Some effects of 2,4,5-trichlorophenoxyacetic acid on the mitotic cycle of lateral root apical meristems of Vicia faba

Roots of Vicia faba were given a one hour pulse label with. ³H-TdR (1 C/ml), either before or after a three hour treatment with a 10^–5 M solution of 2,4,5-trichlorophenoxyacetic acid (TCPA). The durations of the various phases of the mitotic cycle were derived from labeled prophase curves, prepared from autoradiographs of lateral root apical meristems. — TCPA was found to lengthen the duration of the mitotic cycle, primarily because it extended the duration of the period of DNA synthesis (S), though post-synthetic interphase (G₂) was also longer. No measurements could be made with respect to the duration of presynthetic interphase (G₁), because of rapid changes in the lengths of the G₂ and S periods following treatment. — As well as extending the duration of S, TCPA treatment also resulted in at least an initial increase in the rate of DNA synthesis and a decrease in the actual number of cells in S. These results have been discussed with respect to the control of the organization of the root apical meristem.Supported by a grant from the Assistant Professor Research Fund of the University of Missouri.     

Effects of kinetin on cell division,with special reference to initiation and duration of mitosis

Summary Kinetin, a substance recently isolated from DNA preparations, produced polyploidy and various forms of pycnosis in meristematic cells of growing onion roots.Non-toxic concentrations of the substance changed the mean durations of mitosis and interphase as well as the relative durations of prophase, metaphase, anaphase, and telophase in onion root tip cells. It was inferred that the time of the mitotic period was increased, while the duration of interphase was decreased by addition of kinetin to the medium.The phenomena observed are interpreted to be due to (a) a trigger action of kinetin some time during interphase, resulting in premature prophase initiation, and (b) effects of kinetin on the coiling cycle of the chromosomes.It is suggested that the activity of kinetin may in some way be associated with the RNA metabolism of the nucleus.     

Autoradiographic study on the synthesis of nuclear proteins

Experiments were carried out on cultures of Chinese hamster fibroblasts. Cells were short-labelled with H³-Iysine or H³-tryptophane. The kinetics of silver grain number over interphase nuclei, metaphases, and individual chromosomes of the first pair were studied. — The grain count distribution over chromosomes of different groups and over sister-chromatids was analyzed. The data presented allow us to conclude that: 1. There is equal distribution of nuclear and chromosomal proteins between daughter nuclei and sister-chromatids, resp., during each mitotic cycle. 2. The synthesis of chromosome proteins, in general, and nonhistone proteins, in particular, takes place at all stages of the cycle. 3. At each stage of the cycle, proteins are incorporated into chromosomes of different groups simultaneously and at equal rate. The rate of this process in S and G2 is two times that in G1.     

The grasshopper X chromosome

The X chromosome can be identified with the light microscope throughout all stages of the gonial cell cycle (including interphase) in the grasshopper Brachystola magna. At gonial mitotic stages the X chromosome gives the appearance of being undercondensed or negatively heteropycnotic. At interphase the X projects out from the body of the nucleus. — Examination with the electron microscope reveals that the X is compartmentalized at least two gonial cell cycles prior to the entry of the cells into meiotic prophase. The membrane layers that envelope the X chromatin at interphase remain associated with the X chromosome throughout gonial mitotic stages providing the ultrastructural basis for the apparent negative heteropycnosis observed with the light microscope. — The X chromosome is inactive in RNA synthesis during gonial mitotic stages but is hyperactive in RNA synthesis when compared to autosomes at gonial interphase. — X chromosome condensation which reaches its maximum at premieotic interphase is initiated at or prior to the pre-pentultimate gonial division.     

Preprophase bands of microtubules and the cell cycle: Kinetics and experimental uncoupling of their formation from the nuclear cycle in onion root-tip cells

We have studied the timing of preprophase band (PPB) development in the division cycle of onion (Allium cepa L.) root-tip cells by combinations of immunofluorescence microscopy of microtubules, microspectrophotometry of nuclear DNA, and autoradiography of [³H]thymidine incorporation during pulse-chase experiments. In normally grown onion root tips, every cell with a PPB had the G2 level of nuclear DNA. Some were in interphase, prior to chromatin condensation, and some had varying degrees of chromatin condensation, up to the stage of prophase at which the PPB-prophase spindle transition occurs. In addition, autoradiography showed that PPBs can be formed in cells which have just finished their S phase, and microspectrophotometry enabled us to detect a population of cells in G2 which had no PPBs, these presumably including cells which had left the division cycle. The effects of inhibitors of DNA synthesis showed that the formation of PPBs is not fully coupled to events of the nuclear cycle. Although the mitotic index decreased 6-10-fold to less than 0.5% when roots were kept in 20 g·ml^-1 aphidicolin for more than 8 h, the percentage of cells containing PPBs did not decrease in proportion: the number of cells in interphase with PPBs increased while the number in prophase decreased. Almost the same phenomena were observed in the presence of 100 g·ml^-1 5-aminouracil and 40 g·ml^-1 hydroxyurea. In controls, all cells with PPBs were in G2 or prophase, but in the presence of aphidicolin, 5-aminouracil or hydroxyurea, some of the interphase cells with PPBs were in the S phase or even in the G1 phase. We conclude that PPB formation normally occurs in G2 (in at least some cases very early in G2) and that this timing can be experimentally uncoupled from the timing of DNA duplication in the cell-division cycle. The result accords with other evidence indicating that the cytoplasmic events of cytokinesis are controlled in parallel to the nuclear cycle, rather than in an obligatorily coupled sequence.Abbreviations APC aphidicolin - 5-AU 5-aminouracil - DAPI 4, 6-diamidino-2phenylindole - HU hydroxyurea - MI mitotic index - MT microtubule - PMSF phenylmethyl-sulfonyl fluoride - PPB preprophase band - %PPB percentage of cells with PPBs     

Further characterization of the growth inhibitory effect of rotenone on in vitro cultured Ehrlich ascites tumour cells

Summary As an approach for a better understanding of the mode of action of rotenone on mammalian cells we have studied the proliferation properties, metabolism and basic cell composition of Ehrlich ascites tumour cells cultured in vitro in the presence of 2,5 µM rotenone and after removal of the inhibitor.Experiments on asynchronous cells showed a rapid cessation of cell division accompanied by increased glycolytic rate, reduced oxygen consumption, moderate increase in DNA content and a fair increase in protein and RNA content of the cultures. DNA histograms obtained by flow-cytometry revealed an accumulation of cells in the G2 and M phase of the cell cycle. Electron micrographs taken after a 24 h treatment of cells illustrated the formation of giant mitochondria and fragmented nuclei.In order to elucidate the dual effect of rotenone — inhibition of mitochondrial energy metabolism and of mitotic processes — the influence on cells of rotenone at different stages of the cell cycle was tested using Ehrlich ascites tumour cells enriched in G1, S and G2 by centrifugal elutriation. DNA histograms and [³H]thymidine labelling index curves of cells from the different fractions cultured in the presence of 2,5 AM rotenone indicated that in addition to the observed accumulation in G2 and mitotic arrest of cells, the cell cycle progression is delayed in G1 phase. This may be explained by an effect of the inhibitor on the respiratory chain. S phase cells seemed to continue the cycle for several hours at a rate comparable to that of controls.Recultivation experiments on rotenone-treated asynchronous cells in inhibitor-free medium confirmed that some cells reinitiate DNA synthesis without preceeding cell division.Thus it must be concluded that cells at all stages of the cycle are affected by rotenone, but the impairment of cellular metabolism becomes manifest and lethal as soon as the acute block at mitosis is abolished and cells reenter the cycle.Abbreviations EAT cells Ehrlich ascites tumour cells - Hanks' solution Hanks' balanced salt solution - Hepes 4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid     

The mitotic and endomitotic nuclear cycle in Allium carinatum

Nuclear RNA synthesis has been studied in the course of the mitotic and endomitotic cell cycles occurring respectively in the tip meristem and the differentiating (elongating) region of Allium carinatum roots, by means of ³H-uridine autoradiography. The specificity of incorporation was tested by treatments with RNase and DNase, and the labelled RNA was characterized by its sensitivity to -amanitin. RNA synthesis ceases during mitotic chromosome condensation, but continues through the endomitotic structural changes of the chromatin. Nuclei in comparable interphase stages incorporate the more ³H-uridine, the higher the degree of endopolyploidy (ratio about 124). The mean grain numbers counted over nuclei in G₁ and G₂ of the same cycle show, however, ratios of only 11.31. Since the rate of RNA synthesis exhibits a closer relationship to the nuclear volume than to the nuclear DNA content, the synthesis of specific proteins might be necessary to make the reduplicated DNA available as a template. The possible function of the endomitotic cycle in cell differentiation is shortly discussed.     

EXPERIMENTAL CONTROL OF DNA SYNTHESIZING AND DIVIDING CELLS IN EXCISED ROOT TIPS OF PISUM

The number of dividing and DNA-synthesizing cells in excised pea roots can be regulated by eliminating the carbohydrate normally supplied in the culture medium. When the excised roots were allowed to remain for 24 hr in a medium lacking carbohydrate, the number of mitotic figures and tritiated thymidine (H³-T) labeled cells was reduced almost to zero. After an additional 24 hr in the incomplete culture medium, 15% of the interphase cells were H³-T labeled, the percentage of the cells that were dividing never exceeded 1.4, and 30% of these were H³-T labeled. When the roots remained in the deficient medium for 72 hr, neither cell division nor cells synthesizing DNA were observed. Upon addition of 2% sucrose, cell division and DNA synthesis were resumed in the roots that were maintained for 24 or 72 hr without an exogenous carbohydrate supply. It has been hypothesized that some proliferative systems consist of two cellular subpopulations which selectively stop or remain in either the pre-DNA synthetic (G₁) or post-DNA synthetic (G₂) periods of the mitotic cycle. The addition of sucrose, H³-T, and 5-aminouracil to the medium, after the roots had been maintained for 24 hr without a carbohydrate, indicated that most of the proliferative cells in the roots had accumulated in either G₁, a quasi-G₁ condition, i.e., DNA synthesis stopped sometime before completion, or G₂ periods of interphase; the majority, however, were in G₁ or quasi-G₁ conditions. The results suggested that DNA synthesis (S period) and mitosis or the onset of these processes have the highest metabolic requirements in the mitotic cycle and that G₁ and G₂ were the most probable states for proliferative cells in a meristem with a low metabolic level.     

Correlation between the high rate of protein synthesis during mitosis and the absence of G1 period in V79-8 cells

The object of this study was to investigate whether modification of culture conditions would induce G1 and G2 periods in the Chinese hamster cell line, V79-8, which has been reported to exhibit neither of these phases in its life cycle. The results of this study indicate that under optimum culture conditions this cell line multiplies rapidly, with a generation time of about 9.5 h, and exhibits no measurable G1 period. However, under conditions of confluent growth, deprivation of isoleucine or inhibition of polyamine biosynthesis, a significant fraction (44–85%) of the cell population is preferentially arrested in the G1 period. Transient G2 arrest can also be induced in these cells by replacing the amino acid phenylalanine by its analog p-fluorophenylalanine. We have observed that decreasing the concentration of serum in the medium from 16 to 1% resulted not only in the prolongation of generation time but also resulted in a significant increase in the length of G1 period. Culturing cells in medium with 1% serum had no measurable effect on the rate of protein synthesis in interphase cells but a 50% reduction was seen in that of mitotic cells. The ratio between the rates of protein synthesis in mitotic and interphase cells in the line V79-8 is considerably higher (0.373) than that of G1-1 (0.218), a variant of V79-8 that has a G1 period of 4.25 h. These data suggest that cell line V79-8 is unique in retaining a relatively high rate of protein synthesis during mitosis under most favorable conditions. Probably this feature allows the synthesis of the factors necessary for the initiation of DNA synthesis while the cells are still in mitosis. However, under subnormal conditions the protein synthesizing machinery in the mitotic cells becomes inefficient and the cells require a longer time to synthesize the inducers of DNA synthesis; hence a G1 period is expressed.     

Effect of Fluoride on Nucleic Acids and Growth in Germinating Corn Seedling Roots

Corn seeds were treated with 0.01 M sodium fluoride for various time periods. The treated seeds were germinated and grown until the seedling roots reached a standard size of 12±3 mm. Analyses were made for RNA and DNA contents of 3-mm seedling root tips. Determinations also were made for growth rate, rate of cell elongation, cell multiplication, and tissue maturity of 12-mm roots. RNA contents of 3-mm root tips were found to be directly proportional to the growth rates of the entire seedling root of corn seeds treated with sodium fluoride for various periods of time. The RNA content was reduced on a cell basis and was independent of the root tip cell number. The amount of DNA was not related to the growth rate of the intact seedling roots. Since fluoride reduced the number of mitotic figures, it was likely that fluoride inhibited DNA synthesis during the interphase of the mitotic cycle. Growth by cell multiplication was inhibited more than that by cell elongation in the sample treated with fluoride for a shorter period. The two types of growth, however, showed a similar level of growth reduction in the sample treated with fluoride for a longer period. Fluoride seemed to reduce the rates of cellular elongation and multiplication not more than about 40 per cent of the control value in these tissues under present experimental conditions. Fluoride also induced maturity in the seedling roots in proportion to the periods of fluoride treatment.     

Synchronisation of cell division in the shoot apex of Silene in relation to flower initiation

In plants of Silene coeli-rosa, induced to flower by 7 LD, synchronisation of cell division in 20 per cent or more of the cells in the shoot apical dome was found on the 8th and 9th days after the beginning of induction, during the plastochron before sepal initiation. Synchronisation was inferred from the changes in the proportions of cells with the 2C and 4C amounts of DNA, and changes in mitotic index and labelling index. From the peaks of mitotic index a cell cycle of 10 h was measured for the synchronised cells, half that of cells in the apices of uninduced plants in short days. The faster cell cycle and synchronisation in the induced plants was associated with a shortening, of both G1 and G2, suggesting two control points, while S and M remained unchanged. These results are compared with those from other plants in which synchronisation occurs at the beginning rather than the end of evocation.Abbreviations LD long day(s) - SD short day(s) - S DNA synthesis phase of cell cycle - G1 pre-S interphase - G2 post-S interphase - M mitosis     

Nuclear differentiation and nucleic acid synthesis in well-fed exconjugants of Paramecium aurelia

Paramecium aurelia exconjugants contain new macronuclear anlagen and numerous fragments of the old pre-zygotic macronucleus. Macronuclear anlagen develop during the first two cell cycles after conjugation. During this time their volume increases from about 11 m³ to about 3700 m³ and more than 10 doublings of DNA content occur. The rate of DNA synthesis is between two and three times as great as in the vegetative macronucleus. — In macronuclear fragments, however, DNA synthesis is suppressed. The rate of DNA synthesis in macronuclear fragments during the extended first cell cycle after conjugation (11 1/2 hr. vs. 5 1/2 hr. for the vegetative cell cycle) is only about one-third of the rate in vegetative macronuclei and there is only a 65% increase in the mean DNA content of fragments. The rate of fragment DNA synthesis continues to decrease during each of the subsequent two cell cycles. — Unlike the rate of DNA synthesis, the rate of RNA synthesis per unit of DNA is similar in macronuclear anlagen, macronuclear fragments and fully developed macronuclei. Macronuclear fragments continue to synthesize RNA at the normal rate long after the new macronuclei are fully developed. Fragments contribute about 80% of all RNA synthesized during the first two cell cycles after conjugation. RNA synthesis begins very early in the development of macronuclear anlagen and nucleolar material appears during the first half-hour of anlage development. — Chromosome-like structures were never observed during anlage development and there was no evidence of two periods of DNA synthesis separated by a DNA poor stage as has been observed in several hypotrichous Ciliates.     

Effect on the whole alimentary starvation on DNA synthesis in cells precursors in human bone marrow]

The rate of DNA synthesis in cells precursors of the bone marrow of 15 psychic patients was studied by tritiated thymidine autoradiography during different periods of whole alimentary starvation (duration 28 days) which was used with the aim of therapy. It was shown that starvation induced rapid, "leap", "uneven" block of DNA synthesis and decrease in mitotic index. It was found that DNA synthesis block was marked with different degree in different marrow cells. It was supposed that DNA synthesis block occured mainly during G1--S period of interphase. During the initial period of refeeding, intensification of DNA synthesis, which preceded to sharp increase in mitotic index, was observed.     

SELECTIVE INHIBITION OF CELL CYCLE STAGES IN THE ALLIUM ROOT MERISTEM BY COLCHICINE AND GROWTH REGULATORS

The treatment of root tips of Allium carinatum, Allium cepa, and Allium flavum with colchicine, abscisic acid, kinetin, and indole-3-acetic acid, applied in appropriate concentrations, combinations, and durations, makes possible the selective blockade of the cell cycle in G₁, G₂, any mitotic stage, and between karyokinesis and cytokinesis. Moreover, treatment with abscisic acid followed by a recovery period stimulates polyploid nuclei in mature tissues to divide. Colchicine, kinetin, and indole-3-acetic acid applied together cause end-to-end association of metaphase chromosomes. These results together with earlier findings suggest that any step of the cell cycle is independently controlled both by specific balance of the growth regulators and by specific synthesis of the nucleic acids.     

Changes in the Duration of the Mitotic Cycle Induced by Colchicine and Indol-3yl-acetic Acid in Vicia faba Roots

Cells of root meristems of Vicia faba were labelled with tritiatedthymidine and treated with colchicine or IAA or both. The effectsof these compounds on the duration of the mitotic cycle andits constituent phases have been determined using the labelledmitoses wave method of Quastler and Sherman. Colchicine shortensthe mitotic cycle of the cells in interphase at the time oftreatment; it appears to stimulate cells in G1 or early S tocomplete interphase faster than untreated cells. The affectedcells arrive at mitosis 9–12 h after the beginning oftreatment and contribute to the increase in mitotic index seenafter treatment with colchicine. Treatment with IAA did notaffect cells in G2 but it delayed cells in S; this results ina temporary fall in M.I. The effect of IAA in prolonging interphasewas also seen in roots treated with colchicine and IAA; thetetraploid cells induced by colchicine take longer to reachmetaphase than cells treated only with colchicine. The resultssuggest that colchicine and IAA affect different phases of thecell cycle.     

Nucleolar behaviour in Triticum

The maximum number of major nucleoli (macronucleoli) per nucleus of hexaploid, tetraploid and diploid wheat, Aegilops speltoides and Ae. squarrosa corresponded to the number of satellited chromosomes of each species. Smaller nucleoli (micronucleoli) were rare or absent in all of these species except the hexaploid, in which they were predominantly organized on chromosome arm 5D^s. — Fewer than the maximum number of macronucleoli in a mitotic interphase nucleus resulted from fusion of developing nucleoli. Enforced proximity of nucleolus-organizing regions resulted in more frequent fusion of nucleoli. — Analyses of related interphase nuclei showed that nucleoli, and hence probably chromosomes, undergo limited movement during mitotic interphase. These observations also indicate that specific chromosomes do not occupy specific sites in the interphase nucleus.     

Biochemical and Cytological Analyses of RNA Synthesis in Kinetin-treated Pea Root Parenchyma

Excised cortical parenchyma from the pea root (cv. Little Marvel) responds to kinetin/auxin treatment with an increased rate of RNA synthesis well before reinitiating DNA synthesis. Few cells synthesize RNA in the 1st hour of culture. In the presence of kinetin/auxin, the nuclear labeling index increases 2.5-fold as compared to control cultures. The RNA synthesis response has an apparent lag period of 2-4 hours as shown by double label ([³H]adenosine/[¹⁴C]adenosine) experiments. Qualitatively, the RNA synthesized at 4-6 hours sediments between 18S and 5S. The RNA synthesized at 14-16 hours and 24-26 hours is primarily ribosomal RNA when kinetin is present. In the absence of kinetin, no clear pattern of RNA synthesis emerges.     

Some Characteristics of DNA Synthesis and the Mitotic Cycle in Ehrlich Ascites Tumor Cells

In vivo studies of Ehrlich ascites tumor cells during the first 5 days of growth in peritoneal cavities of mice consisted of the following: 1. Determination of growth curves by direct enumeration of cells. 2. Estimation of the duration of each phase of the mitotic cycle based on incidence of cells in different phases. 3. Radioautographic studies to determine the proportion of cells in different phases of the mitotic cycle that incorporate tritiated thymidine during a single brief exposure to this precursor of DNA. 4. Estimation of the rate of incorporation of tritiated thymidine at different times during the period of DNA synthesis by comparison of mean grain counts over nuclei in radioautographs at different times following exposure to tritiated thymidine. The assumptions underlying these experiments and our observations concerning the duration of the period of DNA synthesis and its relation to the mitotic cycle are discussed. It is concluded that DNA synthesis is continuous, occupying a period of 8.5 hours during the interphase and that the average rate of synthesis is approximately constant.     

The cell cycle of epidermal cells during reprogramming in the pupa of Galleria

The epidermal cell cycle of the pupal mesonotum of Galleria was investigated by the determination of mitotic indices, [³H]thymidine incorporation and flow-cytophotometric analysis during the first 48 h after pupation.Immediately after the pupal ecdysis nearly all epidermal cells are arrested in G2. Thereafter only a few mitoses occur, leading to a slow increase in the number of G1 nuclei. With the onset of a mitotic wave at a pupal age of 21 h this increase becomes more rapid. On day 2, the cell population reaches a plateau in the number of G1 (resp. G2) cells, reflecting a steady state between mitotic activity and DNA synthesis.A comparison of these cell cycle changes with known data of the time course of reprogramming and ecdysteroid titre leads to the conclusion that there is no causal relationship between DNA synthesis and cellular determination in the sense of a quantal cell cycle, and that DNA synthesis can precede the definite rise in ecdysteroid titre.     

INCORPORATION OF TRITIUM-LABELED THYMIDINE AND LYSINE INTO CHROMOSOMES OF CULTURED HUMAN LEUKOCYTES

The incorporation of thymidine-H³ and lysine-H³ into human leukocyte chromosomes was studied in order to determine the temporal relationships between the syntheses of chromosomal deoxyribonucleic acid and chromosomal protein. The labeled compounds were incorporated into nuclei of interphase cells. Label from both precursors became apparent over the chromosomes of dividing cells. Incorporation of thymidine-H³ occurred during a restricted period of midinterphase (S) which was preceded by a nonsynthetic period (G₁) and followed by a nonsynthetic period (G₂). Incorporation of lysine-H³ into chromosomal protein occurred throughout interphase. Grain counts made over chromosomes of dividing cells revealed that the rate of incorporation of lysine-H³ into chromosomal protein differed during various periods of interphase. The rate of incorporation was diminished during G₁. During early S period the rate of incorporation increased, reaching a peak in late S. The high rate continued into G₂. Thymidine-H³ incorporated into DNA was distributed to mitotic chromosomes of daughter cells in a manner which has been referred to as a "semi-conservative segregation." No such semi-conservative mechanism was found to affect the distribution of lysine-H³ to the mitotic chromosomes of daughter cells. Therefore, it is concluded that synthesis of chromosomal protein and its distribution to chromosomes of daughter cells are not directly influenced by synthesis and distribution of the chromosomal DNA with which the protein is associated.