Is there "Metabolic" DNA in the Mouse Seminal Vesicle?

This study was designed to answer the question: Is H³-thymidine uptake by nuclei of the mouse seminal vesicle evidence for DNA synthesis and mitosis, or does it signify some "metabolic" function of DNA unrelated to chromosome duplication? Mice were given an intraperitoneal injection of H³-thymidine. Six hours later Feulgen squashes of the seminal vesicle epithelium were made and covered with autoradiographic stripping film. The silver grains above labeled nuclei were counted, and the Feulgen dye contents of these same nuclei were determined photometrically after removal of the grains from the emulsion. Unlabeled nuclei were also measured. The dye contents of non-radioactive nuclei form a unimodal distribution, indicating that polyploidy is absent from this tissue. The radioactive nuclei fall into two groups. In the first, the average dye content is the same as that of the cold nuclei (2C). In the second, the values range from 2C to 4C. In the 2C to 4C group the grain count is proportional to the dye content, showing that incorporation is correlated with synthesis. The radioactive 2C nuclei arose by mitosis during the course of the experiment. This is shown by the following facts: (1) They frequently occur in pairs. (2) They average smaller than unlabeled 2C nuclei. (3) Their average grain count is approximately half that of the 4C nuclei. (4) Labeled division figures are found. (5) A mitotic rate estimated from the number of labeled 2C nuclei accords reasonably well with one based on the number of observed mitoses. Since the incorporation of thymidine accompanies DNA synthesis and precedes mitosis, there is no reason to postulate a special "metabolic" DNA in this tissue.     

STUDIES ON THE GERMINATION OF SEEDS OF THE ROOT PARASITES,ALECTRA VOGELII AND STRIGA GESNERIOIDES. II. DNA SYNTHESIS AND DEVELOPMENT OF THE QUIESCENT CENTER IN THE RADICLE

DNA synthetic activity in the radicle meristem of embryos of germinating seeds of the obligate root parasites, Alectra vogelii and Striga gesnerioides was followed by autoradiography of ³H-thymidine incorporation. Incorporation of ³H-thymidine occurred in the nuclei of cells destined to form the vascular tissues, ground meristem and epidermis. An analysis of the distribution of labeled nuclei demonstrated the presence of a quiescent center of 2-4 cells in the radicle at the beginning of seed germination, becoming more prominent at later stages of germination. During continued growth of the radicle which resulted in a reduction in size of the meristem, cells of the original quiescent center were activated to undergo DNA synthesis.     

THE USE OF TRITIATED THYMIDINE IN THE STUDY OF TISSUE ACTIVATION DURING GERMINATION IN ZEA MAYS

Stein, O. L. (Montana State U., Missoula), and H. Quastler. The use of tritiated thymidinein the study of tissue activation during germination in Zea mays. Amer. Jour. Bot. 50(10): 1006-1011. Illus. 1963.—Corn embryos were exposed to H³thymidine at various times during the first 80 hr of germination. An analysis of labeled nuclei was made from autoradiographs, and the number and position of mitoses were recorded. Those tissues which approach maturity during embryogeny (root cap, coleorhiza, scutellur node) are first to resume DNA synthesis (30 hr after soaking). No mitoses were observed in these tissues. In shoot and root, mitoses usually precede DNA synthesis, indicating that the nuclei of the dormant embryo have a DNA value of 4C(twice the diploid DNA) or more. The shoot begins its activity much later than the root (50 hr). The shoot apex was the last region to boeomo active, some 70 hr after initiation of the soaking treatment.     

DNA MICROSPECTROPHOTOMETRY OF SHOOT APICAL MERISTEM CELL POPULATIONS IN CERATOPTERIS THALICTROIDES (FILICALES)

A microspectrophotometric analysis of the DNA content of cell populations in the shoot apical meristem of young and adult stages of the filicalean fern Ceratopteris thalictroides was conducted to determine if the previously reported uptake of labeled DNA precursors by the apical cell was a consequence of endomitotic DNA replication or of DNA synthesis preceeding mitosis. Results demonstrate that in both the young and adult plants the estimated DNA content of the apical cell nuclei parallels that of the other cells of the meristem. There was no evidence of an “apical zone” of endopolyploid cells.     

The effect of butyrate on the cell cycle in root meristems of Pisum sativum

Sodium butyrate at 5 mM in aerated White's medium reduced the mitotic index in root meristems of seedlings of Pisum sativum to < 1% after 12 h. This effect was lessened as the butyrate concentrations were lowered. The fraction of the root meristem nuclei in G2 increased to ~ 70% after 12 h in butyrate. After 12 h exposure to butyrate, seedlings transferred lo medium without butyrate gradually re-established their normal root meristem mitotic pattern, with a burst of mitosis at 10 h after the transfer. Even a brief exposure to butyrate inhibited DNA synthesis, and nuclei released from butyrate exposure were still unable to resume normal DNA synthesis even after 12 h. This information suggests that butyrate halts progression through the cell cycle by arresting meristem nuclei in G2 and inhibiting DNA synthesis.     

Localization of organelle DNA synthesis within the root apical meristem of rice

DNA synthesis in cell nuclei and organelles in the root apicalmeristem of rice was analysed by anti-BrdU immunofluorescencemicroscopy to determine whether there is a specific order ofthese events in monocot roots. In the root meristem, organelleDNAs were synthesized in a specific region in the distal partof the root apical meristem, and were not synthesized in theroot meristem‘s proximal region or the elongation zone.In contrast, cell nuclear DNA was synthesized throughout theroot apical meristem, except in the quiescent centre. In theroot cap of rice, DNA synthesis in both cell nuclei and organellenucleoids was detected only in the two layers of cells at theproximal end, which is a striking characteristic of monocotyledonousplants. Moreover, to determine quantitatively the activity ofDNA synthesis in cell nuclei and organelle nucleoids in micro-scalesections of plant tissues, we developed novel techniques formicro-scale hybridization and immuno-detection analysis. Atthe distal end of the root apical meristem, DNA levels of plastidsand mitochondria were 4-fold and 5-fold greater than those inthe elongation zone, respectively. Intracellular organelle DNAlevels dropped rapidly as the distance from the root tip increased.The activity of organelle DNA synthesis in the distal end ofthe root apical meristem was about 10-fold greater than thatin the elongation zone. Our present results confirm that nuclearand organelle DNA synthesis are not synchronized, but the latteroccurs preferentially before multiple cell divisions. Key words: Organelle DNA synthesis, organelle nucleoids (organelle nuclei), root apical meristem, anti-bromo-deoxyuridine immunofluorescence microscopy, rice.     

Amplification of nuclear DNA sequences during induced plant cell dedifferentiation

Cell dedifferentiation has been induced in Vicia faba roots by removing the whole meristem (decapitation). When centrifuged to equilibrium in CsCl density gradient, the DNA from dedifferentiating tissues forms a heavier satellite, not occurring in the DNA from differentiated tissues. Most of the radioactivity after [3H]thymidine feeding is found in the satellite DNA. Its sequences have a well defined nuclear localization, as shown by in situ hybridization experiments. These results indicate amplification of G + C-rich nuclear DNA sequences during cell dedifferentiation.     

Rapid Staining of Nuclei and Chromosomes in Root Tips

Feulgen reagent quickly heated to and maintained at 60 C just before immersion of plant material, basic fuchsin in acid alcohol at room temperature, and pinacyanol at room temperature will stain hydrolyzed root tip nuclei and chromosomes in one minute or less. This technic, coupled with fast fixation, can be utilized when uncertainties exist as to when to begin sampling plant meristem cells for mitoses or when time does not allow for standard fixation and Schiff staining.     

HORMONE-INDUCED ENDOREDUPLICATION PRIOR TO MITOSIS IN CULTURED PEA ROOT CORTEX CELLS

One-mm-thick cortical explants excised aseptically from 10-11 mm behind the tip of 3-day-old roots of the garden pea, Pisum sativum, cv. ‘Little Marvel’ were cultured on a synthetic nutrient medium supplemented with auxin or auxin and cytokinin. Nuclear DNA contents were measured in cells of the explants at the outset and at specified times during culture up to seven days. Fixed and sectioned preparations were stained with the Feulgen method using the DNA-specific dye auramin-O. Fluorescent microspectro-photometric measurements of individual nuclei were made from each cortical population. At day zero all cortical nuclei measured were either 2c or 4c with respect to their DNA content. In the presence of the auxins, indoleacetic acid and 2,4-dichlorophenoxyacetic acid, and the cytokinin, kinetin, DNA values increased to multiples of the 2c level with populations at the 8c and 16c level predominating after three days of culture as well as at seven days. In the presence of auxins alone no change in DNA values was observed during three days. Kinetin concentrations as low as 0.01 ppm were already effective. The data are interpreted to show that cytokinin, in the presence of auxin, induces two rounds of DNA synthesis prior to the first mitoses, the first round being connected with chromosome doubling by endoreduplication and the second one with normal mitosis. From this we inferred that tetraploid cells in leguminous root nodules might have arisen in the same way, i.e., by endoreduplication prior to the first mitoses induced by the rhizobial division stimulus, unless the chromosome number of root cortical cells had already been doubled by endoreduplication in the normally differentiating root systems.     

DURATION OF THE NUCLEAR CYC LE IN TRADESCANTIA PALUDOSA ROOT TIPS AS MEASURED WITH H3-THYMIDINE

Wimber , Donald E. (Brookhaven National Lab., Upton, N. Y.) Duration of the nuclear cycle in Tradescantia paludosa root tips as measured with H³-thymidine. Amer. Jour. Bot. 47(10): 828–834. Illus. 1960.—The duration of the nuclear cycle and its various subdivisions were measured in Tradescantia root tips by autoradiographic techniques. H³-thymidine was used as a nuclear label and was supplied to the roots for 0.5 hr. After labeling, the roots were allowed to grow in the absence of label for periods up to 38 hr. By determining the percentage of divisions labeled at the various times of fixation, a reconstruction of the nuclear cycle could be made. The average cycle was determined as 20 hr. in duration, DNA synthesis 10.8 hr., presynthetic interphase 4 hr., postsynthetic interphase 2.7 hr., prophase 1.6 hr., metaphase 0.3 hr. and anaphase-telophase 0.6 hr. Approximate standard deviations for the duration of some of the subdivisions were calculated.     

The relation of DNA synthesis and mitosis in tobacco pith tissue cultured in vitro

Summary DNA synthesis and mitosis were initiated in cultured tobacco pith tissue by means of IAA and kinetin. DNA classes were determined by microspectrophotometric measurements (Feulgen); autoradiographs (tritiated thymidine) served to ascertain whether or not nuclei had undergone DNA synthesis during culture.All mitoses in new cells (resulting from divisions in culture) were diploid and had been preceded by DNA synthesis in culture.Whereas many of the old cells (which had not previously divided in culture) found in diploid or polyploid mitosis had undergone DNA synthesis during culture, others had not. Such non-radioactive mitoses still occurred after 16 days.In view of this, a 4 C nucleus in differentiated tissue should be considered as potentially both diploid and tetraploid, for it appears impossible to predict whether it would, upon restoration of conditions conducive to DNA synthesis and mitosis, enter a diploid mitosis or, after undergoing DNA synthesis, a tetraploid one.A high nuclear DNA content seems to have a much more inhibiting effect on the onset of DNA doubling than on that of mitosis.Somatic polyploidization is understood as the result of two DNA doublings between which mitosis was omitted, or aborted, or in effect undone by a failure of cytokinesis leading to fusion during a later mitosis.This work has been supported by research grants to K. Patau from the U.S. Public Health Service (grant No. C-3313) and the American Cancer Society.     

A cytological analysis of the antimetabolite activity of 5-hydroxyuracil in Vicia faba roots

The effects of 5-hydroxyuracil (5-HU) (isobarbituric acid) upon cell elongation, mitosis, and DNA synthesis were studied in Vicia faba roots. 5-HU had no consistent effect upon root elongation. It blocked DNA synthesis (analyzed by photometric measurements of Feulgen dye in nuclei) during the first 6 hours of treatment; the block spontaneously disappeared by the 12th hour of treatment. Uracil and thymine had no effect upon this block of synthesis. Both thymidine and uridine reversed the block in 6 and 9 hours respectively. In all cases blockage of DNA synthesis was followed by inhibition of mitosis (determined by changes in the percentage of cells in mitosis) and resumption of DNA synthesis was followed by resumption of mitosis. Inhibition indices calculated from the mitotic data indicated a competitive relationship between 5-HU and thymidine and 5-HU and uridine. 5-HU is considered to block DNA synthesis by competing with thymidine for sites on enzymes involved in the synthesis. It is suggested that uridine reverses the block in synthesis by undergoing a conversion to thymidine.     

A Cytological Analysis of the Antimetabolite Activity of 5-Hydroxyuracil in Vicia faba Roots

The effects of 5-hydroxyuracil (5-HU) (isobarbituric acid) upon cell elongation, mitosis, and DNA synthesis were studied in Vicia faba roots. 5-HU had no consistent effect upon root elongation. It blocked DNA synthesis (analyzed by photometric measurements of Feulgen dye in nuclei) during the first 6 hours of treatment; the block spontaneously disappeared by the 12th hour of treatment. Uracil and thymine had no effect upon this block of synthesis. Both thymidine and uridine reversed the block in 6 and 9 hours respectively. In all cases blockage of DNA synthesis was followed by inhibition of mitosis (determined by changes in the percentage of cells in mitosis) and resumption of DNA synthesis was followed by resumption of mitosis. Inhibition indices calculated from the mitotic data indicated a competitive relationship between 5-HU and thymidine and 5-HU and uridine. 5-HU is considered to block DNA synthesis by competing with thymidine for sites on enzymes involved in the synthesis. It is suggested that uridine reverses the block in synthesis by undergoing a conversion to thymidine.     

Nuclear and Cell Sizes in Different Regions of Root Meristems of Zea mays L.

Nuclear volumes and cell areas were determined for seven regionsof the meristem of roots of Zea mays. Roots were fixed in 10per cent neutral buffered formalin, in 3 per cent glutaraldehydeor in acetic acid/alcohol; they were prepared as sections oralls were teased apart. Mean volumes of interphase nuclei weresimilar in all regions of the root except the vascular tissueof the stele. Mean nuclear volumes and the overall range ofvolumes were similar in sub-populations of cells with differentproportions of G¹, S and G² cells, e.g. in row I of root capinitials, whose cells lack a G¹ phase, and in quiescent centrecells, which are mainly in G¹. Nuclear volume does not appearto be closely correlated with DNA content. Nuclear volumes covereda 6 to 12-fold range within a meristem and even within specificregions, in which cells are part of the same cell lineages,there was a 4- to 9-fold range. Nuclear volumes were comparedin sister cells in rows I and II of the root cap initials. In10 per cent of the pairs, sister nuclei had identical volumes;the other pain had different volumes and mean difference was68 µm³. Mechanisms by which this variability could begenerated are discussed, particularly asymmetry, at mitoses,of factors that regulate nuclear growth. Zea mays L., nuclear volume, cell size, root mcristem, DNA content, mitosis     

Die Verteilung der Chromosomen auf die Tochterzellkerne multipolarer Mitosen in euploiden Gewebekulturen von Microtus agrestis

In kidney epithelial cultures from female Microtus agrestis, 3,55% of all mitoses are multipolar, 94% of them tripolar. Feulgen photometric measurements of 21 tripolar mitoses reveal a total DNA amount corresponding to the mitotic diploid value (4c) in 5 cases, and to the tetraploid value (8c) in 16 cases, Diploid tripolar mitoses divide into one daughter nucleus with a diploid DNA value (2c) and two nuclei each with a haploid DNA value (1c). Most tetraploid tripolar mitoses divide into one daughter nucleus with a diploid DNA value (2c) and two nuclei with a triploid DNA value (3c). Also the sex chromosomes are distributed to the daughter nuclei in the relation of 2∶3∶3. This can be seen in anaphase figures as well as in interphase nuclei presumably derived from tripolar mitoses, showing chromocenters according to the number of X-chromosomes. In two cases of tripolar tetraploid mitoses the resulting nuclei have a haploid, a triploid and a tetraploid DNA value. The DNA replication pattern is always identical in the daughter nuclei of diploid and tetraploid tripolar mitoses. — Our observations suggest segregation and distribution of haploid chromosome sets or multiples of haploid sets to the daughter nuclei of multipolar mitoses. They also show a possible way of formation of haploid and triploid cells in a basically diploid tissue. Presumably triploid nuclei (with 3 chromocenters) are capable of DNA synthesis.     

Some cytological aspects of antagonism in synthesis of nucleic acid

Summary The effects of solutions of benzimidazole and 5-aminouracil onAllium cepa root meristems immersed in them have been studied cytologically. Benzimidazole and 5-aminouracil, analogues of purines and pyrimidines respectively, reduce the number of nuclear divisions being initiated. A marked decline in frequency of cells in mitosis occurs at the beginning of treatment with benzimidazole (300 p.p.m.) but not until 6 or more hours later with 5-aminouracil (125 p.p.m.).After treatment with 5-aminouracil, small interstitial portions of chromosomes are Feulgen-negative and become extended during anaphase into thin strands. The distal portion of these chromosomes may lag during anaphase and contribute to malformations of the surface of the sister nuclei and to micronuclei-like bodies lying along their adjacent surfaces. However, stickiness resulting in chromosome bridges was not observed. After treatments with benzimidazole, chromosomes are more contracted at metaphase and anaphase than in the control. At the concentrations used, neither component was observed to affect the spindle mechanism of the divisions in progress at the beginning of treatment nor of the divisions which escape inhibition.Reversal of these effects by the introduction of metabolites which these analogues resemble structurally, was investigated. Adenine sulphate (800 p.p.m.) acts antagonistically to benzimidazole (300 p.p.m.) by delaying the marked decline in frequency of mitoses by more than 6 hrs. By 12 hrs. the frequency of cells in mitoses was very low; thus, reversal is not permanent. Half the inhibition of growth caused by 5-aminouracil (50 p.p.m.) is reversed by thymine (50 p.p.m.) for the 24 hrs. of the test period. When 5 p.p.m. folic acid was added to this mixture, root growth exceeded that of the controls. Feulgen-negative gaps, however, are still present in chromosomes at anaphase.Estimations of Feulgen content of nuclei to determine the effect of these treatments on DNA synthesis were made by means of a microphotometer. In control roots there are two classes of nuclei at interphase according to the amount of DNA per nucleus. The amount doubles during interphase before the next division of the nuclei in the root meristem. In roots treated with 5-aminouracil this doubling is inhibited, so that interphase nuclei with the lower amount of DNA accumulate. In roots treated with benzimidazole the relative frequencies of the two classes of nuclei is unaltered; however, mitotic activity is inhibited. This compound apparently affects not only processes concerned with doubling the amount of DNA per nucleus but also processes necessary to the division itself. There was, however, no unquestionable evidence of a nucleus being reconstituted after it had started a division.Contribution from the Program in Cytology, Department of Botany University of Wisconsin, Madison, supported in part by grants to Dr.C. Leonard Huskins from the American Cancer Society, Rockefeller Foundation, and Research Committee of the Graduate School with funds supplied by the Wisconsin Alumni Research Foundation.     

1,8-Cineole inhibits root growth and DNA synthesis in the root apical meristem ofBrassica campestris L.

1,8-cineole is a volatile growth inhibitor produced bySalvia species. We examined the effect of this allelopathic compound on the growth of other plants usingBrassica campestris as the test plant. Cineole inhibited germination and growth ofB. campestris in a dosedependent manner. WhenB. campestris was grown for 5 days with various concentrations of cineole, the length of the roots was found to be shorter as the concentration of cineole increased, whereas the length of the hypocotyl remained constant up to 400 μM cineole, indicating that cineole specifically inhibited growth of the root. The mitotic index in the root apical meristem of 3-day-old seedlings decreased from 5.6% to 1.6% when exposed to 400 μM cineole, showing that cineole inhibits the proliferation of root cells. We then examined the effect of cineole on DNA synthesis by indirect immunofluorescence microscopy using antibody raised against 5-bromo-2′-deoxyuridine (BrdU, an analogue of thymidine) in thin sections of samples embedded in Technovit 7100 resin. The results clearly demonstrated that cineole inhibits DNA synthesis in both cell nuclei and organelles in root apical meristem, suggesting that cineole may interfere with the growth of other plant species by inhibiting DNA synthesis in the root apical meristem.     

MERISTEMATIC PRECURSORS OF VASCULAR PARENCHYMA DIFFERENTIATE FROM G2 PHASE AFTER REPLICATING DNA DISCONTINUOUSLY

A small population of cells representing 1% or less of those in the root-tip meristem was identified as the precursor of vascular parenchyma and certain root-cap cells in carbohydrate starved cultured pea roots. Autoradiography and cytophotometric measurements of nuclei labeled with [³H]-thymidine showed that in the absence of carbohydrate the precursor cells replicate their DNA discontinuously accumulating temporarily in late S phase prior to differentiating from the G₂ phase. Besides discontinuity of DNA synthesis, the nuclei of precursor cells undergo a change in morphology. The nuclei are shaped round when replicating DNA but later on, while differentiating, they become oblong. This transformation occurs within 72 hr after the starved roots are fed sucrose. Autoradiograms of serial cross-sections of pulse-labeled roots indicate that the cells in late S phase differentiate forming a ring around the stelar cylinder and a ring around the periphery of the root. These observations suggest that during the last half of the final S phase the precursor cells modify their chromosomal DNA and that this modification is associated with the initial steps of differentiation.     

DNA Synthesis, Mitosis and Ploidy of Dividing Cells in Early Crown Gall

DNA synthesis, mitosis and ploidy of dividing cells were studied during 30 h after wounding around wounds inoculated with Agrobacterium tumefaciens, around sterile wounds and in control stems of Vicia faba. DNA synthesis was examined by counting nuclei labelled with ³H-thymidine in slides in which mitoses had been counted and analyzed before autoradiography. The main results were that around inoculated wounds, DNA synthesis and the number of mitoses showed a peak between 12 and 22.5 h. Both types of wounding induced mitoses, many of them polyploid (DNA content higher than 4C), both in the pith and the cortex, whereas in the control stems only diploid mitoses, mostly in the stelar area, were seen. The first polyploid (8C) mitoses around the inoculated wounds took place at 12 h and at 15.5 h 32C mitoses were seen; around the sterile wounds the first 8C divisions occurred at 26 h. The frequency of polyploid mitoses and their degree of ploidy continued to be considerably higher around the crown gall than around the control wounds. When a cell with a higher than 4C content is induced to divide, the 12 chromosomes, as a rule, consist of four, eight, 16 or 32 chromatids, instead of the normal two. The early division of highly polyploid cells around the inoculated wounds is obviously caused by growth factors which are known to be produced by the bacteria. It appears possible that this ability to synthesize excessive amounts of growth factors is subsequently transferred to the host cells through bacterial DNA.     

Nuclear DNA in normal and refed Amoeba proteus

Amoeba proteus synthesizes DNA in G2 phase of the cell cycle upon feeding after starvation. The characteristics of the DNA synthesized in G2 have been studied by microscope photometry of individual Feulgen-stained nuclei and by buoyant density centrifugation of nuclear DNA in CsCl. Amoeba nuclei were found to contain 42.8 pg of DNA. This DNA bands in CsCl at a density of 1.693 g/cm³ with a satellite at 1.714 g/cm³ which makes up 24% of nuclear DNA. DNA from whole cells has an additional non-nuclear satellite at 1.726 g/cm³. When cells are starved and re-fed with food labeled with [³H]thymidine, the DNA synthesized is predominantly the 1.714 satellite. The amount of DNA synthesized in G2 is small since there is no measurable difference in Feulgen dye binding to nuclei of starved vs starved and re-fed cells. The data suggest that refeeding induces a resumption of late S phase DNA synthesis, or the preferential synthesis of specific DNA sequences such as rRNA genes.