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Term Paper on Cell Division

Term Paper Contents:

1. Term Paper on the Introduction to Cell Division
2. Term Paper on the Synchoronised Cell Division
3. Term Paper on the Source of Energy for Cell Division
4. Term Paper on the Blocking Cell Division
5. Term Paper on the Initiation of Cell Division
6. Term Paper on the Nature and Formation of the Mitotic Apparatus
7. Term Paper on the Furrow Formation and Cleavage (Cytokinesis)

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Term Paper # 1. Introduction to Cell Division:

Cells that are growing are destined to divide. Cell division takes place at different intervals which depend upon the species and the environmental conditions. Escherichia coli on a medium containing water, glucose, ammonium chloride, potassium basic phosphate, sodium sulfate and trace metals, kept at 37°C., divides every 45 minutes. If supplied with amino acids and some other organic materials at 37°C., it divides every 20 minutes. Eukaryotic cells divide at a slower rate than bacteria.

It is true that eggs of some marine animals from warm climates divide about every 30 minutes, and those from cooler climates divide at about one hour intervals. However, such cells have reserves and need only convert these to cell constituents before dividing. Protozoan cells divide somewhat more slowly; Tetrahymena pyriformis, under favourable conditions, divides every 3 to 4 hours, and Paramecium aurelia, every 6 hours.

Cells of higher plants and animals divide about once or twice a day. Thus the generation time (time between divisions) for cells of the broad bean Vicia faba is about 14.3 hours; for the cells of Tradescantia paludosa, about 17 hours; for Chinese hamster fibroblasts, 11 hours; and for mouse fibroblasts, 22 hours. Cell division or mitosis, which consists of prophase, metaphase, anaphase and telophase, occupies only a small part of the generation time. The major portion of the division interval is interphase.

The cell, the basic unit of all living systems, has to maintain the integrity from generation to generation for that the heredity material has to be copied accurately, i.e. the cell division must result in two daughter cells similar to each other and resembles the parental cell from which they are produced. However, in cell divisions of sex cells, the daughter cells may be different from parent cell but they possess most of the essential features in common.

There are two kinds of cell division in eukaryotes. Mitosis is a division involved in the develop­ment of an adult organism from a single fertilized egg, in growth and repair of tissues, in regeneration of body parts, and in asexual reproduction. In mitosis, the parent cell produces two “daughter cells” (The term “daughter cell” is conventional, but does not indicate the sex of the offspring cell.),which are genetically identical. Mitosis can occur in both diploid (2n) and haploid (n) cells.

Mitosis and meiosis are similar processes in that they both result in the separation of existing cells into new ones. They differ, however, in their specific processes as well as in their products. The reason for these differences lies in the difference in the class of cells that each process creates. Mitosis is responsible for reproducing somatic cells, while meiosis is responsible for reproducing germ cells.

Interphase consists of three main stages:

a. G₁ (Gap 1) from telophase to the beginning of S (synthesis);

b. S, during which DNA synthesis takes place; and

c. G₂ (Gap 2), from the end of DNA synthesis to the beginning of mitosis (division).

The amount of time for DNA synthesis varies. It is evident that in a multicellular plant or animal cell division goes on until the adult stage is reached. Thereafter, cell division occurs only in germinal tissues such as the cambium in the stem and the root of a plant and the germinal layer of the epidermis, the gametogenic cells in the gonads and blood cell-forming tissues of the animal. Divisions also occur during repair of wounds in both plants and animals.

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Term Paper # 2. Synchoronised Cell Division:

For many of the studies of cell division it has been found desirable to have all of a population of cells in a culture divide at one time. Such synchronised cell division makes possible more effective analysis of the various components of the process.

Eggs of marine animals have been favourite objects for such studies since practically all the eggs divide at the same time in a suspension of healthy and normal cells.

This synchrony in dividing marine eggs implies that some event has occurred to achieve the synchrony, e.g., the almost simultaneous entry of sperm in all eggs starts a train of events that takes about the same amount of time in each of the cells.

Cleavage in the embryo continues in synchrony for several generations unless cells are separated from one another. When the eggs have been affected by unfavourable conditions, e.g., temperature, radiations, pH, division may be delayed, and when it starts, some eggs are found to cleave before others.

The susceptibility of the cells to the unfavourable conditions varies about a mean for the population and a characteristic distribution curve is obtained for onset of division in the population. In a culture of bacteria, protozoa or tissue culture cells the number of cells in division at any one time is limited, being from 5 to 10 per cent. The ratio of the number of cells in division to the total number of cells is called the mitotic index.

The mitotic index appears to be a function of the generation time for a given species under the conditions provided and the actual length of time a cell of this species remains in mitosis. The mitotic index may be increased by selecting a single cell from such an asynchronous culture and using it as the progenitor of a culture.

For a few generations thereafter, synchrony is obtained, but it also gradually disappears. A variation of this technique consists of filtering bacteria of a given size from a mixed population of cells, thus obtaining cells in essentially the same stage of growth which will divide at approximately the same time.

The failure to achieve synchrony of division in a culture of cells is probably a result, in part, of differential exposure to various environmental conditions. In a syncytium (multinucleate cell) in which mixing of all materials occurs and the conditions of the environment are similar, the division of nuclei is usually perfectly synchronised. Also, in the syncytial insect egg nuclear division continues synchronously for as many as eleven generations.

The same is true for the nuclei in the syncytial endosperm of plant embryos. It is thought likely that nuclei in division may secrete division- stimulating materials. To explain the low mitotic index in most cell cultures it has been suggested by some that the division-stimulating material from the few cells in division does not affect many other cells, either because they are distant or because the membranes of such cells are relatively impervious to the hypothetical division-controlling substances.

This concept of control of synchrony by secretion of materials from nuclei has some experimental backing. For example, grafts can be made between two multinucleate amoebas (Chaos chaos), each with nuclei synchronously dividing but out of phase with one another. After one cycle the division of all nuclei from both amoebas now present in one cell is synchronous, the larger piece imposing its time upon the smaller.

There are many examples of synchronised cell division which can be found in cells of higher plants and animals. Cells in synchronous division are found, for example, in spermatogenic cells lining the lumen of a germinative tubule, which are connected to one another by cytoplasmic bridges. Such bridges insure entry of the division-stimulating material from one cell to another.

It is important at this time to emphasize that a suspension of cells, whether they are cells in tissue culture, protozoans, yeast or bacteria, are fundamentally different from a suspension of marine eggs. Marine eggs have undergone a period of growth in the ovary and they have self- contained supplies which will serve for many cell divisions before intake of nutrients need take place.

Cells other than eggs, however, must incorporate nutrients and grow before they can divide. Perhaps not all cells are able to incorporate the same nutrients at the same rate.

Studies on Tetrahymena (ciliate protozoan) show that the mass of the cell increases linearly as measured by oxygen consumption, by increase in volume and by ¹⁴C-methionine incorporation, during the entire synthetic period, (S), from the onset of furrowing in one division until about 10 to 20 minutes before the next division. The division cycle has a generation time of 2.25 to 2.5 hours at 28 to 29°C.

Furthermore, ³H-histidine incorporation (protein synthesis) and ³H- uridine incorporation (RNA synthesis) continue during division with little change. During the 10 to 20 minute predivision period, which precedes furrowing, there is no increase in mass while the cell gets ready to divide. Such a period has also been found in other cells; e.g., in Amoeba it occupies about one-sixth of the generation time.

Studies on Paramecium aurelia show that, the mass, as measured by increase in dry weight, changes little for a period after division, and then it increases almost exponentially. From the data recorded this would appear to be exceptional compared to most cells studied.

On the other hand, Lovlie in a careful study on single Tetrahymena (using the diver technique) has shown that the type of curve obtained for increase in mass with time is related to the conditions of growth. He found three types of curves for Tetrahymena, exponential, linear and linear with a plateau, depending on whether the growth was balanced (doubling during the interdivision period) or unbalanced.

Since it is not possible to achieve synchrony of cell division in cell suspensions other than marine eggs, attempts have been made to shock cells into synchrony.

Two methods have been used:

i. Chemical shock and

ii. Physical shock.

Chemical shock consists of withholding or limiting the supply of some nutrient necessary for division and then supplying it to the culture at one time in a large quantity, inducing in this manner a high level of simultaneous metabolic activity. The physical shock is one that is unfavourable for the act of cell division yet favourable to other metabolic activities preceding division, thus allowing the cells in the pre-division stages of the division cycle to, catch up with those in the later stages of the division cycle.

Only a few experiments using shocks for synchronisation of cell divisions will be considered here. Cell division and DNA synthesis in Escherichia coli T-15, a thymine-requiring mutant (thymineless), are blocked immediately upon transfer of the organism to a thymine-free medium.

However, RNA and protein synthesis continue, apparently at the original rate. When thymine is added 30 minutes later, DNA synthesis is resumed and nearly all the cells are found to divide simultaneously after a lag of 35 to 40 minutes. Similarly, in Lactobacillus acidophilus, synchrony can be induced by the addition of thymidine to a thymidine-starved culture.

Yeast cells starved in succinate buffer until some of the reserves are gone will divide synchronously after return to complete nutrient medium, including carbohydrate and nitrogen sources. Similar results were obtained with a variety of cells needing some particular metabolite.

For example, synchronous division in the cells of the epidermis of the insect Rhodnius follows its periodic ingestion of blood. In Chlorella and various algae, all photosynthetic, the daily periodic lighting regimen makes possible synchronisation of cell division, presumably by periodic accumulation of food reserves during the period of illumination. Lighting, too, may be considered a physical shock, much like temperature; in fact, all the arguments used for temperature apply to light in light- sensitive cells. Environmental changes produce oscillations in growth of many types of cells.

Use of temperature as a physical shock to obtain synchronised division stems from the notion that the processes that occur during the division cycle are differentially sensitive to temperature. If some reaction in the pre-division period is more sensitive to heat than are the reactions in the synthetic period, then a high temperature might prevent division without stopping syntheses.

Thus, cells lagging behind in preparations for division might be given a chance to catch up with the others. As expected, a single temperature shock synchronises only a small fraction of the cell population because it allows only a small proportion of the cells to accumulate in pre-division stages.

On the other hand, a series of temperature shocks which block division-each alternating with an exposure to a near optimal temperature for a period insufficient to allow the cells in the pre-division stages to divide-synchronises most of the cell population, because of the gradual accumulation of a large proportion of the cells in pre-division stages before cessation of heat shocks.

Zeuthen and Scherbaum found that alternate exposure of suspensions of the ciliate Tetrahymena, for half-hour periods at 28 to 29° C. (optimal) and 34° C. (inhibitory) for seven cycles resulted in 85 per cent synchronisation of the cells in division when the cultures were subsequently kept at 28° C. Synchrony persisted for several cell generations and then division became random. Thermal shocks have also been effective for synchronising division of many other kinds of cells, including bacteria.

Cold shocks have also been used to induce synchronisation of cell division in a number of protozoans and bacteria. Nutritional deficiency, coupled with temperature shocks, has also been very effective. X-rays have also been shown to induce synchrony in division, again presumably by holding back the cells in the radiation-sensitive pre-division stages and causing accumulation of the cells which will ultimately divide at nearly the same time.

Much effort has gone into production of synchronised cultures of cells because in such cultures division and growth are essentially uncoupled. Therefore, growth can be studied in the cells prior to division and the process of division and its accompaniments can then be studied in the cells accumulated in the pre-division stages.

The cells shocked into synchrony have accumulated sufficient reserves for a series of divisions, which follow one another more rapidly than the usual generation time for the species until the accumulated reserves have been partitioned among the descendants.

This, of course, is also true of a suspension of marine eggs. Heat-shocked cells thus grow until they are considerably larger than controls. In a heat-shocked culture of Tetrahymena, the generation time for the first few synchronised divisions is about 60 per cent of that required for a culture that was not subjected to heat shock.

Division of heat-shocked Tetrahymena continues even in the presence of inhibitors of synthesis of DNA and steroids. However, certain long chain unsaturated fatty acids must be synthesized, as must certain specific proteins. The specific protein synthesis continues even if the cells are starved.

The specific protein is presumably “division protein” thought to be required for structuring the cell for division. It has been extracted and characterised. Interestingly, DNA synthesis, as measured by ³H- thymidine incorporation is not synchronised with cell division in heat- shocked cultures. It would be of interest to determine the DNA polymerase activity at various times during the cell division cycle.

Synchronised cultures of various cells in suspensions are now being widely used in biochemical and cytochemical research. It is important to note, however, that changes in size and composition of the cells after synchronisation must be taken into consideration. For example, resistance of such cells to ultraviolet radiations is markedly altered.

Synchronisation of cell division in the photosynthetic dinoflagellate Gonyaulax, in the normal diurnal rhythm of day and night, occurs in 85 per cent of all cells destined to divide during a 5 hour period spanning the end of darkness and the beginning of the light period.

Although it might appear that cell division here, as in Chlorella, is related to periodic changes in nutritional conditions, keeping Gonyaulax in continuous dim light only sufficient to maintain nutritional balance in the cells does not stop synchronous division for at least 14 days.

The synchronous division here is also relatively independent of small variations in light intensity and temperature. In high light intensity, however, synchrony is lost in 4 to 6 days. This is an interesting case worthy of detailed study. At present it is interpreted as an example of inherent biological rhythm.

Bruce lists the generation time of cells from a large number of species of plants and animals. In many cases the division of cells appears to be circadian, almost every 24 hours, but it has not yet been determined whether the day-night cycle will synchronise these to a 24 hour rhythm.

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Term Paper # 3. Source of Energy for Cell Division:

Cell division, is a fundamental activity of the living cell. During cell division the cell does work at the expense of energy derived from nutrients, as witness the case just cited of synchronised cell division in yeast obtained whenever the cells are supplied with adequate food. It has also been shown that addition of glucose to isolated epidermal tissue culture results in synchronous division of many cells. Presumably, cell division had previously been blocked by lack of nutrient since such cells store little glycogen. Even if glucose is supplied, division fails if oxygen is not available.

Glucose may be replaced by lactate, glutamate, fumarate or citrate. All the experiments suggest that operation of the Krebs cycle supplies the energy for division of the cells. As expected, mitosis is inhibited by Krebs’ cycle poisons such as malonate, cyanide and carbon monoxide, and by phlorhizin, a phosphorylation inhibitor.

The latter finding suggests that high energy phosphate bonds are an energy source or are involved in building this source. Marine eggs and protozoans require oxygen for division, but frog eggs and many embryonic tissue cells do not, presumably supplying their energy for cell division by glycolysis. Cells which require oxygen for division have a lower rate of glycolysis compared to their rate of respiration.

Cells which divide in absence of oxygen have rates of glycolysis much higher than that of respiration, sometimes several fold greater. As might be expected, in cells which can supply their energy needs for division by glycolysis cell division is very sensitive to glycolytic inhibitors such as iodoacetic acid. In order to have an effect on cell division the energy sources must be supplied early in the cell division cycle during what Bullough calls the antephase.

Once a critical concentration of the energy-rich substances has been built up and the prophase begins, he found that nothing short of killing the cell will stop it from dividing. This has been noticed not only for cells deprived of oxygen or metabolically poisoned, but also for those damaged by radiations or subjected to other injuries; such cells divide and soon thereafter cytolyse. Initiation of mitosis appears to begin a series of concatenated and irreversible reactions which stop only when the cells have divided.

By employing carbon monoxide, working under green light, to block cytochrome oxidase in sea urchin eggs and releasing the block by shifting from green light (which was not thought to be absorbed by the enzyme-carbon monoxide complex) to white light, Swann obtained the following results. If the inhibitor is applied before sea urchin eggs enter mitosis, the first cleavage is delayed by a time about equal to the time of application of carbon monoxide.

If the inhibitor is applied after the cells have entered mitosis, they complete mitosis and cleave with little or no delay, but the second cleavage is delayed for a period which is roughly equal to the period of inhibition. It was suggested that these results can best be explained by a hypothetical energy reservoir which is continually being filled and in which energy is stored for a cell division during the preceding mitosis and cleavage.

Once the energy has siphoned out it carries the cell through mitosis and cleavage; if at this time the cell is poisoned for a period by monoxide and energy is no longer being accumulated in the reservoir, cleavage will nevertheless continue to completion. However, the next cell division is delayed for an interval equal to the period of application of the poison. Lack of oxygen and various other poisons that affect the aerobic enzymes would also affect cell division in a similar manner.

Poisons which do not affect energy-liberating systems act in a different manner. For example, ether (1 per cent) is almost without effect if applied to a suspension of sea urchin eggs before the mitotic spindle has formed, but if applied after that time, it blocks development of the spindle fibers, maintaining the proteins in a solvated state.

If the treated eggs are then washed free of the ether, development proceeds at once, the only delay being the period during which the eggs were subjected to the ether since in this case the energy reservoir, if it exists, is unaffected by ether.

The need for a postulated energy reservoir is difficult to accept on the basis of data collected both on animal and plant cells. Epel found that cleavage rate in the eggs of the purple sea urchin was decreased when the concentration of carbon monoxide was at a level which inhibited respiration. At this time the ATP level was also decreased. When the ATP level had dropped to 50 per cent of normal, mitosis was completely inhibited.

By varying the carbon monoxide concentration, and thus obtaining varying degrees of inhibition of ATP production, Epel demonstrated that the mitotic rate paralleled the ATP level in cells. He also found that division could be blocked at any stage of mitosis if the inhibitor was applied at the appropriate time.

When phosphorylation is uncoupled by dinitrophenol (DNP), ATP fails to accumulate and cell division is also blocked. Such a concentration of DNP, which uncouples phosphorylation, causes an increased rate of respiration, but the respiration is now useless and idling.

This point again at ATP as the likely source of energy for cell division. The discrepancy between the results of Epel and those of Swann perhaps rests upon a difference in experimental technique. Swann did his “dark” experiments in green light, which is theoretically not effective in reversal of carbon monoxide poisoning; he used white light for full reversal of carbon monoxide poisoning.

Epel, on the other hand, put his carbon monoxide experiments in full darkness because he found that in green light the rate of respiration and ATP production were reduced to only 75 per cent of the level obtai­ned by the same concentration of carbon monoxide in full darkness.

Heat shock, does not stop growth although it prevents division, and, consequently, the cells may reach a size four times that of the controls. Returning the cells to the optimal temperature after one or more heat shocks permits cleavage in less than the generation time.

Presumably the mitogenic and growth channels are separate at their definitive ends, though both are using the same sources of energy and materials. When the mitogenic channel is blocked by physical or chemical means, the energy and material which might normally go through this channel pass instead into synthetic reactions leading to extra cell growth. Consequently, after removal of the block, sometime must elapse before the specific molecules necessary for cell division are synthesized to the necessary level.

In Tetrahymena if heat shocks as used by Zeuthen continue well beyond seven cycles, the cells may ultimately divide, even during a heat shock. This is taken to mean that accumulation of products of growth in a cell, and especially of the material needed for cell division has reached a point at which division is initiated. Division can then no longer be prevented by thermal shocks that previously blocked it.

Perhaps an even more clear-cut separation of growth processes from cell division is shown in experiments with Tetrahymena cells which are heat shocked in nutrient medium and then transferred to balanced salt medium with no nutrients. Such cells subsequently divide at least twice in absence of nutrients. Cleavage of such cells under these conditions can be “set back” or delayed by various metabolic poisons or physical shocks.

Experiments with dinitrophenol also distinguish two phases of metabolism in Tetrahymena. One, constituting about 30 per cent of total metabolism, relies on endogenous reserves and can support cell division; the other phase (about 70 per cent of total metabolism) depends upon exogenous supplies and is coupled with growth.

It has become increasingly evident that specific protein (division protein) probably plays an important role in cell division in Tetrahymena. It is thought that a critical concentration of this particular protein is needed for cell division. It has also been shown that ATP, GTP and RNA accumulate in the period just preceding cytoplasmic division and that the changes are reversed at mitosis.

However, such an accumulation of free energy sources is interpretable on the basis of protein synthesis, because protein synthesis decreases for a period beginning just about two thirds of the time to the next cell division. At this time the protein formed on the ribosomes is less readily released than it was previously.

The call for ATP is then less than its concentration in the cell, along with that of GTP, and RNA therefore increases. This change in concentration thus appears to be incidental to the slowing or cessation of protein synthesis rather than due to accumulation of an energy reservoir. The high energy phosphates then play a part in reversing the binding of the protein to the ribosomes.

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Term Paper # 4. Blocking Cell Division:

a. By Radiations:

Blocking of cell division by x-radiation was observed soon after the discovery of x-rays, and this led to the use of ionising radiations in the treatment of cancer. Almost without exception dividing cells are most sensitive to x-rays during a short period just prior to the onset of mitosis. Cells already well along in the division cycle complete the division, but those in earlier stages regress.

The type of cell seems of little consequence provided the cell is growing and dividing either in culture or in the living organism. (The cells in a mature tissue are apparently radio-resistant, i.e., not killed, when they have ceased dividing). As a consequence, x-rays produce a degree of synchronous division of cells in tissue culture. The locus of action of the x-rays is not fully known.

The rate of incorporation of precursors into DNA is decreased or inhibited in cells of tissues subjected to x-rays. Since in most cases assays were made many hours or even days after the irradiation, at which time the cell population may have declined as a result of x-raying, the results from this type of experiment have been questioned.

However, by using cell suspensions, it has been shown that application of a certain x-ray dose, at a time just before synthesis of DNA has begun, and is much more effective in stopping DNA synthesis than if the radiations are applied after synthesis has started. There thus appears to be a system connected to, but not identical with, DNA synthesis that is even more sensitive to x-rays than DNA synthesis itself. Larger doses affect both this process and synthesis of DNA as well.

It is interesting that synthesis of protein and RNA continue in a radiation sterilised cell (a cell that will not divide again). The result is the production of giant cells. Unbalanced growth of this type is followed by death, but the undivided cell may live for a considerable time.

During this time, various materials leak from these cells which are found useful in experiments with tissue culture cells. For example, Puck has used mats of similar non-dividing (sterilised) cells on which to place a single normal un-irradiated human tissue cell. The cell lives using leaking material as nutrient and gives rise to a colony, the cells of which form a clone; that is, they are all the descendants of the single cell placed there.

Ultraviolet radiations also retard or stop cell division, but, because the radiations are absorbed so superficially, they are not suitable for cancer therapy. The action spectrum for cell division-inhibition in a suspension of cells resembles the absorption spectrum of nucleic acids or nucleoproteins. Also, it has been shown that DNA synthesis is retarded by small doses of ultraviolet radiations.

As in the case of x-rays, exceedingly small doses may inhibit division of cells without having any detectable effect on DNA synthesis, indicating the existence of a process even more sensitive than DNA synthesis but probably closely related to it. The action spectrum for the effect implicates nucleic acid.

In general, synthesis of DNA appears more sensitive to ultraviolet radiations than is RNA synthesis or protein synthesis. Since the blockage of DNA synthesis leads to unbalanced growth and larger doses of ultraviolet radiation inhibit RNA and protein syntheses, the interrelations between all these syntheses are complex.

b. By Poisons:

A great number of poisons block cell division, but only those which help identify metabolic reactions or events in the complex which makes up cell division can be mentioned here. The action of such poisons has been studied extensively because of its theoretical interest and its possible use in control of cancer.

Many mitotic poisons interfere with interphase growth. For example, metabolic poisons, such as the inhibitors of respiration and glycolysis or those which uncouple phosphorylations from energy-yielding reactions, prevent the accumulation of high energy compounds and prevent release of energy for synthesis or division. Another group of mitotic poisons-the antimetabolites-interfere with syntheses.

Still others, such as the antibiotic chloramphenicol (isolated from Streptomyces), which resembles phenylala-nine and presumably competes with the latter on the surface of the protein-synthesising enzymes, block protein synthesis.

If applied in sufficient concentration early in the division cycle, chloramphenicol also blocks cell division. Tetracyclines (isolated from Streptomyces) and puromycin interfere, with protein synthesis, although probably in some manner other than that of chloram­phenicol.

A few mitotic poisons block nucleic acid synthesis. These compounds resemble some normal metabolite, and thus compete with the normal metabolite for the surface of the particular cell enzyme (competitive inhibition). For example, azaguanine, which resembles the purine guanine, acts in this manner, and many other purine and pyrimidine analogues have been tested.

Other such substances that have been used extensively are 5-fluorouracil and 5-fluorodeoxyuridine, which at relatively low concentrations interfere with DNA synthesis by inhibiting thymidine synthetase. At higher concentrations these substances also inhibit RNA synthesis and protein synthesis.

However, whereas such agents may act in this manner, it is possible that they also act in other ways. Unfortunately, many such analogues have too great an effect on cell division in germinative areas (e.g., on blood-forming cells of man) to be useful for treatment of cancer. Mitomycin C interferes with DNA synthesis without markedly affecting RNA synthesis or protein synthesis.

Actinomycin D at concentrations which have no effect on DNA synthesis blocks DNA dependent RNA synthesis, probably mainly messenger RNA production. Both antibiotics retard cell division and have been used extensively in recent years as selective inhibitors of the synthesis of the two nucleic acids. Such poisons generally prevent cells from entering mitosis.

Antagonists to folic acid such as amethopterin and aminopterin affect a specific step in the separation of the chromosome between metaphase and anaphase. They have therefore been classed as specific metaphase inhibitors. These experiments demonstrate that one of the B vitamins controls a specific step in the mitoses of the cells (chick fibroblasts) upon which they were tested. However, according to Taylor cells are arrested by such poison in either the G₁ or the early S phase and do not progress unless thymidylie acid becomes available.

Alkylating agents (e.g., those which replace an H atom of a compound with an alkyl group such as the methyl or ethyl) do not affect growth but prevent cell division. The nitrogen mustards (used as mutagens and in the treatment of cancer) and methane sulfonates (e.g., Myleran used in treatment of chronic myeloid leukemia) are such alkylating agents.

The exact manner in which inhibition of cell division occurs is unknown, but some of these alkylating agents cause chromosome breakage or failure of normal chromosome movement similar to the effects following x-radiation. This could presumably occur as a result of translocation of parts of two chromosomes, resulting in two kinetochores (chromosome constrictions for spindle fiber attachment) at two places on a single chromosome which is torn in half during anaphase.

Abnormal disjunction could occur from failure of attachment of kinetochores. These agents also appear to affect the formation of spindle fibers, generally reducing the viscosity of the cytoplasm. Esterification of carboxyl groups of proteins and combinations with nucleic acids have also been demonstrated, but the precise nature of the reactions is unknown.

The antimitotic agents such as colchicine, podophyllin and the urethans inhibit the formation, or the breakdown of the mitotic apparatus. Colchicine apparently inhibits cell division by disorganising the mitotic spindle, without stopping growth or duplication of various organelles in the cell.

Chromosomes duplicate themselves, but the spindle fibers that form are disoriented, resulting in polyploidy which, in some species, persists even after the poison is removed. It does not interfere with the making or breaking of disulfide bonds between proteins forming the spindle fibers, but seems to interfere with the secondary bonding in the formation of an oriented and symmetrical mitotic spindle.

The sulfhydryl reactants (e.g., chloracetophenone), on the other hand, block cell division by interfering with the disulfide and sulfhydryl cycle which is necessary for the formation and dissolution of the spindle fibers. All the other processes continue up to the formation of the spindle.

Another interesting poison which has given considerable insight into mitosis is mercaptoethanol which, among other things, prevents the duplication of mitotic centers (centrioles) of the cell. Centrioles always appear in animal cells in pairs. Each member of a pair must divide before they can act as division centers for the cell. The precise way in which mercaptoethanol affects the pattern of cell division depends upon just when it is applied during the division cycle.

Exposure of the sea urchin eggs to this poison delays cell division to the extent that it delays duplication of the mitotic centers, which divide only after the cells are removed from the poison. In addition to this delay, cell division is made abnormal because, although the division of centrioles is inhibited by mercaptoethanol, the separation of the centrioles from one another is not affected.

Along with all the other processes preparing for cell division, the members of the pair of centrioles must separate and take their positions at the poles of the cell. If this poison is applied to an egg cell just before its first division when it already has two pairs of centrioles and the egg is kept in mercaptoethanol beyond the time for the first division, it will then prepare for the second division except that the required duplication of the centrioles will not occur.

The two pairs of centrioles separate and single centrioles-now occupy four coordinate radii of the egg. Released from mercaptoethanol after being washed in sea water, the centrioles first divide and then the egg divides into four cells at once. The data are of interest because they show that the poison specifically affects only the duplication of centrioles,-not their separation or movement in the cell.

Some antibiotics, such as penicillin, prevent cell division of bacteria by interfering with the incorporation of amino acids into the cell walls and causing some bacteria to develop as protoplasts-without cell walls. Such protoplasts cannot divide-apparently the cell wall is necessary for bacterial cell division. Since animal cells do not form cell walls they are unaffected by this action of penicillin.

A search is being made for naturally occurring substances with dual actions, on the one hand promoting growth, and on the other, retarding growth. Such substances are induced in animals after injection with active cancerous cells. Chemical analysis suggests keto-aldehydes as the compounds produced in response to such injections. Such compounds are being tested as possible regulators of cell division.

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Term Paper # 5. The Initiation of Cell Division:

The cell, as we have seen, grows to a certain stage and then divides. In some unknown way each unit (organelle?) in the cell presents a template upon which duplication occurs. When cell division is completed each daughter cell contains in itself a duplicate of all the varied structures present in the mother cell.

Cell division has been studied in a multitude of cells, and most cells, under normal conditions, are known to divide by mitosis. Chromosomes condense and appear on an equatorial plane. The daughter chromosomes, produced by splitting, separate from one another, and a duplicate set of chromosomes is moved toward each of the two poles. Finally the cytoplasm separates into two portions. The complete process may take no more than half an hour in some marine eggs. The interest here centers on the mechanism initiating cell division.

The trigger which sets off the cell division has been of interest for a long time and numerous suggestions have been made about the nature of this process. One possibility that suggests itself is the doubling in mass of the cell, since this might be the last growth requirement for a series of processes, each resulting in doubling of cell organelles.

Division then occurs because the ratio of cytoplasm to nucleus is upset. Some evidence points this way. When an ameba has doubled its mass it divides, but not immediately. The division takes place after a lapse of time (rest period) during which something occurs which triggers the division of the cell. This time lapse before division is about 4 hours, or about one-sixth of the generation time of 24 hours for Amoeba proteus.

Also, when an ameba about to divide is shaken, it divides unequally, but each daughter ameba grows until it equals the mass of the mother ameba before it divides again. Moreover, amputation of a part of a growing ameba is followed by replacement of the mass removed before division occurs at all. In all these cases division takes place 4 hours after the ameba has doubled its mass. Growth before division in all cells is not alike however. Tetrahymena shows an increase in mass followed by a pre-division “rest period” similar to Amoeba, but in Escherichia coli B, synthesis proceeds in exponential fashion.

Another suggestion for the trigger in cell division is the upsetting of the surface to volume ratio. However, if a piece of cytoplasm is removed at a “critical” time just before an ameba has doubled its mass, division will still occur, even though smaller offspring than normal are produced.

This experiment might be taken to indicate that, by this time, division has been determined and follows even if the ameba is not as large as a typical mother cell about to divide, and that neither the ratio of nucleus to cytoplasm nor the ratio of surface to volume can in itself be the probable trigger for cell division. When cells are starved they may undergo division producing smaller daughters. However, such alterations in cell size are also produced by a number of other changes in environment.

Doubling the DNA content has been suggested as still another possibility. By the use of labeled precursors, it has been shown in most cells, however, that DNA is doubled in the interphase long before division actually occurs, in fact even before the formation of the extra pairs of chromosomes.

Doubling of DNA content is therefore not the likely trigger. As the nucleolus re-forms in the “resting” nucleus, intense protein synthesis appears to occur just outside the nuclear membrane. The interphase is apparently not a period of rest for the cell but rather a period during which the necessary cell constituents are manufactured by it.

The possibility that the nucleolus has a vital role in initiating cell division is suggested by experiments in which microbeam irradiation of a nucleolus (neuroplast of a grasshopper) at certain critical times (telophase to middle prophase) permanently stops mitosis in the cell, while comparable irradiation of the surrounding areas of the cell does not.

It was also thought possible that the chromosomes or other organelles produce a specific substance which induces the division of a cell. One team of workers found that kinetin (6-furfurylaminopurine) serves this function in some plant cells in which division is otherwise infrequent. No convincing evidence has accumulated to show this to be a general phenomenon.

In fact, it has been shown that nuclear structures need not be present for the cytoplasm of the sea urchin egg to divide, since non- nucleated egg fragments can be excited to divide by parthenogenetic agents. However, such cells develop abnormally and eventually die since they have no nuclei.

According to Heilbrunn, cell division is initiated by a coagulative process similar to the formation of a clot in blood, and any stimulus which induces clot formation is likely to initiate formation of spindles and cell division. It has been shown that agents which inhibit clot formation, e.g., heparin, interfere with the division of normal fertilised eggs. Agents which release calcium, which is also required for clotting blood, facilitate cell division, whereas agents which bind calcium, such as oxalate or citrate, inhibit cell division.

Whatever the immediate cause of cell division, it is clear that there are apparently many factors that contribute toward a favourable state for division of the cell and that preparations must be completed along many parallel lines before cell division can occur.

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Term Paper # 6. The Nature and Formation of the Mitotic Apparatus:

That the division spindles are gels has long been known. This was demonstrated by micromanipulative studies and by experiments in which intact mitotic figures were isolated from dividing eggs by the use of mild detergents which solubilise the rest of the cell.

The mitotic apparatus has also been isolated from dividing eggs by changes in pH and from dividing eggs from which the membrane and the hyaline outer cytoplasm has been removed by immersion in Versene (ethylenediamine tetra acetic acid), dextrose and dithioglycol solutions. The mitotic spindle is made up of about 3 to 5 per cent ribonucleic acid (RNA), and the remainder appears to be largely one type of protein.

Electrophoretic diagrams indicate two peaks, one of which is the protein, and the other it’s conjugate with RNA; both act as antigens. Electron microscope studies of fixed cells demonstrate that the spindle gel consists of definitely organised and oriented microtubules of protein, the centers of orientation being the centrioles.

When three pairs of centrioles are present, as in polyspermic eggs, the orientation is toward three poles. Presumably, something in or from the centrioles leads to the development of such oriented tubules, although no evidence for its origin or transport has yet been obtained.

On the basis of chemical evidence, it was suggested that the linkages between protein molecules in the microtubules of the mitotic spindle are disulfide (S-S) bonds. In the preparatory phase the intra-molecular disulfide bonds of protein molecules are presumably reduced by glutathione.

This is considered to take place before the metaphase, at a time when the glutathione content of the egg is known to be declining. When the mitotic apparatus is fully formed and growing, the disulfide bonds are supposedly restored, but now as intermolecular disulfide bonds, binding the protein molecules into tubules and liberating the glutathione.

The concentration of the glutathione in the egg cell was found to increase at this time. Postulation of intermolecular disulfide bonds rests on the assumption that the probability of forming a bond between sulfur atoms on two protein molecules would be greater than the probability of re-forming the original intra-molecular disulfide bonds. In essence, then, the sulfhydryl cycle has converted intra-molecular disulfide bonds into intermolecular disulfide bonds, the glutathione acting essentially like a catalyst, being recovered unaltered at the end of the cycle.

Although the cyclic variation in SH bonds present in dividing cells has been confirmed independently, there is little evidence to back the specific hypothesis just outlined.

If orientation and organisation of the spindle tubules are a prime necessity for cell division, then any agent which interferes with the spindle should interfere with cell division, even if this agent permits gelation. This is exactly what happens when a cell is poisoned with colchicine.

A gel appears, but the secondary bonds between protein molecules fail to develop, the gel is-disoriented and separation of the doubled chromosomes fails to occur (“flabby anaphase”). Because of this, polyploid cells are produced by the use of colchicine. When such cells are washed free of colchicine, they later divide, but the polyploid condition is maintained.

When Hoffmann-Berling placed cells about to divide (i.e., showing spindles) in glycerin solutions, the division process ceased. When they were later placed in a balanced salt solution, the spindles remained, but division still failed to occur. It is perhaps possible that some material necessary for cell division had leached into the glycerin solution. When ATP was applied to such mitotic “models” (glycerin-extracted dividing cells) the spindle tubules contracted and the chromosomes moved toward the poles.

Furthermore, an enzyme for decomposing ATP is present in the spindle. On the other hand, ATP produced no movement when applied to mitotic spindles isolated from sea urchin eggs by mild detergents and subsequently alcohol hardened. Hoffmann-Berling points out that hardening the spindles denatures the proteins of the tubules, making them incapable of contraction in response to ATP, even though hardening permits their separation and purification from other cellular constituents.

It is an oversimplification to consider only the contraction of the spindle tubules. Careful analysis of the movements during mitosis indicates that only the early part of mitosis (to anaphase) occurs by contraction. This is indicated by the broadening of the spindle.

Evidence suggests that in anaphase the two sets of chromosomes in the cell are actually pushed apart by the tubules between the poles. For example, when the spindles of grasshopper neuroblasts are immersed in hypertonic solutions which reduce the volume of the cell and thus crowd the mitotic spindle, the chromosomes are crumpled and spiraled during the anaphase and pushed against the ends of the cell.

The active elongation of the spindle poses the same problems as elongation and relaxation of muscle fibers, both active processes. It has been shown that low concentrations of ATP applied to the mitotic mod­els cause contraction; higher concentrations, elongation.

On the other hand, the spindle fibers appear to consume them as contraction occurs, unlike a muscle which maintains its mass in spite of shortening. A single contraction of the spindle tubules, however achieved, suffices to separate the duplicated chromosomes from one another. It would almost appear as if the chromosomes, as they move toward the poles, release some material which dissolves the spindle tubules.

The fact that glycerin-extracted models of dividing cells contract when placed in ATP, and that the chromosomes on the metaphase plate separate, could conceivably be explained as the result of contraction of the entire cell in such a way as to actually simulate mitosis rather than by contraction of the spindle tubules. The problem of chromosome movement should not be considered solved, despite the appeal of the contractile tubule hypothesis.

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Term Paper # 7. Furrow Formation and Cleavage (Cytokinesis):

The formation of a furrow in a dividing egg or other animal cell appears to be accompanied by increased viscosity in the area of the furrow. High pressures, which liquefy protein gels, also prevent the formation of cleavage furrows and cause such dividing cells to fuse again. The furrow disappears with even a slight pressure applied to both sides with a micro needle, but cell division resumes when the pressure is released.

An egg ruptured at the time of furrow formation disintegrates, but a part of the cortex and the furrow remain intact, indicating that the furrow is apparently a rigid part of the cell. Furthermore, it has been noticed that as the cortex forms in the cleaving eggs, a slight separation between the cortical layers of the two blastomeres (the two celled stage) is maintained for a time, indicating that some change has occurred which prevents coalescence of the two layers.

An attempt has been made to explain the formation of a furrow in a dividing cell by means of the contractile ring theory (or cortical gel contraction theory). According to this theory the belated ring in the cortex of the dividing cell is considered to contract like the non-motile portion of an ameba, and it might be expected, therefore, to decrease the surface area. However, measurement in some cells showed that before division the surface increased by about 26 per cent.

The increase in surface area accompanying cell division does not invalidate contraction theories of cell division. It is essential that the cell elongate before it cleaves, and at this time the surface area increases. But elongation is part and parcel of any contractile system, as already seen in muscle fibers.

When relaxation of muscle cells is better understood it may also be possible to understand cell division better. In modified form the contractile furrow theory, illustrated in Figure 9.10, allows for a passive increase in surface at the poles as a result of active contraction of the furrow region.

The expanding surface theory on the other hand, suggests that a nuclear substance is liberated (probably from chromosomes) which causes expansion of the cellular membrane at the poles. As the polar area expands, the equator contracts, leading to division. This theory is supported by two lines of evidence. First, movement of material on the surface of cells, consistent with such a suggestion, has been observed.

Second, expansion at the poles should weaken the membrane there. The fact that cytolysis of an egg occurs more readily during division than during interphase suggests some change in the cell membrane at the poles. The equatorial part persists after cytolysis, apparently being more rigid.

However, even cells without nuclei or chromosomes cleave, indicating that the excitant may have origins other than the nucleus. Another form of this theory postulates expansion of surface area as a result of growth. It is interesting that the resting potential of a cleaving starfish egg does not change.

The spindle elongation theory assigns a decisive role to the spindle and asters in cell division. The evidence comes from observation of movement, with respect to one another, of kaolin particles attached to the surface of an egg membrane.

The basic observation is that the elongation of-the cell at anaphase is accompanied by a shrinkage at the equator, the two kaolin particles on either side of the equator coming closer together while there is a corresponding stretching at the poles. Since the spindle and asters appear to be rigid structures, as tested with a micromanipulator, the driving force is believed to be the elongation of the spindle tubules which pushes the centers apart, bringing the astral rays attached to the equator close together.

Because a nucleus-free half of a sea urchin egg will cleave when artificially stimulated to divide, the astral relaxation theory, based only on changes produced by the asters, has been proposed to account for cleavage. Surface tension and elasticity measurements show that an egg surface is under uniform tension before cleavage begins. When the asters reach the poles of the egg it is thought that they produce a change (the nature of which is unknown), and the surface tension at the poles is lowered. This permits the furrow region, which maintains its surface tension, to contract while the polar regions expand.

Experiments of Hiramoto, however, in which the mitotic apparatus was either sucked out or displaced by injection of mineral oil into the egg, indicate that even in the absence of spindle tubule attachments (asters), cleavage furrows are formed in the same place as they are when mitotic apparatus is intact and in place.

Also, a piece of cortex of an amphibian egg develops a furrow even in the absence of a nucleus and a spindle. Stiffness of the cortex of the sea urchin egg increases before cleavage furrows form and varies cyclically during the division interval. The contracting power of the equatorial ring of the sea urchin egg reaches a maximum at the middle of the cleavage cycle.

Work with eggs under pressure and subjected to various inhibitory reagents suggests that the ATP system accounts for the energy of whatever movements occur during cleavage, since addition of ATP often relieves the effect of an inhibitor. The energy required for division of an egg has been calculated to be about three times that normally available from respiration.

Since the rate of respiration raises only a few per cent during cleavage, it would appear that the energy for cleavage must come from a specific source. It is suggested that compounds with high energy phosphate bonds, formed by the cell in preparation for each successive cleavage, might well serve this purpose.

It is apparent that all of these theories have much in common and that they are developed primarily from data on the marine egg cell. They do not apply to such phenomena as multiple fission, which occurs in some plant and animal cells, nor do they take into consideration the division of plant cells with cell walls where the laying down of a new wall is of prime importance for cleavage. In bacteria and yeast, removal of the cell wall prevents cell division even though the cells continue to increase in size. We are still without a generalized theory of cytokinesis.