BIOLOGA 2 BACHILLERATO Mitosis When a eukaryotic cell divides into two, each daughter or progeny cell must receive:

a complete set of genes (for diploid cells, this means 2 complete genomes, 2n) a pair of centrioles (in animal cells) some mitochondria and, in plant cells, chloroplasts as well some ribosomes, a portion of the endoplasmic reticulum, and perhaps other organelles

There are so many mitochondria and ribosomes in the cell that each daughter cell is usually assured of getting some. But ensuring that each daughter cell gets two (if diploid) of every gene in the cell requires the greatest precision. This image (provided by J. R. Paulson and U. C. Laemmli) provides a graphic illustration of the problem. It shows a bit (no more than 3%) of the single molecule of DNA released from a single human chromosome. (The chromosome was treated to remove its histones). Remembering that this is 3% of the DNA of only one of the 46 chromosomes in the human diploid cell, you can appreciate the problem faced by the cell of how to separate without error these great lengths of DNA without creating horrible tangles.

The answer: 1. Duplicate each chromosome during the S phase of the cell cycle. 2. This produces dyads, each made up of 2 identical sister chromatids. These are held together by proteins called cohesins. 3. Condense the chromosomes into a compact form. This requires ATP and a protein complex called condensin. 4. Separate the sister chromatids and 1

BIOLOGA 2 BACHILLERATO 5. distribute these equally between the two daughter cells. Steps 3 - 5 are accomplished by mitosis. It distributes one of each duplicated chromosome (as well as one centriole) to each daughter cell. It is convenient to consider mitosis in 5 phases. When a cell is not engaged in mitosis (which is most of the time), it is said to be in interphase. 1. Prophase:

The two centrosomes of the cell, each with its pair of centrioles, move to opposite "poles" of the cell. The mitotic spindle forms. This is an array of microtubules, synthesized from tubulin monomers in the cytoplasm, that develops from each centrosome. The chromosomes become shorter and more compact.

2. Prometaphase:

The nuclear envelope disintegrates. A protein structure, the kinetochore, appears at the centromere of each chromatid. With the breakdown of the nuclear envelope, spindle fibers attach to the kinetochores as well as to the arms of the chromosomes.

The microtubules attached to a kinetochore exert tension on its chromatid. For each dyad, one of the kinetochores is attached to one pole, the second (or sister) chromatid to the opposite pole. Failure of a kinetochore to become attached to a spindle fibers interrupts the process. 3. Metaphase: The tension is proportional to length; thus if a dyad approaches one pole, the tension in the opposite direction increases and the dyad is pulled back to an equilibrium position midway between the poles. In due course, all the dyads reach this position, the equatorial plane or metaphase plate. The chromosomes are at their most compact at this time. 4. Anaphase 2

BIOLOGA 2 BACHILLERATO The sister kinetochores suddenly separate and each moves to its respective pole dragging its attached chromatid (chromosome) behind it. Separation of the sister chromatids depends on the breakdown of the cohesins that have been holding them together. It works like this.

Cohesin breakdown is caused by a protease called separin (also known as separase). Separin is kept inactive until late metaphase by another protein called securin. Anaphase begins when the anaphase promoting complex (APC) destroys securin (by tagging it for deposit in a proteasome) thus ending its inhibition of separin and allowing separin to break down the cohesins.

5. Telophase: A nuclear envelope reforms around each cluster of chromosomes and these return to their more extended form. Cytokinesis: Mitosis is the process of separating the duplicates of each of the cell's chromosomes. It is usually followed by division of the cell. However, there are cases (cleavage in the insect embryo is an example) where the chromosomes undergo the mitotic process without division of the cell. Thus a special term, cytokinesis, for the separation of a cell into two. In animal cells, a belt of actin filaments forms around the perimeter of the cell, midway between the poles. As the belt tightens, the cell is pinched into two daughter cells. In plant cells, a membrane-bounded cell plate forms where the metaphase plate had been. The cell plate, which is synthesized by the Golgi apparatus, supplies the plasma membrane that will separate the two daughter cells. Synthesis of a new cell wall between the daughter cells also occurs at the cell plate. Chromosomes Composition: In eukaryotes, chromosomes consist of a single molecule of DNA associated with:

many copies of 5 kinds of histones. Histones are proteins rich in lysine and arginine residues and thus positively-charged. For this reason they bind tightly to the negatively-charged phosphates in DNA. a small number of copies of many different kinds of non-histone proteins. Most of these are transcription factors that regulate which parts of the DNA will be transcribed into RNA.

Structure:

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For most of the life of the cell, chromosomes are too elongated and tenuous to be seen under a microscope. Before a cell gets ready to divide by mitosis, each chromosome is duplicated (during S phase of the cell cycle). As mitosis begins, the duplicated chromosomes condense into short (5 m) structures which can be stained and easily observed under the light microscope. These duplicated chromosomes are called dyads. When first seen, the duplicates are held together at the centromere. In humans, the centromere contains over 3 million base pairs of DNA. Most of this is repetitive DNA: short sequences (e.g., 171 bp) repeated over and over in tandem arrays. While they are still attached, it is common to call the duplicated chromosomes sister chromatids, but this should not obscure the fact that each is a bona fide chromosome with a full complement of genes. The kinetochore is a complex of proteins (11 in budding yeast) that forms at the centromere and helps to separate the sister chromatids as mitosis proceeds into anaphase. The shorter of the two arms extending from the centromere is called the p arm; the longer is the q arm. Staining with the trypsin-giemsa method reveals a series of alternating light and dark bands called G bands. G bands are numbered and provide "addresses" for the assignment of gene loci.

Meiosis

The Problem Mitosis produces two cells with the same number of chromosomes as the parent cell. Mitosis of a diploid cell (2n) produces two diploid daughter cells. If two diploid cells went on to participate in sexual reproduction, their fusion would produce a tetraploid (4n) zygote. The Solution: Meiosis Meiosis is a process of cell division in eukaryotes characterized by:

two consecutive divisions: meiosis I and meiosis II no DNA synthesis (no S phase) between the two divisions 4

BIOLOGA 2 BACHILLERATO

the result: 4 cells with half the number of chromosomes of the starting cell, e.g., 2n n fusion of two such cells produces a 2n zygote

Meiosis in Animals Used to produced the gametes: sperm and eggs Meiosis in Plants Used to produce spores. Spores are the start of the gametophyte generation which, in time, will produce gametes (by mitosis because the starting cells are already haploid). Meiosis I Prophase of meiosis I (prophase I) is a more elaborate process than prophase of mitosis (and usually takes much longer). Here is a brief overview of the process. A more detailed view is provided below.

When the chromosomes first become visible they are already doubled, each homologue having been duplicated during the preceding S phase. Result: pairs of homologous dyads each dyad consisting of two sister chromatids held together by a shared kinetochore and proteins called cohesins. Synapsis (also called pairing): Each pair of homologous dyads align lengthwise with each other. Result: a tetrad. (These structures are sometimes referred to as bivalents because at this stage you cannot distinguish the individual sister chromatids under the microscope.) The two homologous dyads are held together by o one or more chiasmata (sing. = chiasma) which form between two nonsister chromatids at points where they have crossed over. o the synaptonemal complex (SC), a complex assembly of proteins (including cohesins)

BIOLOGA 2 BACHILLERATO At metaphase I, the microtubules of the spindle fibers attach each kinetochore to opposite poles of the cell. Result: one homologue is pulled above the metaphase plate, the other below. The chiasmata keep the homologues attached to each other, and the cohesins keep the sister chromatids together. At anaphase I,

the cohesins break down allowing the chiasmata to slip apart. Result: the homologous dyads separate and migrate toward their respective poles.

Meiosis II Chromosome behavior in meiosis II is like that of mitosis.

The centromere of each chromatid in the dyad acquires its own kinetochore. At metaphase II, spindle fibers attach one of these to one pole, the other to the opposite pole. At anaphase II, the chromatids separate and (each now an independent chromosome) move to their respective poles.

Genetic Recombination Meiosis not only preserves the genome size of sexually reproducing eukaryotes but also provides three mechanisms to diversify the genomes of the offspring. 1. Crossing Over

Chiasmata represent points where earlier (and unseen) nonsister chromatids had swapped sections. The process is called crossing over. It is reciprocal; the segments exchanged by each nonsister chromatid are identical (but may carry different alleles). Each chromatid contains a single molecule of DNA. So the problem of crossing over is really a problem of swapping portions of adjacent DNA molecules. It must be done with great precision so that neither chromatid gains or loses any genes. In fact, crossing over has to be sufficiently precise that not a single nucleotide is lost or added at the crossover point if it occurs within a gene. Otherwise a frameshift would result and the resulting gene would produce a defective product or, more likely, no product at all. 6

BIOLOGA 2 BACHILLERATO In the diagram above, only a single chiasma is shown. However, multiple chiasmata are commonly found (in humans the average number of chiasmata per tetrad is just over two). In this photomicrograph (courtesy of Prof. Bernard John), a tetrad of the grasshopper Chorthippus parallelus shows 5 chiasmata. 2. Random Assortment of Homologues In meiosis I, the orientation of paternal and maternal homologues at the metaphase plate is random. Therefore, although each cell produced by meiosis contains only one of each homologue, the number of possible combinations of maternal and paternal homologues is 2n, where n = the haploid number of chromosomes. In this diagram, the haploid number is 3, and 8 (23) different combinations are produced. Random assortment in humans produces 223 (8,388,608) different combinations of chromosomes. Furthermore, because of crossing over, none of these chromosomes is "pure" maternal or paternal. So I think it is safe to conclude that of all the billions of sperm produced by a man during his lifetime (and the hundreds of eggs that mature over the life of a woman), no two have exactly the same gene content.

3. Fertilization By reducing the number of chromosomes from 2n to n, the stage is set for the union of two genomes. If the parents differ genetically, new combinations of genes can occur in their offspring. Taking these three mechanisms together, I think that it is safe to conclude that no two human beings have ever shared an identical genome unless they had an identical sibling; that is a sibling produced from a same fertilized egg. 7

BIOLOGA 2 BACHILLERATO Prophase I - a detailed view The lengthy and complex events of prophase I can be broken down into 5 stages. 1. Leptotene

All the chromosomes condense. Homologous dyads (pairs of sister chromatids) find each other and align themselves from end to end with the aid of an axial element (that contain cohesins). How the nonsisters recognize their shared regions of DNA homology is not known. Double-stranded breaks (DSBs) occur in the DNA of the chromatids.

2. Zygotene

The synaptonemal complex begins to form. DNA strands of nonsister chromatids begin the process of recombination. How they are able to do so across the synaptonemal complex, which is over 100 nm thick, is unknown.

3. Pachytene

Synapsis is now complete. Recombination nodules appear (at least in some organisms, including humans). They are named for the idea that they represent points where DNA recombination is occurring. o There must be at least one for each bivalent if meiosis is to succeed. There are often more, each one presumably representing the point of a crossover. o They contain enzymes known to be needed for DNA recombination and repair. [Link] The steps in recombining DNA continue to the end of pachytene.

4. Diplotene

DNA recombination is complete. The synaptonemal complex begins to break down. The chromatids begin to pull apart revealing chiasmata. At first the chiasmata are located at the sites of the recombination nodules, but later they migrate towards the ends of the chromatids.

5. Diakinesis In some organisms, the chromosomes decondense and begin to be transcribed for a time. This is followed by the chromosomes recondensing in preparation for metaphase I. In creatures where this does not occur, diakinesis is simply a transition from diplotene to metaphase I. Meiosis is not flawless 8

BIOLOGA 2 BACHILLERATO It is estimated that from 10-20% of all human fertilized eggs contain chromosome abnormalities, and these are the most common cause of pregnancy failure. These chromosome abnormalities

arise from errors in meiosis, usally meiosis I; occur more often (90%) during egg formation than during sperm formation; become more frequent as a woman ages. Aneuploidy - the gain or loss of whole chromosomes - is the most common chromosome abnormality. Aneuploidy is caused by nondisjunction, the failure of chromosomes to separate correctly o homologues during meiosis I or o sister chromatids during meiosis II Zygotes missing one chromosome ("monsomy") cannot develop to birth (except for females with a single X chromosome). Three of the same chromosome ("trisomy") is also lethal except for chromosomes 13, 18, and 21 (trisomy 21 is the cause of Down syndrom). Three or more X chromosomes are viable because all but one of them are inactivated.

The Centrosome The centrosome is

located in the cytoplasm just outside the nucleus. Just before mitosis, the centrosome duplicates. The two centrosomes move apart until they are on opposite sides of the nucleus. As mitosis proceeds, microtubules grow out from each centrosome with their plus ends growing toward the equatorial plate forming spindle fibers.

The photo (courtesy of Tim Mitchison) shows microtubules growing in vitro from an isolated centrosome. The centrosome was supplied with a mixture of alpha and beta tubulin monomers. These spontaneously assembled into microtubules only in the presence of centrosomes. Spindle fibers have three destinations: 9

BIOLOGA 2 BACHILLERATO

Some attach to one kinetochore of a dyad with those growing from the opposite centrosome binding to the other kinetochore of that dyad. Some bind to the arms of the chromosomes. Other microtubules continue growing from the two centrosomes until they extend between each other in a region of overlap.

All three groups of spindle fibers participate in

the assembly of the chromosomes at the metaphase plate at metaphase o The kinetochores move to the tip (plus end) of the spindle fiber. This requires several proteins, including a kinesin. o The chromosome arms use a different kinesin to move to the metaphase plate. the separation of the chromosomes at anaphase. o The sister kinetochores separate and, carrying their attached chromatid, o move along the microtubules powered by minus-end motors, probably dyneins, while o the overlapping spindle fibers move past each other (pushing the poles farther apart) powered by plus-end motors, the "bipolar" kinesins. o In this way the sister chromatids end up at opposite poles.

Other Functions of Centrosomes In addition to their role in spindle formation, centrosomes play other important roles in animal cells:

signaling that it is o.k. to proceed to cytokinesis. Destruction of both centrosomes with a laser beam prevents cytokinesis even if mitosis has been completed normally. signaling that it is o.k. for the daughter cells to begin another round of the cell cycle; specifically to duplicate their chromosomes in the next S phase. Destruction of one centrosome with a laser beam still permits cytokinesis but the daughter cells fail to enter a new S phase. Segregating signalling molecules (e.g., mRNAs) so that they pass into only one of the two daughter cells produced by mitosis. In this way, the two daughter cells can enter different pathways of differentiation even though they contain identical genomes. [Link to further discussion.]

Centrosomes and Cancer Cancer cells often have more than the normal number (1 or 2 depending on the stage of the cell cycle) of centrosomes . They also are aneuploid (have abnormal numbers of chromosomes), and considering the role of centrosomes in chromosome movement, it is tempting to think that the two phenomena are related. Mutations in the tumor suppressor gene p53 seem to predispose the cell to excess replication of the centrosomes. Chromosome movement in mitosis also involves polymerization and depolymerization of the microtubules. Taxol, a drug found in the bark of the Pacific yew, prevents 10

BIOLOGA 2 BACHILLERATO depolymerization of the microtubules of the spindle fiber. This, in turn, stops chromosome movement, and thus prevents the completion of mitosis. Taxol is being used with some success as an anticancer drug. Centrioles Each centrosome contains a pair of centrioles. Centrioles are built from a cylindrical array of 9 microtubules, each of which has attached to it 2 partial microtubules. The photo (courtesy of E. deHarven) is an electron micrograph showing a cross section of a centriole with its array of nine triplets of microtubules. The magnification is approximately 305,000.

When a cell enters the cell cycle, and proceeds from G1 to S phase, each centriole is duplicated. A "daughter" centriole grows out of the side of each parent centriole. Thus centriole replication - like DNA replication (which is occurring at the same time) is semiconservative. Once formed, most of the functions of the centrosomes can be accomplished without centrioles. However,

Centrioles appear to be needed to organize the centrosome in which they are embedded. Sperm cells contain a pair of centrioles; eggs have none. The sperm's centrioles are absolutely essential for forming a centrosome which will form a spindle enabling the first division of the zygote to take place. Centrioles are also needed to make cilia and flagella.

The Cell Cycle A eukaryotic cell cannot divide into two, the two into four, etc. unless two processes alternate:

doubling of its genome (DNA) in S phase (synthesis phase) of the cell cycle halving of that genome during mitosis (M phase) 11

BIOLOGA 2 BACHILLERATO The period between M and S is called G1; that between S and M is G2. So, the cell cycle consists of:

G1 = growth and preparation of the chromosomes for replication S = synthesis of DNA (and centrioles) [see DNA Replication] G2 = preparation for M = mitosis

When a cell is in any phase of the cell cycle other than mitosis, it is often said to be in

interphase. Control of the Cell Cycle The passage of a cell through the cell cycle is controlled by proteins in the cytoplasm. Among the main players in animal cells are:

Cyclins There are 3 groups: o G1 cyclins o S-phase cyclins o M-phase cyclins Their levels in the cell rise and fall with the stages of the cell cycle.

Cyclin-dependent kinases (CDKs) Again, there are 3 groups: o G1 CDKs o S-phase CDKs 12

BIOLOGA 2 BACHILLERATO
o

M-phase CDKs

Their levels in the cell remain fairly stable, but each must bind the appropriate cyclin (whose levels fluctuate) in order to be activated. They add phosphate groups to a variety of protein substrates that control processes in the cell cycle.

The anaphase-promoting complex (APC) and other proteolytic enzymes. The APC o triggers the events leading to destruction of the cohesins and thus allowing the sister chromatids to separate. o degrades the mitotic (M-phase) cyclins

Steps in the cycle

a rising level of G1 cyclins signals the cell to prepare the chromosomes for replication a rising level of S-phase promoting factor (SPF) prepares the cell to enter S phase and duplicate its DNA (and its centrioles) as DNA replication continues, one of the cyclins shared by G 1 and S-phase CDKs (cyclin E) is destroyed and the level of mitotic cyclins begins to rise (in G2) M-phase promoting factor (the complex of mitotic cyclins with M-phase CDK) initiates assembly of the mitotic spindle breakdown of the nuclear envelope condensation of the chromosomes

these events take the cell to metaphase of mitosis at this point, the M-phase promoting factor activates the anaphase promoting complex (APC) which o allows the sister chromatids at the metaphase plate to separate and move to the poles (= anaphase), completing mitosis o destroys the M-phase cyclins. It does this by conjugating them with the protein ubiquitin which targets them for destruction by proteasomes. o turns on synthesis of G1 cyclins for the next turn of the cycle o degrades geminin, a protein that has kept the freshly-synthesized DNA in S phase from being re-replicated before mitosis. This is only one mechanism by which the cell ensures that every portion of its genome is copied once - and only once - during S phase. Link to discussion.

Some cells deliberately cut the cell cycle short allowing repeated S phases without completing mitosis and/or cytokinesis. This is called endoreplication and is described on a separate page. Meiosis and the Cell Cycle The special behavior of the chromosomes in meiosis I requires some special controls. Nonetheless, passage through the cell cycle in meiosis I (as well as meiosis II, which is essentially a mitotic division) uses many of the same players, e.g., MPF and APC. (In 13

BIOLOGA 2 BACHILLERATO fact, MPF is also called maturation-promoting factor for its role in meiosis I and II of developing oocytes. Checkpoints: Quality Control of the Cell Cycle The cell has several systems for interrupting the cell cycle if something goes wrong.

A check on completion of S phase. The cell seems to monitor the presence of the Okazaki fragments on the lagging strand during DNA replication. The cell is not permitted to proceed in the cell cycle until these have disappeared. DNA damage checkpoints. These sense DNA damage o before the cell enters S phase (a G1 checkpoint); o during S phase, and o after DNA replication (a G2 checkpoint). spindle checkpoints. Some of these that have been discovered o detect any failure of spindle fibers to attach to kinetochores and arrest the cell in metaphase (M checkpoint) o detect improper alignment of the spindle itself and block cytokinesis o trigger apoptosis if the damage is irreparable.

All the checkpoints examined require the services of a complex of proteins. Mutations in the genes encoding some of these have been associated with cancer; that is, they are oncogenes. This should not be surprising since checkpoint failures allow the cell to continue dividing despite damage to its integrity. Examples p53 The p53 protein senses DNA damage and can halt progression of the cell cycle in both G1 and G2. Both copies of the p53 gene must be mutated for this to fail so mutations in p53 are recessive, and p53 qualifies as a tumor suppressor gene. The p53 protein is also a key player in apoptosis, forcing "bad" cells to commit suicide. So if the cell has only mutant versions of the protein, it can live on - perhaps developing into a cancer. More than half of all human cancers do, in fact, harbor p53 mutations and have no functioning p53 protein. A genetically-engineered adenovirus, called ONYX-015, can only replicate in human cells lacking p53. Thus it infects, replicates, and ultimately kills many types of cancer cells in vitro. Clinical trials are now proceeding to see if injections of ONYX-015 can shrink a variety of types of cancers in human patients. In some way, p53 seems to evaluate the extent of damage to DNA, at least for damage by radiation.

At low levels of radiation, producing damage that can be repaired, p53 triggers arrest of the cell cycle until the damage is repaired. 14

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At high levels of radiation, producing hopelessly damaged DNA, p53 triggers apoptosis.

ATM ATM (="ataxia telangiectasia mutated") gets its name from a human disease of that name [Link], whose patients - among other things - are at increased risk of cancer. The ATM protein is involved in

detecting DNA damage, especially double-strand breaks; interrupting (with the aid of p53) the cell cycle when damage is found; maintaining normal telomere length.

MAD MAD (="mitotic arrest deficient") encodes a protein that binds to each kinetochore until a spindle fiber (one microtubule will do) attaches to it. If there is any failure to attach, MAD remains and blocks entry into anaphase. Mutations in MAD produce a defective protein and failure of the checkpoint. The cell finishes mitosis but produces daughter cells with too many or too few chromosomes (aneuploidy). Aneuploidy is one of the hallmarks of cancer cells suggesting that failure of the spindle checkpoint is a major step in the conversion of a normal cell into a cancerous one. Infection with the human T cell leukemia virus-1 (HTLV-1) leads to a cancer (ATL = "adult T cell leukemia") in about 5% of its victims. HTLV-1 encodes a protein, called Tax, that binds to the MAD protein causing failure of the spindle checkpoint. The leukemic cells in these patients show many chromosome abnormalities including aneuploidy. A kinesin that moves the kinetochore to the end of the spindle fiber also seems to be involved in the spindle checkpoint. G0 Many times a cell will leave the cell cycle, temporarily or permanently. It exits the cycle at G1 and enters a stage designated G0 (G zero). A G0 cell is often called "quiescent", but that is probably more a reflection of the interests of the scientists studying the cell cycle than the cell itself. Many G0 cells are anything but quiescent. They are busy carrying out their functions in the organism. e.g., secretion, conducting nerve impulses, attacking pathogens. Often G0 cells are terminally differentiated: they will never reenter the cell cycle but instead will carry out their function in the organism until they die. For other cells, G0 can be followed by reentry into the cell cycle. Most of the lymphocytes in human blood are in G0. However, with proper stimulation, such as encountering the appropriate antigen, they can be stimulated to reenter the cell cycle (at G1) and proceed on to new rounds of alternating S phases and mitosis. 15

BIOLOGA 2 BACHILLERATO G0 represents not simply the absence of signals for mitosis but an active repression of the genes needed for mitosis. Cancer cells cannot enter G0 and are destined to repeat the cell cycle indefinitely. Telomeres Each eukaryotic chromosome consists of a single molecule of DNA associated with a variety of proteins. Example: an average human chromosome contains a single molecule of DNA of about 150 million nucleotide pairs (150 x 106 bp). Stretched to its full length, this molecule would extend 5 cm. (about 2 inches). In the chromosome, this molecule is packed into a much more compact structure. The packing reaches its extreme during mitosis when this chromosome would condense to a structure some 5 m long (a 10,000-fold reduction in length). The DNA molecules in eukaryotic chromosomes are linear; i.e., have two ends. (This is in contrast to such bacterial chromosomes as that in E. coli that is a closed circle, i.e. has no ends.) The DNA molecule of a typical chromosome contains

a linear array of genes (encoding proteins and RNAs) interspersed with much non-coding DNA.

Included in the non-coding DNA are

long stretches that make up the centromere and long stretches at the ends of the chromosome, the telomeres.

Telomere functions Telomeres are crucial to the life of the cell. They

keep the ends of the various chromosomes in the cell from becoming entangled and sticking to each other they assist in the pairing of homologous chromosomes and crossing over during prophase of meiosis I.

The telomeres of humans (and mice) consist of as many as 2000 repeats of the sequence 16

BIOLOGA 2 BACHILLERATO 5' TTAGGG 3' 5'...TTAGGG TTAGGG TTAGGG TTAGGG TTAGGG TTAGGG..3' 3'...AATCCC AATCCC AATCCC AATCCC AATCCC AATCCC..5' Replication of linear chromosomes presents a special problem. DNA polymerase can only synthesize a new strand of DNA as it moves along the template strand in the 3' -> 5' direction. This works fine for the 3' -> 5' strand of a chromosome as the DNA polymerase can move uninterruptedly from an origin of replication until it meets another bubble of replication or the end of the chromosome. However, synthesis using the 5' -> 3' strand as the template has to be discontinuous. When the replication fork opens sufficiently, DNA polymerase can begin to synthesize a section of complementary strand - called an Okazaki fragment - working in the opposite direction. Later, DNA ligase stitches the Okazaki fragments together. In the figure on the right, the horizontal black arrows show the direction that the replication forks are moving. Wherever the replication fork of a strand is moving towards the 3' end, the newly-synthesized DNA (red) begins as Okazaki fragments (red dashes).

This continues until close to the end of the chromosome. However, the molecular requirements of the process are such that 5' end of each newly-synthesized strand cannot be completed. Thus each of the daughter chromosomes will have a shortened telomere.

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BIOLOGA 2 BACHILLERATO It is estimated that human telomeres lose about 100 base pairs from their telomeric DNA at each mitosis.

This represents about 16 TTAGGG repeats. At this rate, after 125 mitotic divisions, the telomeres would be completely gone. Is this why normal somatic cells are limited in the number of mitotic divisions before they die out? Telomeres and Cellular Aging Telomeres are important so their steady shrinking with each mitosis might impose a finite life span on cells. This, in fact, is the case. Normal (non-cancerous) cells do not grow indefinitely when placed in culture. Cells removed from a newborn infant and placed in culture will go on to divide almost 100 times. Well before the end, however, their rate of mitosis declines (to less than once every two weeks). Were my cells to be cultured (I am 70 years old), they would manage only a couple of dozen mitoses before they ceased dividing and died out. Could shrinkage of telomeres be a clock that determines the longevity of a cell lineage? Evidence: Some cells are immortal.

the cells of the germline (the germplasm) unicellular eukaryotes (like Paramecium) some cancer cells

It turns out that these cells are able to maintain the length of their telomeres. They do so with the aid of an enzyme telomerase. Telomerase 18

BIOLOGA 2 BACHILLERATO Telomerase is an enzyme that adds telomere repeat sequences to the 5' end of DNA strands. By lengthening the strand prior to replication, cells with active telomerase are able to compensate for telomere shortening during DNA replication. Telomerase:

is a ribonucleoprotein Its single RNA molecule provides an AAUCCC (in mammals) template to guide the insertion of TTAGGG. Thus telomerase is a reverse transcriptase; synthesizing DNA from an RNA template.

Telomerase is generally found only in

the cells of the germline, including embryonic stem (ES) cells unicellular eukaryotes cancer cells

However, when normal somatic cells are transformed in the laboratory with DNA expressing telomerase, they continue to divide by mitosis long after their normal life span is over. And they do so without any further shortening of their telomeres. This remarkable demonstration (reported by Bodnar et. al. in the 16 January 1998 issue of Science) provides the most compelling evidence yet that telomerase and maintenance of telomere length are the key to cell immortality. Telomerase and Cancer Most cancers arise from somatic cells. But one of the crucial features that distinguishes a cancer cell from a normal somatic cell is its ability to divide indefinitely. It turns out that most cancer cells have regained the ability to synthesize telomerase and thus are able to prevent further shortening of their telomeres. Perhaps agents that prevent the expression of the gene for telomerase - or prevent the action of the enzyme - will provide a new class of weapons in the fight against cancer. Telomerase and transplanted cells One approach to gene therapy it to

remove cells from the patient transform them with the gene for the product that the patient has been unable to synthesize return them to the patient.

One problem with this approach is that the cells - like all normal somatic cells - are mortal. After a series of mitotic divisions, they die out. That is the reason the children described in the link above required periodic fresh infusions of their transformed T cells.

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BIOLOGA 2 BACHILLERATO What if their cells could be transformed not only with the therapeutic gene but also with an active telomerase gene? This should give them an unlimited life span. But if cancer cells regain the ability to make telomerase, might not the reverse be true; that cells transformed with an active telomerase gene might become cancerous? Perhaps not. The cells described by Bodnar et. al. in the 16 January 1998 issue of Science have continued to grow in culture for another year and have been subjected to a number of tests to see if they have acquired any properties of cancer cells in culture. The results are encouraging. While these cells continue to divide indefinitely as cancer cells do,

They still show contact inhibition as normal cells do when grown in culture. They do not grow into tumors when injected into immunodeficient mice (as cancer cells do). They are still fussy about their diet - unable to grow on the simple media that supports cancer cells in culture. They still retain a normal karyotype; something that cancer cells seldom do.

Telomeres and Cloning The now-famous sheep Dolly was cloned using a nucleus taken from an adult sheep cell that had been growing in culture. The cell donor was 6 years old, and its cells had been growing in culture for several weeks. What about Dolly's telomeres? Analysis of telomere length in Dolly's cells reveals that they are only 80% as long as in a normal one-year-old sheep. Not surprising, since the nucleus that created Dolly had been deprived of telomerase for many generations. Two other sheep - cloned from embryonic, not adult, cells - also had shortened telomeres although not as short as Dolly's. Perhaps the length of time the cells spent in culture before they were used accounts for this. Does this mean that Dolly is doomed to a shortened life? Probably not. She seems healthy and has even had babies of her own. She will probably live a normal life span before her chromosomes give out. But her short telomeres do add another question to the debate about cloning mammals from adult cells.

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