THE REPLICATION TIME AND PATTERN OF THE LIVER CELL IN THE GROWING RAT

Three-week-old male rats of the Wistar strain were given tritiated thymidine, 1 µc/gm body weight, intraperitoneally and were killed at intervals from 0.25 to 72 hours later. Autoradiographs were made from 5 µ sections, stained by the Feulgen method. The replication time and its component intervals were determined from the scoring of the labeling of interphase nuclei as well as of prophase, metaphase, anaphase, and telophase nuclei. Absorption of the intraperitoneally injected label is rapid and is attended by "flash" labeling during interphase. The results show that at any one time about 4 per cent of the liver cells are synthesizing DNA preliminary to cell division. These cells alternate with waves of other cells and it is estimated that about 10 per cent of the liver cell population is engaged in cell duplication. The replication time is about 21.5 hours, and its component intervals occupy the following times: DNA synthesis, 9 hours; post-DNA synthesis gap, 0.50 hour; prophase, 1.3 hours; metaphase, 1.0 hour; anaphase, 0.4 hour; telophase, 0.3 hour; postmitosis gap, 9.0 hours. A group of liver cells has been recorded in at least 3 successive replication cycles.     

THE REPLICATION TIME AND PATTERN OF CARCINOGEN-INDUCED HEPATOMA CELLS

The replication time and pattern have been investigated in hepatoma cells induced by feeding 3'Me-DAB to male rats for 5 months. With the use of tritiated thymidine as a DNA label along with autoradiography, mitotic nuclear labeling has been studied 0.5 to 72 hours after the administration of the label. The following time intervals have been estimated: replication time, 31 hours; DNA synthesis, 17 hours; G₂ plus Mitosis, 2 hours; G₁, 12 hours. Only about 8 per cent of the tumor cell (interphase) population is "flash" labeled, following a single dose of 50 µC of H³TDR. This group of cells has been followed through three cycles of division. The repeated rhythmic passage of tumor cells through cell division is similar to that previously reported for normal liver cells in the growing rat. However, tumor cells have longer replication and DNA synthesis times. In addition, the several time intervals studied vary more in the tumor cell population than they do in the growing normal cell population.     

MACRONUCLEAR EVENTS IN SYNCHRONOUSLY DIVIDING TETRAHYMENA PYRIFORMIS

The macronuclei of synchronously dividing mass cultures of Tetrahymena pyriformis (strain WH₆) were examined with the electron microscope for changes during two division cycles. Samples were prepared at 30-minute intervals for a period of 8½ hours which included the time required to induce synchrony by five heat shocks (4½ hours). The interphase macronucleus contains peripheral, crescent-shaped nucleoli and evenly distributed chromatin bodies. Centrally located RNA bodies, composed of fibers, appear 1 to 2 hours following the initial heat shock. They are completely destroyed with ribonuclease whereas the nucleoli are only partially so. Following the third heat shock the RNA bodies move to the periphery and disintegrate; the nucleoli aggregate and form blebs which protrude into the cytoplasm where they appear to pinch off and may contribute to the cytoplasmic ribonucleic acid. Cytokinesis does not occur at this time. Instead the nuclear events are repeated during the 4th and 5th hours, even though the heat shocks are terminated at 4½ hours. Cytokinesis takes place at about 6 hours. The second division occurs about 2½ hours later during which all the macronuclear events noted above are repeated.     

MICRONUCLEAR RNA SYNTHESIS IN PARAMECIUM CAUDATUM

In a generation time of 8 hr in Paramecium caudatum, the bulk of DNA synthesis detected by thymidine-³H incorporation takes place in the latter part of the cell cycle. The micronuclear cycle includes a G₁ of 3 hr followed by an S period of 3–3½ hr. G₂ and division occupies the remaining period of the cycle. Macronuclear RNA synthesis detected by 5'-uridine-³H incorporation is continuous throughout the cell cycle. Micronuclear RNA synthesis is restricted to the S period. Ribonuclease removes 80–90% of the incorporated label. Pulse-chase experiments showed that part of the RNA is conserved and released to the cytoplasm during the succeeding G₁ period.     

THE TIMING OF MEIOSIS AND DNA SYNTHESIS DURING EARLY OOGENESIS IN THE TOAD, XENOPUS LAEVIS

Recently metamorphosed female Xenopus laevis toads were injected with tritiated thymidine. Animals were kept at 20°C and were sacrificed 1–23 days after isotope injection. Radio-autographs of squash preparations of the ovaries were made. The progress of labeled germ cell nuclei was followed to obtain information on the time course of early meiosis and extra-chromosomal DNA synthesis. Premeiotic S was estimated to take not more than 7 days. Leptotene takes 4 days, zygotene takes 5 days, and pachytene was estimated to be completed in about 18 days. The major period of amplification of the extrachromosomal DNA occurs in pachytene and takes about 13 days. A low level of synthesis was observed before and after this period, in zygotene and late pachytene-early diplotene, extending the total time for extrachromosomal DNA synthesis during meiosis to about 18 days. These data allowed the calculation to be made that one round of replication of the amplified DNA takes between 1.2 and 3.0 days. It was also found that in both oogonial and premeiotic interphases, the nucleolus-associated DNA shows asynchronous (probably late) labeling with respect to the chromosomes.     

THE RELATIONSHIP BETWEEN DEOXYRIBONUCLEIC ACID REPLICATION AND CELL DIVISION IN HEAT-SYNCHRONIZED TETRAHYMENA

The effect of supraoptimal temperature on macronuclear DNA synthesis in Tetrahymena was studied by radioautography during prolonged heat and heat-shock synchronization treatments. Prolonged heat treatments (34°C) delayed the initiation of S, but did not appreciably delay DNA synthesis in progress. Return to optimal temperature (28°C) 50 or 100 min later resulted in initiation of S, in delayed cells, at a rate greater than in controls. During the synchronization treatment, most cells were unable to enter S during a heat shock, but initiated S with a slight delay during the following intershock period. These cells were not appreciably delayed in completion of S by subsequent heat shocks. Supraoptimal temperature appears to affect the DNA synthetic cycle near the G₁ to S transition. Cells subjected to the heat-shock treatment in early G₁ all participated in one S period, and many underwent a succession of two S periods. DNA synthesis occurred in about 50% of the cells between EST and the first synchronous division, with the likelihood of DNA synthesis becoming greater the longer the interval between these two events. In some cells no detectable DNA synthesis occurred between EST and the second synchronous division. It was concluded that a precise temporal alternation of DNA replication and cell division is not obligatory in Tetrahymena.     

THE EFFECT OF UNILATERAL ULTRAVIOLET LIGHT ON THE DEVELOPMENT OF THE FUCUS EGG

1. When Fucus eggs which have been fertilized for a sufficient length of time are irradiated unilaterally with monochromatic ultraviolet light (λ2804 Å) of adequate dosage, 97–100 per cent form rhizoids on the halves of the eggs away from the source of radiation (see Figs. 1 and 2). 2. The responsiveness of the eggs increases gradually after fertilization and does not reach a maximum until about 7 hours at 15°C. (see Fig. 3). The first rhizoids begin to form in a population at about 12 hours after fertilization. The responsiveness remains maximal until at least 11 hours after fertilization. 3. It is suggested that the low responsiveness of a population of eggs at an earlier period is due to recovery from the effects of irradiation before the rhizoids begin to form. 4. The response of eggs to λ2804 Å is proportional, over a wide range, to the logarithm of the dosage (see Fig. 1). Dosage was regulated by the duration of exposure during the period of maximum response. 5. High dosages of λ2804 Å, of the order of 10,000 ergs per mm.², cause the rhizoids to form fairly precisely away from the source of radiation (see Fig. 2). Twice this dosage inhibits rhizoid formation altogether without causing cytolysis. 6. Other wave-lengths which have also been shown to be effective are: 3660, 3130, 2654, 2537, 2482, and 2345 Å. Only exploratory measurements have been made to test the effectiveness of these wave-lengths, but they show that much greater energy is necessary to obtain a strong response with λ3130 and 3660 Å, especially the latter. The wave-lengths shorter than 2804 Å, on the other hand, show the same order of effectiveness as λ2804 Å. Some may be more effective. 7. A beam of λ2804 Å which is incident on a single layer of Fucus eggs is completely extinguished at 2, 3, 6, or 6½ hours after fertilization. About 85 per cent of a beam of λ3660 Å is extinguished. The wave-length 3660 Å is thus not so completely absorbed as λ2804 Å, but the difference in proportion absorbed by the egg is not nearly so great as the difference in effectiveness.     

THERMAL DEPOLARIZATION OF FLUORESCENCE FROM POLYTENE CHROMOSOMES STAINED WITH ACRIDINE ORANGE

The degree of polarization of fluorescence from stretched Chironomus thummi polytene chromosomes, stained with low concentrations of acridine orange (AO), decreases with increasing temperature. The "half temperature" of this decrease (T_½R) is lower than the expected DNA thermal denaturation temperature (T_m) by about 20°C. T_½R is lowered as histone is removed from chromosomes. Balbiani ring regions of the fourth chromosome have T_½R's much lower than other regions, and nearly as low as chromosomes which had been extensively pretreated with trypsin to remove histone and other proteins. Measurements of the thermal change in the rotational diffusion rate of AO in solution with DNA indicate that the temperature at which the DNA-AO bonding changes from a "rigid" to a "loose" mode varies with the GC percentage of the DNA, and in the same fashion as T_m, although 20°C lower.     

STUDIES OF NATIVE GLYCOGEN ISOLATED FROM SYNCHRONIZED TETRAHYMENA PYRIFORMIS (HSM)

Native glycogen was isolated from Tetrahymena pyriformis (HSM) by isopycnic centrifugation in cesium chloride density gradients. A density of 1.62 to 1.65 was isopycnic for glycogen. Most of the banded glycogen existed as 35 to 40 mµ particles which had a sedimentation coefficient of 214. These particles were composed of aggregates of 2 to 3 mµ spherical particles. Extraction of glycogen with hot alkali reduced the sedimentation coefficient of native glycogen from 214 to 64.7 and the particle diameter from approximately 40 to 20 mµ and smaller. Cell division was synchronized by a repetitive 12-hour temperature cycle, and glycogen was measured at several times during the cell cycle. The temperature cycle consisted of 9.5 hours at 12°C and 2.5 hours at 27°C. Approximately 90 per cent of the cells divided during the last 1.5 hours of the warm period. The carbohydrate/protein ratio of cells at the end of the cold period was 0.27 and was reduced slightly during the warm period. Glucose was incorporated into glycogen during both periods, although the rate of incorporation was greater during the warm period. No preferential incorporation on the basis of particle size was noted. Incorporation was measured in both native glycogen and KOH-extracted glycogen. Tetrahymena glycogen is compared with rat liver glycogen previously isolated by similar procedures, and the significance of using combined rate-zonal and isopycnic centrifugation for isolating native glycogen is discussed.     

MINOR COMPONENTS OF THE DNA OF PHYSARUM POLYCEPHALUM : Cellular Location and Metabolism

DNA metabolism in the slime mold Physarum polycephalum was studied by centrifugation in CsCl of lysates of cultures labeled with radioactive thymidine at various times in the cell cycle. During the G₂ (premitotic) phase of the cell cycle, two components of the DNA are labeled. One component is lighter (buoyant density 1.686 g/cc) than the mean of the principal DNA (1.700 g/cc), and one is heavier (approximately 1.706 g/cc). The labeled light DNA was identified chemically by its denaturability, its susceptibility to DNase, and the recovery of its radioactivity in thymine. Cell fractionation studies showed that the heavy and the principal DNA components are located in the nucleus and that the light DNA is in the cytoplasm. The light DNA comprises approximately 10% of the DNA. About ⅓–½ of the light DNA is synthesized during the S period, and the remainder is synthesized throughout G₂ (there is no G₁ in Physarum). The light DNA is metabolically stable. A low, variable level of incorporation of radioactive thymidine into the principal, nuclear DNA component was observed during G₂.     

The Metabolism of Serum Proteins in Neonatal Rabbits

1. Incorporation of S³⁵-labeled amino acids into serum proteins has been studied in neonatal and developing rabbits. It was found that, per unit weight, neonatal rabbits synthesized only about 1/36 of the gamma globulin, 1/7 of the beta globulin, ½ of the alpha globulin, and ⅛ of the albumin that an adult synthesized. The growing rabbit developed the ability to synthesize various serum proteins at different times. 2. Plasma volumes and serum protein concentrations were determined at different times during the growth period of the rabbit. Plasma volumes were found to be 1 and ½ times larger in newborn animals than in adults, with a gradual decline to the adult level. The total serum protein concentration at birth was about 60 to 65 per cent of the adult value and gradually increased with growth as the plasma volume decreased. 3. Half-lives of homologous albumin and gamma globulin were studied. The half-life of albumin in neonates was nearly twice as long as the half-life in adults, the latter value being reached at 1 month of age. The half-life of gamma globulin in neonates was more than twice as long as the half-life in adults and reached adult values at 2 to 3 months. 4. Attempts were made to alter serum protein metabolism. Gamma globulin synthesis early in life was augmented with antigen injections.     

THE MECHANISM OF 5-AMINOURACIL-INDUCED SYNCHRONY OF CELL DIVISION IN VI CIA FABA ROOT MERISTEMS

Cessation of mitosis was brought about in Vicia faba roots incubated for 24 hours in the thymine analogue, 5-aminouracil. Recovery of mitotic activity began 8 hours after removal from 5-aminouracil and reached a peak at 15 hours. If colchicine was added 4 hours before the peak of mitoses, up to 80 per cent of all cells accumulated in mitotic division stages. By use of single and double labeling techniques, it was shown that synchrony of cell divisions resulted from depression in the rate of DNA synthesis by 5-aminouracil, which brought about an accumulation of cells in the S phase of the cell cycle. Treatment with 5-aminouracil may have also caused a delay in the rate of exit of cells from the G₂ period. It appeared to have no effect on the duration of the G₁ period. When roots were removed from 5-aminouracil, DNA synthesis resumed in all cells in the S phase. Although thymidine antagonized the effects of 5-aminouracil, an exogenous supply of it was not necessary for the resumption of DNA synthesis, as shown by incorporation studies with tritiated deoxycytidine.     

STUDIES ON NUCLEAR STRUCTURE AND FUNCTION IN TETRAHYMENA PYRIFORMIS : I. RNA Synthesis in Macro- and Micronuclei

Tetrahymena in the log phase of growth were pulse labeled with uridine-³H, fixed in acetic-alcohol, extracted with DNase, and embedded in Epon. 0.5-µ sections were cut, coated with Kodak NTB-2 emulsion, and developed after suitable exposures. Grains were counted above macronuclei, above 1000 micronuclei, and above 1000 micronucleus-sized "blanks" which were situated next to micronuclei in the visual field by means of a camera lucida. An analysis of grain counts showed that micronuclei were less than ½₀₀₀ as active as macronuclei on the basis of grains per nucleus. Since micronuclei contained, on the average, about ½₀ as much DNA as macronuclei, micronuclear DNA had less than 1% of the specific activity of macronuclear DNA in RNA synthesis. However, even this small amount of apparent incorporation was not significantly different from zero. Comparisons of the frequency distributions of labeled micronuclei with those of micronuclear "blanks" showed no evidence of a small population of labeled nuclei such as might be expected if micronuclei synthesized RNA for only a brief portion of the cell cycle. We conclude from these studies that there is no detectable RNA synthesis in Tetrahymena micronuclei during vegetative growth and reproduction.     

Electrochemical Polarization-Induced Changes in the Growth of Individual Cells and Biofilms of Pseudomonas fluorescens (ATCC 17552)

The effect of surface electrochemical polarization on the growth of cells of Pseudomonas fluorescens (ATCC 17552) on gold electrodes has been examined. Potentials positive or negative to the potential of zero charge (PZC) of gold were applied, and these resulted in changes in cell morphology, size at cell division, time to division, and biofilm structure. At −0.2 V (Ag/AgCl-3 M NaCl), cells elongated at a rate of up to 0.19 μm min⁻¹, rendering daughter cells that reached up to 3.8 μm immediately after division. The doubling time for the entire population, estimated from the increment in the fraction of surface covered by bacteria, was 82 ± 7 min. Eight-hour-old biofilms at −0.2 V were composed of large cells distributed in expanded mushroom-like microcolonies that protruded several micrometers in the solution. A different behavior was observed under positive polarization. At an applied potential of 0.5 V, the doubling time of the population was 103 ± 8 min, cells elongated at a lower rate (up to 0.08 μm min⁻¹), rendering shorter daughters (2.5 ± 0.5 μm) after division, although the duplication times were virtually the same at all potentials. Biofilms grown under this positive potential were composed of short cells distributed in a large number of compact microcolonies. These were flatter than those grown at −0.2 V or at the PZC and were pyramidal in shape. Polarization effects on cell growth and biofilm structure resembled those previously reported as produced by changes in the nutritional level of the culture medium.     

CELL DIVISION AND DNA SYNTHESIS IN TETRAHYMENA PYRIFORMIS DEPRIVED OF ESSENTIAL AMINO ACIDS

The question of amino acid requirements for DNA synthesis and cell division has been studied in Tetrahymena pyriformis by depriving cells of histidine and tryptophan at defined stages in the interdivision interval. Deprivation any time before DNA synthesis does not prevent the initiation of such synthesis but completely inhibits the following division and limits the increase in DNA, as measured microspectrophotometrically, to 20 per cent. H³-thymidine added to the medium is not incorporated during the 20 per cent increase. Deprivation after DNA synthesis is initiated does not prevent the continuation (to completion) of DNA synthesis, and cell division ensues. H³-thymidine added to the medium under these conditions is incorporated into macronuclear DNA. The data indicate that some amino acid-dependent event occurs, about the time of the beginning of the DNA synthesis period, which is not essential for initiation of DNA synthesis but which is essential for the maintenance of synthesis once it has begun. These results are further discussed in terms of enzymes required to convert thymidine (and possibly the other three deoxyribonucleosides) to the immediate precursor of DNA synthesis.     

Long-Term Single Cell Analysis of S. pombe on a Microfluidic Microchemostat Array

Although Schyzosaccharomyces pombe is one of the principal model organisms for studying the cell cycle, surprisingly few methods have characterized S. pombe growth on the single cell level, and no methods exist capable of analyzing thousands of cells and tens of thousands of cell division events. We developed an automated microfluidic platform permitting S. pombe to be grown on-chip for several days under defined and changeable conditions. We developed an image processing pipeline to extract and quantitate several physiological parameters including cell length, time to division, and elongation rate without requiring synchronization of the culture. Over a period of 50 hours our platform analyzed over 100000 cell division events and reconstructed single cell lineages up to 10 generations in length. We characterized cell lengths and division times in a temperature shift experiment in which cells were initially grown at 30°C and transitioned to 25°C. Although cell length was identical at both temperatures at steady-state, we observed transient changes in cell length if the temperature shift took place during a critical phase of the cell cycle. We further show that cells born with normal length do divide over a wide range of cell lengths and that cell length appears to be controlled in the second generation, were large newly born cells have a tendency to divide more rapidly and thus at a normalized cell size. The platform is thus applicable to measure fine-details in cell cycle dynamics, should be a useful tool to decipher the molecular mechanism underlying size homeostasis, and will be generally applicable to study processes on the single cell level that require large numbers of precision measurements and single cell lineages.     

EFFECT OF HIGH PRESSURE ON GERMINATION OF SEEDS (MEDICAGO SATIVA AND MELILOTUS ALBA)

An increase in percentage germination is obtained with seeds of Medicago sativa exposed for 1 to 10 minutes at 2000 atmospheres hydraulic pressure at 20°C., dried, and germinated after 30 days; and from seeds of Melilotus alba under the same conditions of pressure, when exposed for 5 to 30 minutes, dried, and germinated 30 days later. Exposures to 500 atmospheres pressure was less advantageous for germination; the vitality of seeds normally germinating was more rapidly destroyed than the hard impermeable seeds rendered permeable by the pressure treatment. At 0°C., it required approximately 2½ times the exposure to 2000 atmospheres for seeds of Medicago sativa, and approximately 5 times the exposure for seeds of Melilotus alba, as it did at 20°C.     

Different Rates of Synthesis and Degradation of Two Chloroplastic Ammonium-Inducible NADP-Specific Glutamate Dehydrogenase Isoenzymes during Induction and Deinduction in Chlorella sorokiniana Cells

The kinetics of accumulation (per milliliter of culture) of the α- and β- subunits, associated with chloroplast-localized ammonium inducible nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase (NADP-GDH) isoenzymes, were measured during a 3 hour induction of synchronized daughter cells of Chlorella sorokiniana in 29 millimolar ammonium medium under photoautotrophic conditions. The β-subunit holoenzyme(s) accumulated in a linear manner for 3 hours without an apparent induction lag. A 40 minute induction lag preceded the accumulation of the α-subunit holoenzyme(s). After 120 minutes, the α-subunit ceased accumulating and thereafter remained at a constant level (i.e. steady state between synthesis and degradation). From pulsechase experiments, using ³⁵SO₄ and immunochemical procedures, the rate of synthesis of the α-subunit was shown to be greater than the β-subunit during the first 80 minutes of induction. The α- and β-subunits had different rates of degradation during the induction period (t_½ = 50 versus 150 minutes, respectively) and during the deinduction period (t_½ = 5 versus 13.5 minutes) after removal of ammonium from the culture. During deinduction, total NADP-GDH activity decreased with a half-time of 9 minutes. Cycloheximide completely inhibited the synthesis and degradation of both subunits. A model for regulation of expression of the NADP-GDH gene was proposed.     

Intercellular Correlations: Relation between DNA Synthesis and Cell Division in Early Stages of in Vitro Bud Neoformation

As well as showing the existence, during the first stages of in vitro bud neoformation, of cell populations in a tissue composed of a single cell layer (stem epidermis of Torenia fournieri Lind), some new physiological characteristics of mitosis are defined. Most of the cells which divide during organogenesis synthesize their DNA between 20 and 48 hours of culture. On an epidermal strip (10 × 2.5 millimeters composed of about 5,500 cells) 20% of the original cells enter the S-phase. The first division takes place at the 20-hour stage after the entry into the S-phase of a cell population of about 25 cells. Almost none of the cells of this population divide. The greatest percentage of divisions occurs in cells which synthesize DNA near the 48-hour stage. The relation [Formula: see text] has a value of about 25 at the beginning of cell division (20 hours) and falls to a value of about 1.4 for cells which synthesize DNA near the 48-hour stage. A hypothesis of the existence of a mitotic stimulant in the epidermis is put forward; this stimulant, at first weak, increases progressively.     

Two components of auxin transport

The transport of indoleacetic acid-1-¹⁴C out of sunflower stem sections has been analyzed by a compartmental analysis procedure in which the radioactivity moving out of the tissue (log per cent) is plotted against time. The analysis indicates that indoleacetic acid is transported via a fast transport system (t_½ of about 30 minutes) and a slow transport system (t_½ about 10 hours). While we do not know the sources of these two pools, by analogy with ion transport studies, the fast efflux is characteristic of transport from the cytoplasm across the plasmalemma and the slow efflux is characteristic of transport across the tonoplast and thus out of the vacuole. Both components of transport are inhibited by 2,3,5-triiodobenzoic acid.