Cell Division and Chromosome Theory of Inheritance PDF

6 Cell Division and Chromosome Theory of Inheritance The period that follows the formation of a cell by division of its mother cell until the time when the cell divides again to form two daughter cells is called the cell cycle. The cycle consists of four...

6 Cell Division and Chromosome Theory of Inheritance The period that follows the formation of a cell by division of its mother cell until the time when the cell divides again to form two daughter cells is called the cell cycle. The cycle consists of four phases, G1, S, G2 and M. The first three phases (G1, S and G2) comprise interphase while M constitutes cell division (mitosis or meiosis). The S phase lasts about 6–8 hours in mammalian cells, G2 about 3–4 hours, while the length of G1 is variable. Whereas DNA synthesis is restricted to the S (synthetic) phase, protein synthesis takes place throughout interphase. A cell entering mitosis or meiosis has double the quantity of DNA and chromosomal proteins. The cell organelles such as mitochondria and ribosomes are assembled throughout interphase in the cytoplasm and passed on to the two daughter cells. MITOSIS Cell division in the somatic or body cells of diploid organisms takes place by mitosis (Fig. 6.1) in successive stages described below. resting nucleus prophase metaphase anaphase telophase Fig. 6.1 Diagrams showing successive stages of mitosis. For convenience only 4 chromosomes are represented. 43  44 GENETICS Prophase The DNA in the diffuse chromatin of the resting nucleus in interphase has been duplicated in S phase preceding this cell division. The extended state of interphase chromatin allows transcription and replication of DNA, but is not suitable for division into two daughter cells. Therefore, prophase, the first stage of cell division shows contraction and condensation of chromatin into shorter, thicker fibres. By mid-prophase, the nucleolus starts to disappears and nuclear membrane breaks down, so that chromatin lies free in cytoplasmic space. Shortening of chromatin fibres continues to yield thicker, somewhat rod-like chromosomes. The breakdown of the nuclear envelope involves the enzyme Cdk kinase which is activated just before initiation of mitosis, in G2 phase of cell cycle. The inner face of the nuclear envelope is lined by a layer of fibrillar proteins of the cytoskeleton (called intermediate filaments), termed nuclear lamina. The enzyme Cdk kinase phosphorylates the lamin filament molecules, causing disassembly of nuclear lamina. The entire nuclear envelope that surrounds the condensing chromatin then breaks up into small vesicles which disperse into the cytoplasm. The nuclear pore complexes in the nuclear envelope also dismantle during fragmentation of the nuclear envelope. Metaphase The dissolution of the nuclear envelope at the end of prophase is described by some authors as the prometaphase stage. At metaphase, maximum condensation of chromatin fibres has been achieved giving rise to distinct rod-like chromosomes. Metaphase represents the most condensed state of chromatin in a cell. The molecular mechanisms responsible for chromosome condensation are still poorly understood. The process seems to involve the DNA untangling enzyme topoisomerase II (Chapter 14). The thick rod-like chromosomes begin to align themselves in the centre of the cell on what is conventionally referred to as the equatorial plate or the metaphase plate. Structure of Metaphase Chromosome: Each chromosome consists of two chromatids and a region of central constriction called centromere or primary constriction. Evidence has established that each chromatid consists of a single duplex DNA molecule. The centromere region contains many copies of highly repeated DNA sequences (details in Chapter 19). A small nodule-like structure called the kinetochore is present at the outer surface of the centromere in each chromatid. The kinetochore functions as the site of attachment of microtubules, a bundle of fibres making up a spindle fibre, all the fibres together constituting the mitotic spindle apparatus. Microtubules attached to the kinetochore are called chromosomal or kinetochore microtubules. The centromere divides longitudinally, and the two centromeres pull the chromatids apart to the two poles by means of spindle fibres. The division of the centromere into two at metaphase of mitosis is a key event that segregates accurately, half the genetic material for one daughter cell and half for the other daughter cell. This is ensured by the fact that the two chromatids contain duplicated DNA acquired from DNA synthesis in S phase of cell cycle preceding mitosis. Alignment of metaphase chromosomes in the equatorial region of the cell is followed by beginning of separation of chromatids to opposite poles. Misalignment of chromosomes in equatorial region arrests cells at metaphase and failure to segregate genetic material to the two daughter cells. The protein encoded by a gene MAD2 is normally localised at the kinetochores of prometaphase chromosomes and misaligned metaphase chromosomes, but is not present on chromosomes that have become properly aligned at the metaphase plate. Cells that possess mutant copies of a gene MAD2 fail to become arrested at metaphase when their chromosomes are misaligned. The presence of MAD2 at the kinetochores seems to provide a ‘‘wait’’ signal  CELL DIVISION AND CHROMOSOME THEORY OF INHERITANCE 45 that delays progression into anaphase. As each chromosome becomes aligned at the metaphase plate, its kinetochore loses all of its MAD2 molecules. It is only after MAD2 protein is absent from all of the chromosomes that anaphase can begin. Microtubule Organisation and Centrosome: In most cells microtubules extend outward from a microtubule-organising center, which in animal cells is called the centrosome. Centrosomes are absent in plant cells. During mitosis, microtubules extend out from the duplicated centrosomes to form spindle fibres. Thus the centrosome seems to play a key role in determining the intracellular organisation of microtubules, but details of its function are not known. The centrosomes contain a pair of centrioles, oriented perpendicular to each other, and surrounded by pericentriolar material. Centrioles are cylindrical structures consisting of nine triplets of microtubules, similar to the basal bodies of cilia and flagella. During spindle formation, microtubules emerge from the pericentriolar material around a centrosome and form a star-like aster. These are referred to as astral microtubules. The pericentriolar material acts as the nucleating site for microtubules of the aster. The fast growing ends of the microtubules, denoted plus ends, are away from the centrosome. The microtubule initiating activity appears to be stimulated by the Cdk protein. Following aster formation, the two centrosomes separate and move to opposite poles, while microtubules stretching between them increase in number and elongate. These are called polar microtubules. The two centrosomes establish two poles of the mitotic spindle. After mitosis, one centrosome is distributed to each daughter cell. Centrosomes are not essential components of mitotic spindles in all cells. Many animal cell types do not have centrosomes, nor do higher plants. The minus ends of the microtubules are thought to be gathered into a cluster at each spindle pole through the activity of motor proteins described later. Anaphase The two sister chromatids of each chromosome split apart and start moving towards opposite poles. There is rapid degradation of an inhibitory protein that acts a proteinaceous ‘‘glue’’ holding the two chromatids together, that facilitates the onset of anaphase. The degradation of anaphase inhibitory proteins occurs in response to activity of the mitotic Cdks. The separation of sister chromatids requires activity of topoisomerase II. The chromatids (actually chromosomes now) start moving towards the poles, accompanied by shortening of microtubules attached to their kinetochore. Shortening of the microtubule results from the loss of subunits at the kinetochore (minus end) during anaphase. When the chromosomes reach the poles, there is elongation of the mitotic spindle, resulting in a simultaneous movement of the spindle poles further away from each other. This is accompanied by the addition of tubulin subunits to the plus ends of the polar microtubules. The movement of chromosomes to the poles has been observed to be completed within 2 to 60 minutes. The forces that bring about movement are discussed later in this chapter. Telophase Chromosomes at the poles organise into a mass of chromatin at each pole that marks the beginning of telophase. The process of formation of nuclear envelope begins when membranous vesicles start fusing with one another to produce a double-membraned envelop surrounding the chromatin. The nucleolus reappears. Thus, two daughter nuclei that are identical in genetic constitution to the parent nucleus are formed. Cytoplasmic membranous organelles such as Golgi complex, endoplasmic reticulum reform in each daughter cell.  46 GENETICS Cytokinesis In animal cells division of the cytoplasm of the parent cell is initiated at late anaphase as an indentation of the cell surface that appears as a band around the cell. The band deepens to form a furrow, the plane of the furrow lying in the same plane as the equatorial or metaphase plate on which chromosomes were aligned. Further deepening of the furrow splits the parent cell into two daughter cells. In plants a cell plate consisting of polysaccharides starts depositing in the central region of the parent cell. This gives rise to the first wall or middle lamella between the two cells. Later on a primary wall is deposited towards the inside of the middle lamella of each daughter cell. MEIOSIS The division which takes place in cells of the germ line is called meiosis (Fig. 6.2). It results in four products of a parent cell with half the amount of genetic material because DNA is duplicated only once and there are two cell divisions, meiosis I and II. Leptotene Zygotene Pachytene Diplotene Diakinesis Metaphase I Anaphase I Telophase I Prophase II Metaphase II Anaphase II Telophase II Fig. 6.2 Diagram illustrating stages of meiosis. Only four chromosomes are shown. Prophase I is of very long duration and consists of five substages. Leptotene shows very long, thin thread-like chromosomes. Zygotene marks the initiation of pairing of homologous chromosomes. By some force of attraction identical partners are drawn towards each other.  CELL DIVISION AND CHROMOSOME THEORY OF INHERITANCE 47 Condensation and shortening of chromosomes is visible. By pachytene pairing is complete and stabilised. Due to the intimate nature of pairing or synapsis and continued shortening of chromosomes, thick ribbon-like bivalents are formed. Pairing is exact so that chromomeres and centromeres of homologues lie against each other. The number and arrangement of bead- like chromomeres, position of centromeres and arm length are distinct for each bivalent and allow mapping of pachytene chromosomes. The nuclear membrane and nucleolus disappear. At diplotene a force of repulsion between the paired chromosomes tends to draw them apart. They are held together at positions of chiasmata where genetic crossing over or exchange of segments takes place. Continued condensation of chromosomes through diakinesis gives rise to the short thick cross-shaped configurations of chromosomes. By Metaphase I maximum condensation has been achieved giving rise to short rod-like bivalents which move towards the centre of the cell and align in the equatorial plane. Their centromeres become attached to spindle fibres running to opposite poles and homologues separate. Due to separation of homologues, half the number of chromosomes will reach one pole and one half of the other pole. Consequently Metaphase I is referred to as the reductional division. The beginning of separation of homologous is also the beginning of Anaphase I. When an entire set of chromosomes reaches either pole we call it Telophase. The nuclear membrane and nucleolus are reorganised to form two daughter nuclei. The newly formed nuclei go through a short rest period or interkinesis before entering the second meiotic division. Prophase II of meiosis is initiated by condensation of chromosomes and is completed rapidly as in mitosis. Metaphase II shows the equatorial alignment of rod like chromosomes which have reached their maximum limit of contraction. The centromere of each chromosome now divides longitudinally. The daughter centromeres are attached to spindle fibres and separate to the poles as in mitosis. Thus in meiosis, metaphase II is an equational division. Anaphase II and telophase II are completed as in meiosis I and in mitosis. At the end of meiosis II four products are formed each with half the amount (haploid) of genetic material that was contained in the original parent nucleus. Occurrence of Meiosis In sexually reproducing diploid plants meiosis takes place in the anther and the ovule. One or more specialised cells of the germ line known as archesporium divide mitotically to produce the sporogenous cells. In the case of male, sporogenous cells multiply and increase in number. After a certain stage they stop dividing, are ready to enter meiosis and are called microspore mother cells. The four products of meiosis are united in a tetrad but later separate as uni- nucleate microspores. Thickenings deposited on the microspore wall produce uninucleate pollen grain which soon becomes binucleate and a large vegetative cell and a small generative cell are organised. In many plants pollen grains are shed in this stage. The nucleus of the generative cell divides usually after pollen germination has begun on the stigma resulting in two male gametes. One male gamete fertilises the egg to form a zygote: the second male gamete fuses with the secondary nucleus in the embryo sac and gives rise to the endosperm which provides nutrition to the growing embryo. Similarly in the case of female, the sporogenous cell in the ovule enlarges to become a megaspore mother cell which divides meiotically to form a tetrad of four megaspores. Usually three megaspores degenerate and one enlarges into the embryo sac. By three mitotic divisions the nucleus of the megaspore forms 8 nuclei (haploid) of which one organises as the egg. In the male in higher animals (mammals and man) the spermatogonial cells in the testis increase in number by mitosis. When they are ready to divide meiotically they are called  48 GENETICS primary spermatocytes. During meiosis II they are called secondary spermatocytes. After meiosis they organise into the elongated spermatids which finally produce sperms after undergoing some changes (head, middle piece and tail are formed; some histone proteins are changed; motility is acquired). In the female of mammalian species including humans, and in contrast to the males meiosis takes place during the embryonal life of an individual. During the first few months after conception in human beings, the primary oocytes in the foetal ovary start undergoing meiosis. After meiosis I the primary oocyte produces a secondary oocyte which alone will produce the functional ovum, and a small first polar body. The polar body gives rise to two more polar bodies: eventually all three polar bodies degenerate. A unique feature of ovarian meiosis is that it stops at about metaphase II stage in the secondary oocyte. In this condition the secondary oocytes (about 400 in number) remain suspended for 40 to 50 years of life after birth. At puberty ovulation starts when one oocyte gets released from the ovary wall each time and is discarded. If an oocyte gets a chance to meet a sperm, then before fertilisation it completes the remaining stages of meiosis (anaphase II to telophase II). Out of the four resulting cells three are polar bodies which will degenerate; the remaining cell enlarges and functions as ovum during fertilisation. Genetics of Meiosis There are two special features of meiosis: production of haploid gametes containing recombined genetic material of the two parents; the process of genetic exchange and recombination in homologues. Some aspects of recombination are discussed in Chapters 8, 19 and 22. Genetic events in meiosis have also been studied from mutants. Mutations affecting meiosis lead to abnormalities in genetic recombination or in chromosome segregation. Many such mutants have been isolated in the lower eukaryotes like yeast, Neurospora, and a few higher plants. The work on yeast mutants has shown that recombination is regulated at two levels: control of overall frequency of crossing over; and controls which influence the frequency of crossing over only in particular regions. Some mutations are known to affect the subsequent stages of chromosome segregation. There are also mutations which suppress initiation of meiosis. Detailed studies of meiotic mutants have been carried out in Drosophila. Meiosis is unusual in normal Drosophila males due to absence of crossing over. The females show cross- overs in all chromosome pairs except number IV. Mutations in male meiosis affect chromosome segregation. One such effect is nondisjunction due to which chromosomes fail to disjoin and move to opposite poles either at meiosis I or II. Most meiotic mutants of D. melanogaster interfere with meiosis I either in male or in female, but not in both. This implies that control of meiosis I is different in male and in female flies. Some mutations affecting meiosis I in Drosophila males have been investigated. The mutant segregation distorter (SD) is due to a dominant gene on chromosome II. The heterozygous males (SD/ ±) transmit the mutant SD gene to 50% of the progeny; the homozygous (SD/SD) males are sterile. Another mutant in males called recovery disrupter (RD) is due to an X-linked gene. It causes fragmentation of Y- bearing sperm resulting in excess of female flies in the progeny. A few more mutations in males such as mei-S8 and mei-081 cause nondisjunction at meiosis in Drosophila females such as c(3) G, and mei-218 either reduce recombination or interfere with chromosome segregation. Those that affect recombination also cause increased nondisjunction of all chromosomes. The Spindle Apparatus Electron microscopy has shown that spindle fibres are made up of bundles of parallel filaments called microtubules. The microtubules are assembled from cytoplasmic proteins namely α and β tubulin, and have an outside diameter of 24 nm, a central lumen of 15 nm across, and variable  CELL DIVISION AND CHROMOSOME THEORY OF INHERITANCE 49 length. In cross section a microtubule appears circular in outline, the circle itself being composed of about 13 smaller circles. These small circles represent cross sections of long strands called protofilaments which are assembled from the globular protein subunits, the α and β tubulin present in the cytoplasm. One molecule each of α and β tubulin become associated to form a dimer. The dimers are arranged in linear order to form protofilaments (Fig. 6.3). Each dimer appears to have a specific binding site for colchicine and another for vinblastine (Weisenberg, 1972), both of which inhibit spindle formation by preventing the assembly of microtubules. 8 nm a-tubulin subunit b-tubulin subunit (a) (b) Fig. 6.3 Cross sectional and side views of spindle microtubules. The microtubules are supposed to be present in the form of a cytoplasmic network in a resting cell. During cell division they become organised as spindle fibres. They are disassembled after cell division. It seems that for normal mitosis there must be a state of equilibrium between microtubules and free subunits of tubulin present in the cytoplasm. Low temperatures disturb the equilibrium and dissociate microtubules. The alkaloid colchicine obtained from the corms of a liliaceous plant Colchicum autumnale binds to tubulin and prevents formation of spindle fibres. Due to the resulting failure of chromosome movement, cell division becomes arrested at metaphase. Such cells may either degenerate, or the duplicated chromosomes may form a nucleus which is polyploid. The effect of colchicine can be reversed by depriving the cells of colchicine. A few other chemicals such as nitrous oxide, acenaphthene, chloral hydrate, vinblastine and podophyllotoxin have the same effect as colchicine. Forces Required For Chromosome Movement The forces powering chromosome movement have not been understood. A variety of different molecular motors have been identified in a wide variety of species. All of the motors thought to be involved in chromosome movement are microtubule motors that include some kinesin-related proteins and cytoplasmic dynein. Kinesin and motor proteins dynein move along microtubules in opposite directions, kinesin toward the plus end and dynein toward the minus end. Kinesin translocates along microtubules in only a single direction, toward the plus end. The kinesin molecule is 380 kDa in weight, consists of two heavy chains of 120 kDa each, and two light chains, 64 kDa each. The heavy chains have long α-helical regions wound around each other in coiled coil manner. The amino-terminal globular head domains of the heavy chains are the motor domains of the molecule. They bind to both microtubules and ATP, the hydrolysis of ATP providing the energy required for movement. The tail portion of the kinesin molecule consists of the light chains in association with the carboxy-terminal domains of the heavy chains. This portion of kinesin is  50 GENETICS responsible for binding to other cell components, such as organelles and membranous vesicles that are transported along microtubules by the action of kinesin motors. Dynein is a very large molecule, up to 2000 kDa, consisting of two or three heavy chains, complexed with various light polypeptides. In dynein too, the heavy chains form globular ATP- binding motor domains that are responsible for movement along microtubules. The light chains of this molecule bind to organelles and membranous vesicles. All members of the dynein family move toward the minus ends of microtubules. Regulation of the Cell Cycle Most eukaryotic organisms duplicate cells by following a more or less similar cell cycle. Since diverse organisms follow a similar pattern for cell duplication, it implies that the cell cycle is under genetic control. Disruption of the genetic controls leads to uncontrolled cell proliferation, as seen in malignancy. Regulation of the cell division cycle involves extracellular signals from the environment as well as internal signals that exert their effect on processes during different cell cycle phases (G1, S, G2 and M). In addition, cellular processes such as cell growth, DNA replication and cell division must be coordinated for progression of cell cycle. This is accomplished by a number of control points that check and regulate progression through the different phases of cell cycle. An important cell cycle regulatory point occurs late in phase G1 and controls progression from G1 to S. This regulatory point was first found in yeast (Saccharomyces cerevisiae), where it is referred to as START (Fig. 6.4). After passing START, cells become committed to go through one cell division cycle. In yeast, the passage through START is controlled by external signals such as availability of nutrients. If there is shortage of nutrients, yeast cells become arrested at START and enter a nondividing resting stage. START is also the point at which cell growth is coordinated with DNA replication and cell division. Budding yeasts which produce progeny cells of different sizes, cell size is monitored by a control mechanism. Accordingly, each cell must reach a minimum size before it can pass through START. Mother cell Daughter cell + M End of To cross START M phase mother cell needs G2 cell size, nutrients mating factors G1 S START Bud formation initiated Fig. 6.4 Cell cycle of yeast is regulated at a point START in late G1. After mother cell crosses START, bud formation begins, and is completed after mitosis. The size of the bud reflects the position of the cell in cell cycle.  CELL DIVISION AND CHROMOSOME THEORY OF INHERITANCE 51 In eukaryotes, cell proliferation is regulated at the G1 phase of cell cycle called restriction point. In contrast to yeasts, the passage of mammalian cells through cell cycle is regulated by extracellular growth factors that signal cell proliferation, instead of availability of nutrients. When the appropriate growth factor is present, cells pass the restriction point and enter S phase. Once it has passed through the restriction point, the cell becomes committed to proceed through S phase and complete the cell cycle, even in absence of further growth factor stimulation. Progression through cell cycle stops at the restriction point if appropriate growth factors are not available in G1. Thus cells become arrested at a quiescent stage called G0 (G zero). Cells in G0 are metabolically active, but have reduced rates of protein synthesis. It has been noted that many animal cells remain in G0 unless induced to proliferate by appropriate growth factors or other extracellular signals. A good example is afforded by skin fibroblasts that remain arrested in G0. When they are required to repair damage resulting from a wound injury, they are stimulated to divide. The proliferation of fibroblasts is signalled by platelet-derived growth factor released from blood platelets during clotting. An example of cell cycle control in G2 is provided by vertebrate oocytes. Oocytes can remain arrested in G2 for very long periods of time, until they are triggered by hormonal stimulation to proceed to M phase. In human female, oocytes become arrested in G2 during fetal life for several decades until stimulated to complete the meiotic cell cycle. Checkpoints in Cell Cycle The coordination between the different phases of the cell cycle depends on a system of checkpoints as well as feedback controls that prevent entry into the next phase of the cell cycle until events of the previous phase have been completed. To ensure that incomplete or damaged chromosomes are not replicated and passed on into daughter cells, there are several cell cycle checkpoints. One well defined checkpoint occurs in G2 which prevents initiation of mitosis until DNA replication is completed. The G2 checkpoint senses unreplicated DNA, then generates a signal that leads to cell cycle arrest. Thus the G2 checkpoint prevents the initiation of M phase before completion of S phase. The cells remain in G2 until the entire genome has been replicated. The checkpoint then releases inhibition of G2, allowing the cell to progress into mitosis, and completely replicated chromosomes are passed on into daughter cells. Progression into the cell cycle is also arrested at the G2 checkpoint in response to DNA damage caused by radiation. In this case, arrest in G2 allows time for repair of damage. DNA damage can also arrest cell cycle progression at a checkpoint in G1, that allows repair of damage to take place before the cell enters S phase. Thus, replication of damaged DNA is prevented. In eukaryotes, arrest at the G1 checkpoint is mediated by the action of a protein called p53 which is rapidly induced in response to damaged DNA (Fig. 6.5). The gene encoding p53 is frequently mutated in human cancers. Mutations in this gene lead to loss of function of p53 that prevents G1 arrest, in response to DNA damage. Thus, damaged DNA is replicated and passed into daughter cells without being repaired. Through damaged DNA the daughter cells inherit an increased frequency of mutations and instability of the genome which contribute to cancer development. Mutations in p53 gene are most common genetic alterations in human cancers. The third cell cycle checkpoint occurs toward the end of mitosis. This checkpoint ensures alignment of chromosomes on the mitotic spindle, so that a complete set of chromosomes is distributed into the daughter cells. If one or more chromosomes fail to align correctly on the spindle fibres, the checkpoint leads to arrest at metaphase. The chromosomes do not separate at the equator until a complete complement of chromosomes has been organised for accurate distribution to the two poles.  52 GENETICS DNA damage p53 increase Cell cycle arrest in G1 check point G1 G1 check point MG2 S Fig. 6.5 Involvement of p53 in G1 arrest induced by DNA damage. When DNA is damaged by irradiation or any other means, p53 levels increase and signal cell cycle arrest at the G1 checkpoint. Mechanisms for Regulation of Cell Cycle Studies on molecular mechanisms that control the progression of mammalian cells through the division cycle have revealed that the cell cycle is controlled by a set of protein kinases which are responsible for triggering the major cell cycle transitions. Cdc2 and Cyclin Studies on frog oocytes indicated that the oocytes that are arrested in G2 phase of cell cycle could be stimulated to enter into the M phase of meiosis by hormonal stimulation. Later investigations showed that oocytes arrested in G2 could be induced to enter M phase by microinjection of cytoplasm taken from oocytes that had been hormonally stimulated. Thus, a cytoplasmic factor present in hormonally stimulated oocytes would allow oocytes that had not been exposed to hormone, to progress from G2 to M. This cytoplasmic factor was called maturation promoting factor (MPF). Further studies showed that MPF is not specific to oocytes, but appeared to act as a general regulator of the transition from G2 to M in somatic cells as well.  CELL DIVISION AND CHROMOSOME THEORY OF INHERITANCE 53 Studies on temperature-sensitive mutants of yeast Saccharomyces cerevisiae that were defective in cell cycle progression also contributed to understanding of regulation of cell cycle. These mutants called cdc (cell division cycle mutants) were remarkable as they showed growth arrest at specific points in the cell cycle. For example, the mutant designated cdc28 replicates normally at the permissive temperature, but at the nonpermissive temperature there is arrest of cell cycle at START, thus indicating that the cdc28 protein is necessary for passage of cells through the regulatory point START in G1. Further investigations on cdc2 brought to light two important points. First, that cdc2 encodes a protein kinase, indicating the role of protein phosphorylation in cell cycle regulation. Second, a gene related to cdc2 was identified in humans and shown to function in yeasts, implying that the cell cycle regulatory activity of this gene is conserved. Studies on protein synthesis in sea urchin embryos provided further insights into cell cycle regulation. After fertilisation, sea urchin embryos go through rapid cell divisions. However, the entry of embryonic cells into M phase requires new protein synthesis. Two proteins were then identified that accumulate during interphase and are degraded at the end of each mitosis. These proteins were called cyclin A and cyclin B. It seemed that cyclins might be able to induce mitosis by controlling entry and exit from M phase. It was then demonstrated that cyclins control G2 to M transition. Subsequent investigations on MPF, the regulator of cell cycle gave interesting results when it was shown that MPF is composed of two subunits, cdc2 and cyclin B. Cyclin B is required for the catalytic activity of the cdc2 protein kinase. Thus, MPF activity is controlled by the periodic accumulation and degradation of cyclin B during cell cycle progression. Further studies have demonstrated regulation of MPF by phosphorylation and dephosphorylation of Cdc2 protein. Cyclin B synthesis takes place in S phase, and it forms complexes with Cdc2 protein throughout S and G2. During this time, Cdc2 is phosphorylated at two regulatory positions, one on threonine-161 (required for Cdc2 kinase activity), the other of tyrosine-15 catalysed by a protein kinase called Wee1 that inhibits Cdc2 activity resulting in accumulation of inactive Cdc2/cyclin B complexes during S and G2. The transition from G2 to M takes place by activation of the Cdc2/cyclin B complex brought about by dephosphorylation of threonine- 14 and tyrosine-15 by a protein phosphatase called Cdc25. After activation the Cdc2 protein kinase phosphorylates a variety of target proteins for entry into M phase. Furthermore, Cdc2 activity also stimulates degradation of cyclin B by ubiquitin-mediated proteolysis. Degradation of cyclin B inactivates Cdc2, causing the cell to exit mitosis, undergo cytokinesis and enter interphase. The Cyclins The characterisation of the Cdc2/cyclin complex have provided insights into the regulation of the cell cycle. Both Cdc2 and cyclin B are found to be members of large families of related proteins, with different members of these families controlling distinct phases of the cell cycle. As already stated, in the case of yeasts, Cdc2 controls passage through START and entry into mitosis. By associating with distinct cyclins, Cdc2 is able to phosphorylate different substrate proteins required during specific phases of the cell cycle. In higher eukaryotes, cell cycles are controlled not only by multiple cyclins, but also by multiple Cdc2-related protein kinases. Referred to as Cdks (cyclin-dependent kinases). The original member of this family, Cdc2, is known as Cdk1 with others following up to Cdk8. Members of the Cdk family associate with specific cyclins to accomplish progression through different stages of the cell cycle. Briefly, progression from G1 to S is regulated by Cdk2 and Cdk4 in association with cyclins D and E; Cdk4 and Cdk6 control progression through restriction  54 GENETICS point in G1 in association with cyclins D1, D2, and D3; Cdk2 and cyclin E complexes are required for G1 to S transition as well as initiation of DNA synthesis in S; Cdk2 with cyclin A control progression of cells through S phase; Cdc2 complexed with cyclin B plays a role in transition from G2 to M. The activity of Cdks during cell cycle is regulated by at least 4 molecular mechanisms. The first level of regulation involves formation of Cdk/cyclin complexes. Second, activation of Cdk/cyclin complex by phosphorylation of a Cdc threonine residue at position 160. The third involves inhibitory phosphorylation of tyrosine residues near the Cdk amino terminus. Fourth, binding of inhibitory proteins called Cdk inhibitors or CkIs to Cdk/cyclin complexes. The combined effects of these multiple mechanisms of Cdk regulation accomplish control of cell cycle progression in response to both checkpoint controls and to the large number of extracellular stimuli that regulate cell proliferation. The D-Type Cyclins The proliferation of animal cells is regulated by a variety of extracellular growth factors that control progression of cells through the restriction point in late G1. In absence of growth factors, cells are not able to progress through the restriction point, become quiescent or enter G0. When stimulated by growth factor, the cells can re-enter the cell cycle. The study of D-type cyclins has provided an important link between growth factor signalling and cell cycle progression. Cyclin-D synthesis is induced in response to growth factor stimulation and continues as long as growth factors are present. Growth factors must necessarily be present through G1 to allow complexes of Cdk 4, 6/cyclin D to drive cells through the restriction point. But if growth factors are removed prior to G1, the concentration of D-type cyclins falls rapidly. The cells are then unable to pass through G1 and S, become quiescent and enter G0. Thus, D-type cyclins play a role in growth factors control of progression of cells through G1. The insights gained on growth factors and cyclin D led to a most important finding that defects in cyclin D regulation contribute to the loss of growth regulation that is characteristic of cancer cells. Indeed, many human cancers have been found to arise as a result of defects in cell cycle regulation, whereas many other cancers result from abnormalities in the intracellular signalling pathways activated by growth factor receptors. Therefore, mutations which result in the continuous unregulated expression of cyclin D1 lead to development of several human cancers, including lymphomas and breast cancers. Furthermore, a key substrate protein of Cdk4, 6/cyclin D complexes is found to be frequently mutated in a variety of human tumors. This protein is called Rb since it was first identified as the product of a gene responsible for retinoblastoma, a childhood eye tumor. It was later found that if Rb is rendered nonfunctional due to mutation, it could result in a variety of human cancers, besides retinoblastoma. Rb seems to be the prototype of a tumour suppressor gene, a gene whose inactivation leads to development of tumors. Subsequent investigations have revealed that Rb plays a significant role in coupling the cell cycle machinery to the expression of genes required for cell cycle progression and DNA synthesis. Rb’s function is regulated by changes in its phosphorylation as cells traverse the cell cycle. For example, Rb becomes phosphorylated by Cdk 4, 6/cyclin D complexes as cells pass through the restriction point in G1. When Rb is underphosphorylated (present in G0 or early G1), Rb binds to members of the E2F family of transcription factors, which regulate expression of several genes involved in cell cycle progression, such as gene encoding cyclin E. Details of Rb’s action indicate that Rb acts as a molecular switch that converts E2F from a repressor to an activator of genes required for cell cycle progression.  CELL DIVISION AND CHROMOSOME THEORY OF INHERITANCE 55 Inhibitors of Cell Cycle Agents that cause damage to DNA result in arrest of cell cycle. Cell contacts between cancerous cells in culture arrest division (contact inhibition). A number of extracellular factors inhibit cell division, frequently mediated by regulators of the cell cycle machinery, usually by induction of Cdk inhibitors. For example, arrest of cell cycle in response to DNA damage is mediated by protein p53. Protein p53 a transcriptional regulator that stimulates expression of the Cdk inhibitor known as p21. The p21 inhibits several Cdk/cyclin complexes. Following DNA damage, p53 induces synthesis of p21 and brings about cell cycle arrest. Protein p21 can also directly inhibit DNA replication. A well characterised extracellular inhibitor of animal cell proliferation is the polypeptide factor TGF-β, that inhibits the proliferation of a variety of types of epithelial cells by arresting cell cycle progression in G1. This action of TGF-β is mediated by induction of Cdk inhibitor p15 which blocks Cdk4 activity. Then Rb is not phosphorylated and the cell cycle becomes arrested in G1. THE CHROMOSOME THEORY OF INHERITANCE In order to understand the role played by chromosomes as carriers of hereditary material, we must turn the clock backward to some landmarks in the history of genetics. The earliest records date back to the work of the 18th Century plant hybridisers. One of them Kölreuter had some theoretical knowledge on the basis of his practical experience in hybridisation work on Nicotiana. He crossed Nicotiana rustica with N. paniculata and found that for all 13 characters studied by him, the resulting hybrid was intermediate between the two parents. The results of his reciprocal crosses were the same. Thus he was first to suggest that the hereditary contribution of the two parents to their offspring was equal. The later half of the 19th century then covered a few more milestones in genetics. Oscar Hertwig in 1876 studied fertilisation in sea urchins while Strasburger (1877, 1884) and Schmitz (1879) made similar observations in plants. Hertwig noted the presence of two nuclei in the fertilised egg and concluded that fertilisation involved fusion of nuclei from two parental gametes. Working with seed plants, Strasburger in 1884 could clearly show that in the orchid Orchis latifolia the pollen tube travels downward through the pistil and enters the embryo sac. But the existence of a hereditary substance inside the nucleus was first postulated by August Weismann working as Professor at the University of Freiburg. He called the substance germplasm. The next problem was to understand the continuity of the germplasm i.e. its transmission from parent to offspring. It was Schneider in 1873 who first demonstrated the continuity of germplasm (nucleus) through cell division by observing condensing chromosomes and their movements in dividing eggs of the flatworm Mesostomum. Walter Flemming in 1878 was first to study mitosis in detail and coined the term. He could observe metaphase chromosomes longitudinally split in half and their movement apart to each of the two daughter nuclei. In this way a parent cell could pass on two identical groups of chromosomes to its two daughter cells. Another evidence for the role of chromosomes in heredity came when E. Van Beneden and Weismann showed that gametes contained only half the number of chromosomes present in somatic tissues and that the somatic number was restored at fertilisation. In 1883 Wilhelm Roux went a step further by suggesting that cell division not only divides the quantity of nuclear material, but also its properties or individual qualities (hereditary determinants of a trait).  56 GENETICS The cytological studies on chromosomes conducted till 1900 constitute the first or premendelian phase in the development of the Chromosome Theory of Inheritance. But after the rediscovery of Mendelism, all efforts were directed towards determining the relationship between Mendelian factors and chromosomes. In Mendel’s crosses, the F2 progeny segregated in typical ratios as expected on theoretical grounds. But the cytological basis of Mendelism was not understood until the behaviour of chromosomes during cell division was known. In 1902 Correns provided evidence that segregation of Mendelian factors occurred during meiotic division. In 1901 Montgomery made the important observation that during meiosis maternal chromosomes paired only with paternal chromosomes. He studied a very favourable material Ascaris megalocephala var. univalens which has only two chromosomes. Obviously when there is pairing it must involve a maternal and a paternal chromosome. In 1902 McClung could associate a specific heritable trait in grasshopper with a specific chromosome. In the same year Theodor Boveri studied multipolar mitoses in sea urchin embryos and concluded that the individual chromosomes are qualitatively distinct from each other and carry different hereditary determinants. Boveri’s conclusion was further strengthened by the observations of Walter Sutton in 1903 who could demonstrate morphological differences between the 23 chromosomes of the grasshopper Brachystola. Sutton was a graduate student of Wilson at Columbia University and is credited for demonstrating a parallel between meiotic behaviour of paired chromosomes and the behaviour of pairs of Mendelian factors. He could explain Mendel’s principle of segregation by showing cytologically that in meiosis one member of a pair of homologous chromosomes goes to one daughter cell, the other to the second daughter cell. Mendel’s second principle of independent assortment found cytological proof from the fact that members of one pair of homologous chromosomes move to the poles independently of the members of another pair. In this way the final mixture of chromosomes (paternal and maternal) at a pole is different from one cell to another. In other words, segregation of paired homologues at Metaphase I occurs at random. This phenomenon was also demonstrated explicitly by Carothers, a student of McClung, in grasshopper in which one pair of homologous chromosomes is such that one member is larger than the other partner. Moreover there is one unpaired chromosome in the grasshopper. She observed separation of the two unequal chromosomes to the two poles in about 300 cells and found that the unpaired chromosome passed to one pole with the larger homologue in about 50% of cells, and with the smaller homologue in the remaining 50% cells. From this she inferred that different chromosome pairs assort independently. Boveri’s work on sea urchins was confirmed later by Blakeslee in 1922 while working with Datura (Jimson weed). The normal diploid chromosome number in Datura is 24 which form 12 pairs in meiosis. Some plants however have 25 chromosomes (designated trisomics today). An interesting feature of the 25th chromosome is that it could be identical to any one of the 12 pairs so that Datura plants could be classified into 12 types. Blakeslee found that the shape and size of the fruit capsule was different in all the 12 groups of plants. This proved that the 12 chromosomes differed from each other qualitatively and each produced a morphologically different capsule. The work of Boveri and Sutton provided excellent correlation between Mendelian factors and chromosomes and became known as the Sutton-Boveri theory. Their experiment led to the discovery of the following characteristic features of chromosomes:  CELL DIVISION AND CHROMOSOME THEORY OF INHERITANCE 57 (a) Continuity of chromosomes from one cell division to the next, (b) Qualitative differences between individual chromosomes, (c) Pairing of maternal with paternal chromosomes, (d) Segregation of chromosomes at random i.e. independent assortment of chromosomes. Sutton in 1903 found that the number of factors obeying Mendel’s law were more than the number of chromosome pairs in the cell. This means that there were many genes on a single chromosome and Mendel’s law of independent assortment could not be applied to them (due to phenomenon of linkage described in Chapter 8). T.H. Morgan in 1909 showed that in Drosophila the gene for white eye colour was linked to the sex chromosome. He crossed a white-eyed male fly with normal red-eyed female and obtained an F1 progeny of red-eyed flies only. The F1 red-eyed flies when mated amongst themselves produced F2 progeny in the ratio of 3 red to 1 white. But the striking feature was that all the F2 white-eyed flies were males. In this way Morgan demonstrated that the gene for eye colour was present on the X-chromosome. The following year he showed that the genes for yellow body colour and for miniature wings were also carried on the X-chromosome. By performing dihybrid crosses (involving eye pigment and body colour) he could show that crossing over could change positions of linked genes (incomplete linkage). C.B. Bridges, a student of Morgan provided support to the chromosome theory from his studies on nondisjunction. When a white-eyed female is crossed with a red-eyed male, normally the F1 consists of red-eyed females and white eyed males. Bridges found that some-times red- eyed males and white-eyed females were also present. He found a cytological explanation by which the two X-chromosomes failed to separate at Metaphase I of meiosis. Thus both X’s passed together into 50 per cent of resulting eggs; the remaining 50 per cent of eggs did not receive an X-chromosome, a phenomenon known as primary nondisjunction. If an egg with two X-chromosomes is fertilised by a normal Y carrying sperm, the resulting zygote has XXY chromosome constitution and is female. Bridges could demonstrate cytologically that such a female indeed had XXY chromosomes thus establishing the validity of the chromosome theory of inheritance. QUESTIONS 1. A preparation of metaphase cells showed 13 chromosomes. Which explanation/s given below could be correct: (a) The cells are at metaphase I of meiosis; (b) The cells are in mitosis; they belong to an individual with XO sex chromosome constitution; (c) Impossible to explain; the cell has an uneven number of chromosomes; (d) The cells are at metaphase II of meiosis. 2. How many chromatids in a zygote nucleus would be: (a) of maternal origin? (b) of paternal origin? 3. Distinguish between: (a) mitotic metaphase and meiotic metaphase stages; (b) mitotic prophase and mitotic telophase.