Completion Of Cell Division Research Articles

A method for the immobilization of living Tetrahymena

The surfaces of plastic (polystyrene) Petri dishes from several suppliers were discovered to have the useful property of immobilizing cells of the ciliate Tetrahymena thermophilia upon contact in nutrient-free buffer (10 mM Tris, pH 7.4). The procedure works with cells in both logarithmic and stationary growth phase, so long as they are first transferred to nutrient-free buffer, and then added to dishes already containing buffer to a depth of 2–10 mm. Dish surfaces specially treated for tissue cultures are unsuitable for this purpose. Cells can be released from the dish surfaces by the simple addition of growth medium (1% proteose peptone). Immobilized cells are fully competent to complete conjugation or cell division. The technique offers promise for facilitating experiments requiring microinjection, microsurgery, or simply detailed observation of living protozoan cells.

Separation of cell enlargement and division in bean leaves

A method is presented for inducing cell enlargement in intact leaves and leaf strips of Phaseolus vulgaris L. without the complication of cell division. Primary bean leaves complete cell division and stop growing after 10 d in dim red light. Transfer to white light induces expansion (50% in 24 h) which is entirely the consequence of cell enlargement. Leaf strips from red-light-grown seedlings placed in white light and provided external solutes (10 mM KCl+10 mM sucrose) expand at the same rate as intact leaves in the light. This system makes possible future investigation of the mechanism of leaf cell enlargement.

Photolability of Photosynthesis in Two Separate Mutants of Scenedesmus obliquus

Two separate mutants of the green alga, Scenedesmus obliquus, are described in which photosynthesis is sensitive to moderate intensities of white light (100 mw cm(-2)). Heterotrophic cultures of both mutants lose photosynthetic activity when exposed to white light; the site of at least the initial phase of this inactivation is within photosystem I. Although all whole cell and cell-free reactions typical of photosystem I examined are inhibited by irradiation, the principal component of photosystem I affected is P-700. In light-sensitive-4 the inactivation of P-700 activity is restored during the subsequent dark period. This recovery is prevented by various antibiotics and by anaerobic conditions. In light-sensitive-41 P-700 activity is recovered only after a complete cell division and new growth. Irradiation periods which inhibit photosynthesis in both mutants are without effects upon the activity or presence of ferredoxin, ferredoxin-NADP(+) oxidoreductase, plastocyanin, cytochrome f(552), cytochrome b-562 or cytochrome b-559.Prolonged irradiation of cells of light-sensitive-41 causes the disappearance of photosystem II activity, alpha-tocopherol, and plastoquinone. Some decrease of both the chlorophylls and carotenoids occurs but there is no preferential deletion of any particular carotenoid.

The Fine Structure of the Sheath of Volvox Carteri f. Weismannia

Spheroids of the green colonial alga Volvox consist of biflagellate Chlamydomonad-like cells embedded in a transparent sheath. The sheath, important as a substance through which metabolic materials, light, and the sexual inducer must pass to and from the cells, has been shown to have an ordered structure (1,2). It is composed of both protein and carbohydrate (3); studies of V. rousseletii indicate an outside layer of sulfated polysaccharides (4).Ultrastructural studies of the sheath material in developmental stages of V. carteri f. weismannia were undertaken employing variations in the standard fixation procedure, ruthenium red, diaminobenzidine, and high voltage electron microscopy. Sheath formation begins after the completion of cell division and inversion of the daughter spheroids. Golgi, rough ER, and plasma membrane are actively involved in phases of sheath synthesis (Fig. 1). Six layers of ultrastructurally differentiated sheath material have been identified.

Ultrastructure of transmitting tissue of Lycopersicon peruvianum style: Development and histochemistry

The development of the transmitting tissue of the style of Lycopersicon peruvianum goes, after the completion of cell division and cell wall formation, through two distinct phases. During the first phase, the cells enlarge and the main part of the intercellular substance, consisting of pectins, is formed. During the second phase, the cells form an extensive rough endoplasmic reticulum (RER) and proteins are incorporated in the intercellular substance. A possible role of these proteins in the incompatibility reaction is proposed.

An analysis of the spatial distribution of ciliary units in a ciliate, Euplotes minuta

Six to eleven longitudinal ciliary rows are arrayed over the dorsal surface of the ciliate Euplotes minuta. Forty-two to 129 ciliary units are distributed among these rows. The number of rows depends on genotype, clonal age, and vegetative ancestry, while the total number of units is controlled partly by the number of rows and also by the separate action of genetic and environmental factors. 2. The pattern of distribution of units among different rows of non-dividing cells can be analysed on the basis of the percentage of the total unit complement of the cell that is found in each individual row. If the assumption is made that ciliary rows are uniformly spaced over a dorsal field whose width is independent of the number of rows, then it can be shown that units are distributed among rows according to a relatively invariant spatial pattern. The form of this pattern remains the same in the face of variation in the absolute number of rows and of units. 3. Prior to cell division new units develop anterior and posterior to old units situated within the equatorial zone of each row. About one-half of the original units are included within this zone. The cell fission line develops within this zone such that the total number of units passed to the anterior and posterior division products are about equal. 4. The pattern according to which units of different zones (proliferating and non-proliferating) are distributed among different rows has been mapped in cells that have completed the process of proliferation of units but have not yet completed cell division. The results of this mapping show that the pattern of distribution of units in the equatorial zone at the conclusion of proliferation is not the same as the overall pattern in non-dividing cells. Further analysis indicates that the geometry of proliferation can be most simply represented as a result of two superimposed processes, one of which is the recruitment of old units into the zone of proliferation, while the other is the intensity of proliferation, i.e. the number of new units formed adjacent to each old unit. Both recruitment and intensity have constant values in the central region of the dorsal field, while recruitment is higher and intensity lower near both margins. The recruitment and intensity distributions are mutually nearly reciprocal, with slight asymmetries that formally account for the more dramatic asymmetry of the pattern of non-dividing cells. 5. A dualistic hypothesis is formulated for the control of the formation of new ciliary units within ciliary rows. The position of each new unit is largely controlled locally in relation to pre-existing units, while the decision of whether or not new units will develop at all, and how many will be formed, depends on superimposed positional systems operating within the context of the entire dorsal surface.

Ultrastructure of a temperature-sensitive rod- mutant of Bacillus subtilis.

Mutant 168ts-200B, resulting from nitrosoguanidine treatment of Bacillus subtilis 168 (trp(-) C2), exhibits a rod-to-sphere morphogenetic interconversion when the incubation temperature is 30 or 45 C, respectively. Ultrathin sections of rods grown at 30 C, after glutaraldehyde-osmium uranium-lead fixation and staining, show trilaminar cell walls with a well-developed underlying periplasm as in wild-type cells. However, the outer wall layer is irregular, and abnormal protrusions of wall material occur at the cross-walls. In contrast, cells growing at 45 C become rounded and are intersected randomly by irregular cross-walls which fail to split normally, resulting in large spherical masses. In these, the outer and inner wall layers and periplasm are lost, and the wall consists only of irregularly thickened and loosely organized middle layer. Wall ultrastructure is reversible in either direction as cell shape changes during temperature shifts. Mesosomes are rare and atypical at either temperature. It thus appears that cell wall ultrastructure is altered by the conditional (temperature-sensitive) mutation, and that loss of normal wall and submural organization is correlated with changes in cell size and shape as well as with inability to complete cell division. Preliminary studies after transformation of the mutant locus to another strain and growth at 45 C showed an increase in mucopeptide, loss of wall teichoic acid, failure of phage adsorption, and identical ultrastructural changes. The site of expression of the basic defect-be it in wall, submural region, or membrane-is undetermined.

Role of the Endocrine System in Controlling Growth and Division Synchrony of Plasmodium berghei in Mice

The malaria parasite of mice may be induced by certain photoperiodic rhythms to show an augmented growth and division synchrony. This process is mediated by the testes and pineal but not by other endocrine organs. Certain steroids will compensate for the missing testes. Four hydroxy-17alpha-methyl testosterone and 4-chlorotestosterone produced an effect nearly equal to that seen in the intact animal. Estrogen, progesterone, and testosterone used alone and in combination were not able to replace the testes. In addition to the partial control of growth and division synchrony, the testes also influence the vascular capture and release of mature parasites along the walls of the blood vessels. Hypophysectomy had no effect on growth and division synchrony or on parasite sequestration even after partial gonadal atrophy had occurred. Neither cyclic peaks in concentration of replacement androgens nor the photoperiodic rhythm are the time clue to which the growth and division synchrony of the parasite is entrained. The nature of the time clue is still unknown. Malaria infections have a striking dependence on the solar day. This is apparent from the fact that the generation time of most species of parasite is an integral multiple of 24 hr. In addition, many species of malaria are so closely tied to a diurnal rhythm that all parasites of the species are found in growth and division synchrony in the same host individual. A species of rodent malaria, Plasmodium berghei, offers certain insights into the mechanism of parasite entrainment to a host rhythm. In this system of mouse-with-parasite, the entrainment only occurs when the host mouse is exposed to sunlight or a nearly equivalent light for at least 16 hr a day. Parasite division occurs between 12 midnight and 6 AM during the time of entrainment (Arnold et al., 1969a). The timing of cell division is not by the timing of the light rhythm. The on-off cycle of the light can be moved in any fashion around the clock as long as the duration of light is at least 16 hr. The parasites still divide from 12 midnight to 6 AM solar time. In some respects, P. berghei is a convenient indicator of the host rhythm. The parasite has a 24-hr generation time. There are no problems with multiple broods of parasites. There are convenient morphologic indicators of the Received for publication 1 April 1969. * This study supported by U. S. Army Contract No. DA-49-193-MD-2545 from the U. S. Army Research and Development Command, Office of the Surgeon General. This paper is contribution No. 561 from the Army Research Program on Malaria. age of the parasite. On the other hand, there are problems with sequestration (the vascular capture and release of mature parasites). Vascular sequestration is another curious feature of the host-parasite relationship. In many species of malaria, including P. berghei, the older and dividing forms of the parasite, together with the host red cell, stick to the blood vessel surfaces, until such time as cell division is complete (Garnham, 1966). On completion of cell division, the young forms usually circulate freely in the blood until they reach a degree of maturity appropriate for sequestration. The cycle is then repeated. In preliminary work from this laboratory, we have found that the pineal body is part of the mediating system for both growth and division synchrony and for vascular sequestration (Arnold et al., 1969b). The effect of the pineal body on growth and division synchrony can be replaced in part by vitamin K, vitamin E, and ubiquinone (Arnold et al., 1969c). Although the presence of an intact pineal or its chemical alternates, vitamin K, vitamin E, or ubiquinone, is needed for growth and division synchrony of P. berghei, none of these factors appears to act as the master clock. The diurnal growth rhythm of the parasites remains on the same time schedule regardless of the time of administration of the ubiquinone or of the phase relationship to the photoperiodic rhythm. In addition, the photop2riodic rhythm must be maintained if chemical replacement is to work in the pinealectomized animal. For this reason, a continued search for other medi-

Macromolecular synthesis, differentiation and cell division in Tetrahymena pyriformis, mating type I variety 1.

AbstractIn Tetrahymena pyriformis, mating type I, variety 1, cycloheximide rapidly and completely inhibited incorporation of 14C‐L‐leucine into protein. Actinomycin D (25 μg per ml) inhibited incorporation of 14C‐uracil into cold‐TCA‐insoluble material, after a 5–10 minute lag. Frequently a subsequent decline in the amount of radioactivity was observed. Protein synthesis continued in actinomycintreated cultures for a variable time after cessation of RNA synthesis.Oral development was affected by cycloheximide virtually immediately, and by actinomycin D after a 10–15 minute lag. Cells affected by either drug before the onset of oral membranelle formation were permanently arrested in the stomatogenic field phase. Cells affected in the early and middle stages of membranelle formation completed development of membranelles, but did not invariably complete cell division. Cycloheximide, when added at the beginning of membranelle formation, brought about arrest or resorption of membranelles after they were completed. Actinomycin did not elicit resorption, but sometimes brought about blockage during cell division. Cells affected by either drug after membranelles were fully formed (and cell division was just beginning) completed oral development, nuclear divisions, and cell division. These results suggest that concurrent RNA and protein synthesis are essential for the initiation but not for the completion of membranelle differentiation. The results also suggest that a specific messenger RNA(s) with a very short half‐life is required for the synthesis of proteins involved in the initiation of membranelle differentiation.

Thymidine as a synchronizing agent. I. Nucleic acid and protein formation in synchronous HeLa cultures treated with excess thymidine.

AbstractSynchronous cultures of HeLa cells obtained by selective detachment of mitoses were treated with high concentrations of thymidine. The inhibitor was added soon after completion of cell division and rates of cell enlargement and accumulation of DNA, RNA and protein were compared for untreated and thymidine‐treated cultures at various points of the cell cycle. It was found that concentrations of thymidine which in randomly growing cultures inhibit the rate of cell division by more than 90% allowed a considerable degree of DNA synthesis and did not affect the rate of accumulation of RNA and protein, when applied to cells in the G1 phase of synchronous culture. Treated and untreated cells enlarged at the same rate throughout their life cycle. The results show that concentrations of thymidine commonly employed to produce cell synchrony do not arrest the cells at the G1‐S boundary, but allow slow progress through S in respect to DNA synthesis, and near‐normal progress towards G2 as regards RNA and protein accumulation and cell enlargement.

The fine structure of compensatory growth in the rat kidney after unilateral nephrectomy

AbstractThe structural changes in the nephrons of the rat kidney during compensatory growth were observed for 96 hours following unilateral nephrectomy. Hypertrophy of tubular epithelial cells involves dilation of the cisternae of rough endoplasmic reticulum, proliferation of Golgi membranes and agranular reticulum, and increase in number of cytoplasmic ribosomes. The appearance of absorption droplets and microtubules, that are polarized along the cell axis, coincides with maximal hypertrophy of proximal tubule cells. Premitotic dedifferentiation of these cells involves flattening of the basal infoldings of the plasmalemma, decrease in number and disorientation of mitochondria, loss of apical compartments, and the reduction in number of microvilli. Cilia appear on the apical borders of some cells. Bundles of 30–50 Å filaments occupy the cytoplasm at the base of proximal tubule cells.In distal tubule cells that are undergoing mitosis, numerous mitochondria remain intimately associated with infoldings of the basal plasmalemma. This persistence of the differentiated basal region prevents complete cell division, and binucleate and multinucleate cells arise as a result of failure of cytoplasmic cleavage after normal karyokinesis. During mitosis, the chromosomes and apical vesicles are restricted to organelle‐free masses of cytoplasm that project into the lumen.In collecting tubules, both intercalated and lining cells undergo mitosis during compensatory growth. By 72 hours, a less differentiated cell type, that is active mitotically, appears in the cortical portion of collecting tubules. The number of binucleate epithelial cells increases in the compensating kidney tubules. Unilateral nephrectomy also induces distinct alterations in the fine structure of the glomerulus and the papilla of the contralateral organ.

Growth and Synthesis of Nucleic Acid and Protein by Excised Radish Cotyledons

Nutritional and light requirements for growth and synthesis of RNA, DNA, and protein by cotyledons excised from 5-day-old seedlings of Raphanus sativus L. were investigated, and the course of synthesis was followed through the cell cycle. The minimum requirements for a net increase in nucleic acid and protein were sugar, nitrate, and light. The cotyledons used nitrite at low concentration, but not ammonium ion. Light was required for preliminary steps in synthesis of RNA, DNA, and protein, but the actual polymerization reactions occurred in the dark. The cotyledons contained sufficient endogenous growth factors for about half of the cells to complete 1 cycle on a medium of 1% sucrose, 80 mm KNO(3). The increase in DNA was limited to about 50% and was accompanied by a comparable increase in cell number. Fresh weight, RNA, and protein tended to increase in proportion to DNA. Growth of the isolated cotyledons commenced with cell enlargement. RNA began to increase after about 4 hours, DNA after about 12. The major increase in protein also began at about 12 hours. The maximum rate of increase for all 3 occurred between 12 and 16 hours. Cell counts indicated that by 28 hours most of the cells which had replicated DNA had also completed cell division.

Fission and Morphogenesis in a Marine Ciliate Under Osmotic Stress*

SYNOPSIS. Condylostoma magnum can be greatly yet reversibly swollen or shrunken by altering the salinity of the medium. These ciliates survive well in 10% to 400% sea water. Swollen animals can initiate and complete cell division, with important implications for the nature of the division furrow and the origin of the division primordium. Reproduction is also possible in 400% sea water though the animals are then tiny and vitreous. Normal oral regeneration at the extreme salinities mentioned also shows that morphogenesis is remarkably unaffected by extraordinary changes in the colloidal state of the cytoplasm.