5. REPLICATION AND CELL DIVISIONS. The mechanism of replication. Concatamer and the rolling circle. Headful and site specific packaging. Mitosis and meiosis. Nondisjunction. Aneuploidy and consequences. INTRODUCTION The similarity of the offspring in the subsequent progenies clearly shows that DNA, the material for inheritance, is passed from cell to cell, generation to generation. Inheritance of the DNA assumes its replication. How does DNA replicate? How is it transmitted to the coming generations? What are the consequences of improper DNA/chromosome transmission? The next chapter aims to answer the above questions. REPLICATION The semiconservative nature of replication In principle, there are three possibilities for the replication of a double stranded DNA molecule. (1) The conservative mechanism assumes preservation of the original double stranded DNA and the synthesis of its exact copy. (2) According to the semiconservative model, strands of DNA become separated and in turn each of them will function as a template for the synthesis of the complementary DNA strand. (3) The dispersive model assumes random composition of the original and the newly synthesized DNA in both strands. As shown by M. Meselson and F. Stahl (1958), replication of DNA follows the semiconservative mode.

Fig. 5.1. Schematic illustration of semiconservative replication.

Meselson and Stahl grew bacteria over several generations in culture medium with 15N, the heavy nitrogen isotope. 15N becomes incorporated into DNA rendering the DNA “heavy”. The heavy DNA sediments near the bottom in the centrifuge tube during gradient centrifugation (Fig.5.1). (At the level, where densities of the DNA and the medium are equal.) The heavy bacteria were transferred into a medium with 14N, a light nitrogen isotope, for one generation (ca. 20 min). (Of course, DNA with light 14N will form a band at the top level in the centrifuge tube.) The DNA was isolated from the bacteria and its density studied in order to determine whether the DNA was light, medium or of heavy density. The DNA formed only a single discrete band of medium density in the centrifuge tube showing that one strand of the DNA was light with 14N the other was heavy with 15N (Fig. 5.1). Results of the experiment proved that DNA replication proceeds the semiconservative way. Had replication been conservative, a heavy (the old DNA) and a light band (the new DNA) would have formed. If DNA replication was dispersive, formation of a single band with large smear would have been expected around the average density value. Meselson and Stahl kept another sample of the heavy bacteria in light medium for two generations. They isolated and analyzed the DNA subsequently. Two discrete bands of equal size formed in the centrifuge tube: one at medium and one at light densities (Fig. 5.1). (In the case of conservative replication a heavy and a light band would have formed, the light one has three times more DNA than the heavy one. In the case of dispersive replication formation of a single smear would be expected.)

Fig. 5.2. Replication of the eukaryotic chromosomes is semiconservative. Herbert Taylor (1958) proved first that replication of eukaryotic DNA is also semiconservative (Fig. 5.2). Taylor transferred mammalian cells for a single cell cycle (ca. 24 hours) into a medium labelled with 3H thymidine. (Since thymidine is only incorporated into DNA, the 3H thymidine will specifically label DNA.) Taylor prepared the metaphase chromosomes

from the cells and stained the chromosomes to see them. The preparations were covered with a thin layer of photo emulsion. Decay of the 3H will mark the emulsion which is detectable in the process called autoradiography. There was uniform label over both of the sister chromatids, showing that both of the sister chromatids contained 3H labelled DNA strand synthesized in the 3H thymidine medium (Fig. 5.2). (In the case of conservative replication, only one of the sister chromatids would have been labelled. In case of dispersive replication, formation of sister chromatids with complementary spots of labelled and non labelled areas would be expected.) Taylor transferred a sample of the cells with 3H labelled DNA for one generation into medium without 3H thymidine, and carried out the above procedure. Following the second round of replication, one of the sister chromatids possessed 3H labelling the other did not. The pattern shown on Fig. 5.2. can appear only if replication is semiconservative. Replication is bidirectional

Fig. 5.3. Replication commences a singly site in prokaryotes (A) and a several sites in eukaryotes (B).

Fig. 5.4. Replication proceeds in both directions in both pro- and eukaryotes.

John Cairns and his co-workers studied, using electron microscope, DNA in the process of replication (Fig. 5.3). They showed that replication of the bacterial chromosome (also of plasmids) commences at a single site. In eukaryotes replication begins at several sites in eukaryotes (Fig. 5.3; see also chapter 22). D. Prescott and R. G. Wake transferred bacteria for a short period of time into 3H thymidine containing medium (others eukaryotic cells). They isolated DNA from the bacteria for electron microscopy and covered the chromosomes with photo emulsion to make 3H thymidine containing DNA visible. As shown on Fig. 5.4, the label was symmetrically distributed showing that replication of DNA is proceeds in both directions. A brief molecular mechanism of replication

Fig. 5.5. Schematic illustration of the molecular mechanism of replication. Replication of the DNA is carried out by several enzymes. Replication commences at the site of replication initiation. The two DNA strands must be unwound. (Speed of unwinding is impressive in bacteria: 104 rotation/min.) Following unwinding and separation, both strands serve as a template for the synthesis of the complementary DNA strand (Fig. 5.5). Synthesis of the new DNA strand is fast: 105 and 104 nucleotides per minute in pro- and eukaryotes, respectively. The new DNA strand is synthesized by the DNA polymerase enzyme complex. The DNA polymerase can add nucleotides only to the 3' end of pre-existing nucleotide polymers, implying that synthesis of DNA strands (in fact of any nucleic acids) proceeds in the 5'→3' direction. For synthesis of nucleic acids the DNA polymerase requires a so-called primer, a short

B

A

stretch of an olygonucleotide. The primer is a ca. 30 nucleotide long RNA in E. coli synthesized by RNA polymerase or by the primase enzyme, based on the DNA template. The primer will eventually be removed by a polymerase reaching from the 5' direction and being replaced by DNA. The DNA polymerase synthesizes DNA based on the DNA template from four types of deoxy nucleotides (dATP, dTTP, dCTP and dGTP). While synthesis of the so called continuous or leading strand proceeds continuously in the 5'→3' direction, synthesis of the discontinuous or lagging strand is achieved in the so called Okazaki fragments (Fig. 5.5). The Okazaki fragments are sealed by the ligase enzyme. (Details of replication are described in handbooks of genetics and molecular biology.) The concatamer DNA is linear in several species of phages and replicates in a special way. Following one round of replication, the 3' ends remain free and are complementary to each other (Fig. 5.6). The complementary 3' ends pair, become sealed by ligase and as a result, a DNA molecule forms with two copies of the phage genome. The process continues, and the number of genome copies increase geometrically with the subsequent rounds of replications. In the long run a concatamer forms, which is a DNA molecule with a tandem arrangement of several phage genomes.

Fig. 5.6. The mechanism of concatamer formation. The rolling circle mode of replication The rolling circle mode of replication is an effective way in case of pages and plasmids (Fig. 5.7). One strand of the ring shaped double stranded DNA is first cleaved by an endonuclease enzyme. While the 5' end becomes free, the DNA polymerase will synthesize DNA on the 3' and using the intact strand

as a template. While the circular template DNA is rolling during DNA synthesis, the newly synthesized DNA becomes longer and longer, and will become a template for the synthesis of more DNA. In the long run a long, double stranded DNA will be synthesized, a concatamer that contains as many genomes as the number of rotations (Fig. 5.7).

Fig. 5.7. The rolling circle replication results in the formation of a concatamer. TRANSMISSION OF CHROMOSOMES FROM CELL TO CELL, GENERATION TO GENERATION Site-specific and headful packaging of phage chromosomes Genomic size fragments are generated from the concatamers through two mechanisms. In the site specific packaging mode the concatamer is cleaved at specific sites by endonucleases. One fragment contains one phage genome. The genome size fragments are incorporated into the protein phage heads. In headful packaging one end of the concatamer fills up a phage head, and the DNA is cleaved once, the phage head is full. The phage head can accept as much DNA as the genome. Should headful packaging start at whichever phage gene, the entire genome will be present in every phage.

The transmission of bacterial chromosomes

Fig. 5.8. Separation of bacterial chromosomes during cell division. The bacterial chromosomes are attached to the cell membrane at one point (Fig. 5.8). (The chromosome membrane attachment site is near the site of replication.) Division of the bacterial cells starts at the point of chromosome cell membrane attachment. (Do you remember the mesosome?) The chromosomes are pulled into different cells along the separation of the two newly formed bacterial cells (Fig. 5.8). DIVISION OF THE EUKARYOTIC CELLS I. MITOSIS

Fig. 5.9. Schematic illustration of mitosis.

Growth and division follow in a cycle of the eukaryotic cells. DNA of the cells replicate during the S (synthesis) phase of the cell cycle. (Details of the cell cycle will be covered during the 23rd lecture.) During mitosis, two daughter cells will be derived from a single mother cell. The two daughter cells are genetically equal with each other and the mother cell. Mitosis is subdivided into four stages, depending on the events seen in light microscopes (Fig. 5.9). Prophase. Every chromosome is composed from two sister chromatids. The sister chromatids are attached at their centromeres and become shortened. The centriole divides. The centrioles migrate to opposite poles over the nuclear envelope. The nucleolus and the nuclear envelope disappear. Metaphase. The centrioles organize three types of microtubules (Fig. 5.10). (1) The polar microtubules overlap in the equatorial plane of the cell. Mechanoenzymes (also called motor protein molecules) connect the polar microtubules over the overlapping region. The motor proteins push away the polar microtubules and indirectly keep the centrosomes away. (2) The kinetochore microtubules attach to the kinetochores. The kinetochore is composed from three layers of proteins arranged in a sandwich fashion (Fig. 5.11). The kinetochore attaches to the centromere. Each kinetochore is connected with 30-40 microtubules, establishing contact with the kinetochore microtubules and the chromosomes. The two sister chromatids are attached to kinetochore microtubules connected to different centrosomes. As far as we know, the kinetochore is attached to the microtubules through motor protein molecules (Fig. 5.11).

Fig. 5.10. Three types of microtubules in a cell. Dyneins and kinesins are the two groups of motor proteins. Pulling action of the motor molecules arranges the chromosomes into the equatorial plane. Colchicine, an alkaloid of meadow saffron, prohibits microtubule function through binding to tubulin molecules and brings about arrest in the metaphase stage of mitosis, allowing karyotyping. (Colchicine

has been used for treatment of gout since the times of ancient Egyptian.) (3) The astral microtubules radiate away from the centrosomes and presumably keep the centrosomes in fixed positions of the cytoplasm.

Fig. 5.11. The relationship of the centromere, kinetochore, motor protein and the kinetochore microtubules. Anaphase. The centromeres are separated and the chromosomes reach to the opposite poles. Movement of the chromosomes is achieved by the motor protein molecules. The motor molecules attach to both the kinetochores and the microtubules and while migrate towards the poles move chromosomes (Fig. 5.12). It was measured that for carrying one chromosome energy of 20 ATP molecules are required. Telophase. Mitosis is completed. The nuclear envelope and the nucleolus reforms. The mitotic spindle apparatus is disassembled. Cytokinesis. While cells become separated from each other, they share cytoplasm of the mother cell. In animal cells a contractile ring composed from actin microfilaments and myosin molecules ensure separation of the daughter cells. In plants, a cell plate forms in the equatorial plane perpendicular to spindle apparatus. The cell plate derives from the microtubules and materials delivered in small vesicles from the Golgi apparatus. The vesicles contain building materials for the new cell membrane and the cell wall.

Fig. 5.12. Chromosomes are transported along the kinetochore microtubules. Mitotic nondisjunction Mitosis take place with great fidelity: (1) the segregation of sister chromatids into different daughter cells and (2) keeping the chromosome number constant. Chromosome number of the daughter cells is seldom different from that of the mother cell. Occasionally a chromosome may be lost during mitosis. Sometimes unequal distribution of the chromosomes may happen between the daughter cells, i.e. nondisjunction takes place. As a consequence of mitotic nondisjunction, usually one of the daughter cells contains one more (2n+1, called trisomy). Here, n stands for the haploid and 2n for the diploid chromosome numbers, respectively. The other cell contains one fewer chromosome than normal (2n-1, monosomy). Cells carrying unusual numbers of chromosomes (usually by only one) are aneuploid. Viability of the aneuploid cells is reduced as compared to the normal diploid cells due to genetic imbalance, the altered gene dosage and gene product concentrations. The genetic imbalance leads to cell death, especially when larger chromosomes are involved. Viability of the trisomic cells is better than that of the monosomic cells. The smaller the extra or missing chromosome is, the better viability of the aneuploid cell will be. Mitotic nondisjunction takes place usually in single somatic cells and, when cells survive, the aneuploid condition may be propagated for the progeny cells, giving rise to a clone of aneuploid cells. The aneuploid cells may be starting points for tumor development (see chapter 23). There are several environmental pollutants known that induce mitotic nondisjunction. (See chapter 24 for details.) Naturally nondisjunction may happen not only during mitosis but also during meiosis.

ATP-driven microtubule motor protein

Kinetochore microtubule

Kinetochore

Chromosome

ATP-driven chromosome movement drives microtubule disassembly

Direction of chromosome movement

II. MEIOSIS

Fig. 5.13. Schematic representation of meiosis by two pairs of homologous chromosomes.

One meiosis is composed from basically two subsequent cell divisions leading to the generation of haploid (germ) cells with only one set of the homologous chromosomes in every cell (Fig. 5.13). The gonial cells are diploid and proliferate mitotically. The gonial cells are called oo- and spermatogonial cells in females and in males, respectively. The oo- and the spermatogonial cells commence the first meiotic division. Cells engaged in the first meiotic division are called primary oo- or spermatocytes. Primary oocytes from in human fetuses during the 3rd to 8th months of gestation and are arrested in prophase of the first meiotic division till puberty. Meiosis continues in a few primary oocytes from sexual maturation upon hormonal signals. (It is worth mentioning that the stage of meiotic arrest may be different in females of different species. In most vertebrate species meiosis is haltered in metaphase of the second meiotic division.) Meiosis continues only following fertilization. Daughter cells that form at the end of the first meiotic division enter the second meiotic division promptly. Cells engaged in the second meiotic division are called secondary oo- and spermatocytes. Cells that may differentiate to germ cells form at the end of the second meiotic division.

Fig. 5.14. The time course of the first meiosis prophase. The first meiotic (reductional) division The first meiotic division, like mitosis, is divided into stages. Prophase I. Prophase of the first meiotic division is subdivided into five stages (Fig. 5.13). During leptotene already replicated chromosomes condense and become visible in microscopes. The sister chromatids are tightly paired. (The number of chromatids is, of course, 4n.) During zygotene the homologous chromosomes with two chromatids each, pair with each other and form the synaptonemal complex (Fig. 5.14). (Geneticists call the paired homologous chromosomes bivalents. The four chromatids compose one tetrad.) The chromatids are told to synapse. During pachytene crossing over takes place, i.e. the physical exchange between chromatids of the homologous chromosomes. The crossing over always includes the non-sister chromatids and is exact. Unequal crossing overs are very seldom. The basis of crossing over is breakage and union of the DNA molecules. Crossing over is one component of

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recombination: reciproc exchange of parts of the maternally and the paternally derived chromosomes provides new genetic combinations. (Detailed molecular mechanisms of crossing over are described in handbooks of genetics.) During diplotene, separation of the homologous chromosomes begins, each with two chromatids. Chromatids are arranged in X fashion at the site of crossing over. The X arrangement is called chiasma (a crosspiece of wood; Fig. 5.15). The chiasma is the cytological sign of crossing over. During diakinesis, the chromatids become condensed and the nuclear envelope begins to break down.

Fig. 5.15. Chiasma forms at the site of crossing over. Metaphase I. The nuclear envelope and the nucleolus are decomposed. Microtubules form and establish contact to the kinetochores. The homologous chromosomes are aligned in the equatorial plane. Anaphase I. One of the homologous chromosomes with both chromatids moves to one, the other to the other spindle pole (Fig. 5.13). The maternally abd the paternally derived chromosomes assort randomly to one or the other spindle poles. (Basis of the 3rd Mendelian law; see chapter 7.) Random combination of the maternal and the paternal chromosomes is the "meaning" of meiosis, an essential factor of recombination, a basis of genetic variability. (Crossing over is another one.) Recombination is the basis of variability of the organisms, the foundation for adaptivity of the organisms to the changing life conditions. (Please note that a human may produce germ cells with as many as 223 = 8.4x106 different chromosome combinations due to the random assortment of chromosomes during anaphase I. The 8.4x106 value is elevated on the average of 23 crossing over per chromosome set and also by mutations.) Telophase I lasts only for a very short period of time. The nuclear envelope forms and a very short interphase follows. The interphase is unusual: the chromosomes are not decondensed and replication does not take place. The second meiotic (equational) division The second meiotic division is basically a mitosis Fig. 5. 13). Prophase II. Lasts only for a few minutes while the nuclear envelope disintegrate. Metaphase II. The chromosomes (one of the homologous with two chromatids) are aligned in the

equatorial plane in tight contact with the kinetochore microtubules. Anaphase II. The sister chromatids become separated and the chromosomes move to opposite spindle poles. The amount of DNA reaches the value characteristic for the haploid cells. Telophase II. Four haploid cells form. The nuclear envelope forms once again. The haploid cells mature to oocytes and polar bodies in females and to sperm in males. Meiotic nondisjunction Naturally nondisjunction, the unequal distribution of chromosomes between daughter cells, may and does happen during both the first (reductional) and the second (equational) meiotic divisions. Autosomes as well as sex chromosomes may be involved. Following fertilization of an aneuploid oocyte with sperm and a normal chromosome set, or vice versa, every cell of the zygote will be aneuploid. Obviously, organisms with all cells aneuploid are more striking than a clone of aneuploid cells in an otherwise normal organism. (Rules for the viability of the aneuploid organisms are the same as discussed for aneuploid cells). The best known type of autosome aneuploidy is Down's syndrome, i.e. trisomy 21. The frequency of giving birth to children with trisomy 21 increases dramatically in women over the age of 40: the frequency is less than 1% up to the maternal age of 30, and is over 20% over 44 years of age. Nondisjunction involves not only the autosomes, but also the sex chromosomes (Fig. 5. 16).

Fig. 5.16. Formation of aneuploid germ cells as the consequence of primary nondisjunction in the course of the meiotic divisions. The sex chromosomes are involved. Viability of individuals with abnormal numbers of sex chromosomes is only slightly reduced as compared to normal individuals. When germ cells with unusual numbers of sex chromosomes participate in fertilization, children are born who possess characteristic syndromes (Table 5.1; for details see the human genetics course). Nondisjunctions that happen in diploid cells are called primary nondisjunction. In secondary nondisjunction, aneuploid germ cells are produced

from already aneuploid cells. The extra chromosome will remain in the aneuploid cells during the two meiotic divisions and end eventually in the germ cells (Fig. 5.17).

Fig. 5.17. During the secondary non- disjunction, aneuploid germ cells derive with high frequency from the already aneuploid germ line cells. Table 5.1. Abnormal sex chromosome complements in humans. Chro- mosome

Birth frequency

Sex Features Barr body

X0 Turner syndrome

0.04 % Female Sterile. Short stature, broad neck. Reduced intelligence.

0

XYY 0,125 % Male Normal 0 XYY Klinefelter syndrome

0,14 % Male Sterile. Tall. Some breast development. Reduced intelligence.

XXX 1,10 % Female Reduced fertility and intelligence.

2

XXXX <0,03 % Female Normal 3 Prenatal diagnosis Prenatal diagnosis is a procedure to detect chromosome abnormalities (both numerical and structural) especially when abnormal fetal development is to be expected (e.g. for pregnant women over 40; Fig. 18). (We shall see in chapter 16 that during prenatal diagnosis DNA of fetus can also be analyzed). During prenatal diagnosis, karyotype of the fetus is established. The fetal cells may derive from the amniotic fluid, from chorionic villi and from the umbilical vein.

Fig. 5.18. Relationship between maternal age and the chance to have a Down syndrome child born. SUMMARY Replication of the material for inheritance is semiconservative. The replicated genetic material is equally shared between the two daughter cells following mitosis. Although rather different mechanisms evolved during evolution, a common feature of the transmission of chromosomes is that the transmitted genetic material is a unit of one genome. It is an essential feature of the genetic material that it supports, thorough different mechanisms, supporting high variability of the organisms, ensuring evolution and survival of the species. Change in the amount of genetic material in cells has serious consequences: lethality or reduced viability. REFERENCES 1. Principles of Genetics. Fristrom and Clegg, 1989, pp. 88-146. 2. Life: the Science of Biology, V.; 1998, pp. 200- 215, 250-256. 3. Molecular Cell Biology, 1995, pp. 177-188, 365-385 and 1090-1106. 4. Molecular Biology of the Cell, 1994, pp. 98-105, 357-365, 911-946 and 1011-1021. 5. Genetic analysis. Griffith et al., 1996, pp. 55-62 and 321-339.

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