template for the repeat sequence in telomeric DNA. The enzyme uses the 3' hydroxyl (Figure 1) on the end of the chromosomal DNA as a primer. It aligns the portion of its RNA that contains the telomeric sequence (usually 5–10 nucleotides, depending on the species of organism) with the end of the DNA and copies this RNA into DNA attached to the chromosome. This process is known as reverse transcription and is analogous to the process used by retroviruses and retrotransposons to copy their RNA into DNA for insertion into chromosomes. The most notable difference is that telomerase copies only a small specific sequence from the middle of its RNA and then repeats the process to form a string of identical repeats. See also:Cellular RNAs: varied roles;Regulatory RNA
Reverse transcription of an RNA template is obviously a good way to replace a sequence that has been lost from the end. What is surprising is that organisms have so many copies of the repeats on the end of each chromosome. For example, human chromosomes in germline cells have about 15 000 base pairs of these repeats on each chromosome end. Chromosomes of the laboratory mouse, Mus musculus, have 10 times more telomeric DNA, although the closely related Mus spretus has telomeres of approximately the same length as human telomeres. All of these telomeres are much longer than necessary to protect the genes from loss by underreplication, providing telomerase is active when germ cells are made. Even if there were no new additions to these arrays, it would take several lifetimes of loss from the ends before important genes would be exposed. The excess telomere sequences are thought to be necessary for the other roles of telomeres. See also:Human chromosomes
Telomeres keep true ends from fusing and from activating cell cycle checkpoints
The realization that telomeres are something more than just ends of chromosomes came from work by two giants in the field of chromosome studies, Herman Muller and Barbara McClintock, both later Nobel Laureates. Muller studied the genetics of fruitflies (Drosophila) and showed that mutations could be produced by X-rays. Studying the products of radiation, he found that chromosomes could be broken and rejoined to delete small parts of the chromosome or to join the fragments in new ways, giving what geneticists now call deletions, inversions and translocations. Many of these aberrations required that a chromosome undergo at least two breaks and then rejoin in a different order. One striking finding concerned the behaviour of chromosome ends: every broken chromosome acquired a preexisting chromosome end. These ends had variable amounts of chromosomal material attached, but all were linked to broken chromosome pieces in the proper orientation. None of the chromosome fusions involved inserting an end interior to the fused product; nor did ends fuse together like beads on a string. Muller concluded that chromosome ends were special structures, which he called telomeres (‘end-parts’). These structures were found only at chromosome ends and acted like ‘caps’; furthermore, all chromosomes needed these end caps. See also:Muller, Hermann Joseph;McClintock, Barbara;DNA damage
McClintock studied broken chromosome ends in corn (maize). In elegant studies, she forced two chromosomes to recombine in a way that produced one chromosome with two centromeres (a dicentric chromosome). These chromosomes broke when the two centromeres tried to go to separate poles at cell division, forming a ‘bridge’. McClintock observed that the resulting broken ends fused with other broken ends to give new dicentric chromosomes. These new chromosomes then repeated the cycle, which she named the ‘bridge-breakage-fusion’ cycle. Without the telomeric ‘cap’, the chromosome ends were sticky, causing problems when the cell divided. McClintock studied bridge-breakage-fusion cycles in corn endosperm (corn kernels) but noted that these cycles stopped and chromosome ends ‘healed’ when the chromosome was passed into the sporophyte (the plant that grew from the kernel). See also:Chromosome rearrangements;Cell cycle
These cytogenetic studies preceded molecular studies of telomeres by nearly 40 yr but their conclusions are still valid, although today we have a better understanding of the underlying mechanisms. The results suggest that, in both animals and plants, telomeres have similar roles in protecting chromosome ends. Fusions of broken ends, like those found by McClintock, can be seen in both plant and animal cells. More recent findings shed new light on their cytogenetic results. For example, we now know that many cells have checkpoint mechanisms that stop them from moving through the cell cycle if they have a broken chromosome. This is thought to explain the failure to recover terminally deleted chromosomes in Muller’s experiments. In addition, we know that, when it is activated, telomerase can add telomere sequences to broken ends of chromosomes. This addition ‘heals’ the chromosome by providing a new telomere (however, the healed chromosome may have lost some genes if a piece of the chromosome was broken off). This is thought to explain the ‘healing’ that McClintock saw in the sporophyte. See also:Checkpoints in the cell cycle
Telomeres can direct the positioning of chromosomes within the nucleus
Organisms with multiple chromosomes face the problem of coordinating chromosome movement at each cell division. In mitotic divisions, each of the two daughter cells must get a complete set of chromosomes. Meiotic divisions that produce eggs and sperm present problems that are even more complex. In the first meiotic division, the number of chromosomes is halved and every gamete must get one member from each pair of homologous chromosomes. Accurate segregation of chromosomes is accomplished by pairing each chromosome with its homologue just before the first meiotic division and then segregating one member of each pair to each daughter cell. See also:Mitosis;Meiosis
Early cytologists noted that telomeres appeared to have a special role in the nucleus as the chromosomes prepared for the first meiotic division. During the time when each chromosome is pairing with its homologous partner, telomeres are closely associated with the nuclear membrane and gradually move to form a cluster on one side of the nucleus. The chromosomes stay arranged in this ‘bouquet’ configuration until after pairing is complete; they then lose their attachment to the nuclear membrane and move to the centre of the nucleus. The early observations could only be made on large chromosomes, such as those found in multicellular animals. Recently, high-resolution techniques have made it possible to study the same stage in fission yeast, Schizosaccharomyces pombe. Although distantly related to mammals and having a very small genome, this organism undergoes a very similar meiotic organization of its chromosomes, led by telomeres. The similarity between this organization in yeast and eukaryotes argues that this is a general solution to the complex problem of finding partner chromosomes in a crowded nucleus. In addition, mutations in genes coding for telomere-associated proteins in yeast disrupt this process, showing genetically that telomeres are involved in this process, as had been suggested by cytological observations on other organisms. See also:Chromosome mechanics
The involvement of telomeres in organizing the meiotic nucleus is readily apparent because these cells have distinctive morphology. It remains an open question whether telomeres have similar roles in other cell types.
Role of Telomeres in Ageing and Cancer
Telomere length and length regulation appear to be vitally important to the cell. Early experiments suggesting that telomere length is related to the ability of cells to proliferate has led to extensive study of telomeres in humans, mice and yeast. In all three organisms, there is experimental evidence that when telomeres become too short, a critical point is reached after which cell viability is rapidly lost.
In yeast, telomere length does not change with age. Telomeres in old cells are approximately the same length as those in younger cells. It has been possible to see the effect of telomere shortening by experimentally knocking out the gene that encodes the telomerase enzyme. When this is done, the telomeres gradually shorten. There is no obvious effect on the yeast until after many cell divisions. Telomeres become critically short after about 50 generations and cells die.
Human telomere length regulation is quite different from regulation in yeast. In humans, telomeres in most somatic cells shorten with age. These cell types do not have enough telomerase activity to maintain their original telomere length. As a result, telomeres shorten with each division. If human somatic cells are put into culture, they cease to divide after a number of divisions characteristic of the cell type. It is generally thought that division stops because their telomeres have shortened to the critical point. (The few cells that survive this crisis generally regain telomerase activity and become immortal.) We do not know how cells sense that the telomere length has become too short. See also:Cell senescence: in vitro;Somatic cell genetics;Primary cell cultures and immortal cell lines
It is thought that the downregulation of telomerase activity that occurs in many types of human cells may have evolved as a protection against the unregulated cell division of cancer. This suggestion is supported by evidence that telomerase has been reactivated in most tumour cells. Other support came from experiments in which active telomerase genes were put into cultured human cells. In the initial experiments, these added genes produced telomerase activity that made the cells immortal. This apparently straightforward experiment became more complex when more cell types were tested and not all responded positively. It appears that, while telomerase gives the cell the ability to expand telomeres, utilization of this ability depends on whether some, as yet unknown, number of other genes are active in the cell. Genes that are known to affect the outcome of telomerase activity include several already known to be involved in checkpoints that regulate movement through the cell cycle.
Further important information on telomeres has come from studies using mice in which the telomerase RNA gene had been knocked out. Their telomeres gradually shorten, but not until the sixth generation does the shortening produce dramatic effects in the mice. These effects are found in the testis and ovary, the blood-forming tissues and the immune system. It was not surprising that these tissues are affected: all undergo much cell division and would be expected to have undergone more telomere shortening than tissues where cells replicate less frequently; however, it was a great surprise that cells from these mice can develop into tumours even though they lack telomerase. It has been suggested that, because of their short life span, mice may not need the stringent control that humans have evolved to limit conversion to cancer cells. See also:Mouse knockouts
Clearly, we have more questions than answers about how telomeres affect cell ageing and cancer. Nevertheless, the evidence is that there is a significant connection and that study of telomeres will help us to understand these important problems of human health. See also:Ageing - future directions for research in the biology of ageing
Further Reading
de Lange T and DePinho RA (1999) Unlimited mileage from telomerase? Science 283: 947–949.
de Lange T and Jacks T (1999) For better or worse? Telomerase inhibition and cancer. Cell 98: 273–275.
Greider C (1998) Telomerase activity, cell proliferation, and cancer. Proceedings of the National Academy of Sciences of the USA 95: 90–92.
Pardue M-L and DeBaryshe PG (1999) Telomeres and telomerase: more than the end of the line. Chromosoma 108: 73–82.
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