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.     

Human hepatocyte polyploidization kinetics in the course of life cycle

The processes of polyploidization in normal human liver parenchyma from 155 individuals aged between 1 day and 92 years were investigated by Feulgen-DNA cytophotometry. It was shown that polyploid hepatocytes appear in individuals from 1 to 5 years old. Up to the age of 50 years the accumulation rate of binucleate and polyploid cells is very slow, but subsequently hepatocyte polyploidization is intensified, and in patients aged 86–92 years the relative number of cells with polyploid nuclei is about 27%. Only a few hepatocytes in the normal human liver reach 16C and 8C×2 ploidy levels for mononucleate and binucleate cells respectively. Using a mathematical modeling method, it was shown that during postnatal liver growth the polyploidization process in human liver is similar to that in the rat, and that polyploid cells are formed mainly from binucleate cells. As in rats, prior to an increase in ploidy level, diploid human hepatocytes can pass several times through the usual mitotic cycles maintaining their initial ploidy level. After birth, only one in ten hepatocytes starting DNA synthesis enters the polyploidization process. At maturity about 60% of 2C-hepatocytes starting DNA synthesis divide by conventional mitosis, the rest dividing by acytokinetic mitosis leading to the formation of binucleate cells. During ageing the probability of hepatocyte polyploidization increases and in this period there are two polyploid or binucleate cells for every diploid dividing by conventional mitosis.     

Cell cycling and DNA replication in a mutant blocked in cell division in the nematode Caenorhabditis elegans

The postembryonic development of the nematode Caenorhabditis elegans has been described at the level of individual cell lineages. A mutant of postembryonic development, lin-5 II, causes a failure of postembryonic nuclear and cell divisions. Mitosis in living animals is seen by light microscopy to proceed through prophase and nuclear envelope breakdown, but an abnormal-looking metaphase plate forms in the mutant, after which the interphase nuclear morphology reappears until the next attempted round of division. The precursor cells which give rise to the ventral nerve cord have been studied in lin-5. In the wild type these cells divide asymmetrically to give six descendants (one hypodermal cell and five neurons). In the mutant these precursors accumulate approximately six times the diploid quantity of DNA within a single nucleus, while attempting mitosis up to three times. These polyploid cells display characteristics of the cells they would have produced ordinarily.     

Developmental changes in the ploidy of mouse implanting trophoblast cells in vitro

Shortly after the onset of implantation, polar mouse trophoblast cells proliferate and give rise to the ectoplacental cone, constituted by two distinct cell populations: undifferentiated, diploid cells and giant cells. Giant cells characteristically exhibit exaggerated dimensions and polyploid nuclei. In this study, we employ ectoplacental cones as a dynamic source of trophoblast giant cells to analyze cell proliferation, cell death, and ploidy under in vitro conditions. Our results show that DNA synthesis and the increase in the cell number are relevant only during the first 24 h of culture. Subsequently, DNA synthesis still occurs, mainly in the giant cell compartment, while the number of cells gradually decreases. Cell death by injury and apoptosis was also observed in the non-giant cell compartment of the ectoplacental cone. These findings suggest that the first 24 h of culture are crucial to the mitotic activity of the ectoplacental cone cells that gradually ceases, favoring the endoreduplication process. The DNA synthesis index during the subsequent experimental intervals emphasizes accumulation of DNA for the polyploidization. There was clear correlation between DNA content and nuclear dimension. The ploidy values for the trophoblast giant cells varied from 2C up to 368C in the giant cells, but were not as expressive as those known from in vivo conditions, probably due to the absence of regulatory factors specific to the embryonic-maternal interface. In situ hybridization and histochemistry for the nucleolus-organizing region showed that trophoblast nuclei have only two marker signals, indicative of a typical polytenic process. This present study elucidates important aspects of trophoblast behavior and provides new information on trophoblast physiology in vivo and in vitro.     

Occurrence of ploidy shift in a strain of the imperfect yeast Candida albicans

A clinical isolate of Candida albicans, a member of the Fungi Imperfecti, was polyploid as shown by the fact that it contained two kinds of nuclei, one of diploid and one of tetraploid DNA content. These determinations were made by fluorescence microscopy-photometry. The nucleus-associated organelles (NAOs), or spindle pole bodies, of yeast cells in this isolate were classified into two groups, one diploid and the other tetraploid, according to their dimensions as determined by serial thin-sectioning electron microscopy. A ploidy shift from diploid to tetraploid was found in individual cells of a culture of this isolate undergoing diphasic growth in minimal salts medium. A process of shift-down or reduction of ploidy from tetraploid to diploid was also observed by electron microscopy during these growth conditions: this appeared to occur in large cells which showed multiple spindle formation during nuclear division, a phenomenon apparently similar to the process of meiosis II during sporogenesis of Saccharomyces cerevisiae, but differing in that it produces diploid daughter nuclei by the vegetative process.     

Uncoupled cell cycle without mitosis induced by a protein kinase inhibitor, K-252a

The staurosporine analogues, K-252a and RK-286C, were found to cause DNA re-replication in rat diploid fibroblasts (3Y1) without an intervening mitosis, producing tetraploid cells. Analysis of cells synchronized in early S phase in the presence of K-252a revealed that initiation of the second S phase required a lag period of 8 h after completion of the previous S phase. Reinitiation of DNA synthesis was inhibited by cycloheximide, actinomycin D, and serum deprivation, but not by Colcemid, suggesting that a functional G1 phase dependent on de novo synthesis of protein and RNA is essential for entry into the next S phase. In a src-transformed 3Y1 cell line, as well as other cell lines, giant cells containing polyploid nuclei with DNA contents of 16C to 32C were produced by continuous treatment with K-252a, indicating that the agent induced several rounds of the incomplete cell cycle without mitosis. Although the effective concentration of K-252a did not cause significant inhibition of affinity-purified p34cdc2 protein kinase activity in vitro, in vivo the full activation of p34cdc2 kinase during the G2/M was blocked by K-252a. On the other hand, the cyclic fluctuation of partially activated p34cdc2 kinase activity peaking in S phase still continued. These results suggest that a putative protein kinase(s) sensitive to K-252a plays an important role in the mechanism for preventing over-replication after completion of previous DNA synthesis. They also suggest that a periodic activation of p34cdc2 is required for S phases in the cell cycle without mitosis.     

Cytogenetics of protoplast cultures of Brachycome dichromosomatica and Crepis capillaris and regeneration of plants

Summary Callus derived protoplasts of Brachycome dichromosomatica (2n=2x=4) and Crepis capillaris (2n=2x=6) have been regenerated into karyologically normal plants, i.e. plants without visible alterations of the diploid chromosome set. However, metaphase analysis of protoplast cultures derived from both callus as well as mesophyll cells showed karyological changes in the overwhelming majority of cells in both species leading to multinucleated, polyploid and aneuploid cells. Furthermore, callus derived protoplasts sometimes exhibited changes at the chromosome level as indicated by translocations. The vast majority of aberrant karyotypes arose from failures during mitosis and cytokinesis, pointing to inadequate microtubules as a possible underlying cause. Karyological events of the kind described herein greatly affect the plating efficiency of isolated protoplasts and the viability of protoplast derived calli. Plant regeneration, although demonstrated in this study for the first time in both species, seems to be limited to rarely occurring, protoplast-derived colonies with a relatively stable genome. Our experiments, performed with chromosomal model species, emphasize the need for controlled, non-mutagenic culture conditions.     

Radioautographic and cytofluorimetric analysis of DNA synthesis during the pre-implantation period of rat and mouse embryonic development]

The patterns of DNA synthesis and kinetics of cell population in the rat and mouse embryos were studied by means of 3H-thymidine autoradiography and cytofluorimetry. The rat and mouse embryos during the period of cleavage consist of a heterogenous population of blastomeres. At all the stages under study, all phases of the cell cycle occur in the blastomeres: G1, S, G2 and mitosis. The embryonic cells were distributed into groups containing 2c, 3c, 4c and more DNA. The ratio of cell number in these groups differed in the mouse and rat embryos. The mouse embryos are characterized by the appearance of a considerable amount of polyploid cells in S phase at the morula stage. The stage and species specific quantitative and qualitative patterns were established for DNA synthesis and kinetics of the cell population of blastomeres.     

Nuclear RNA quantification in protoplast cell-cycle phases

Using acridine orange staining and flow cytometry the DNA and RNA levels (arbitrary units) of individual cells may be established. Here, this method has been applied to nuclei isolated from plant protoplasts during culture. The specificity of the technique has been validated for such plant material; ribonuclease markedly reduced nuclear staining without modifying the DNA histogram; ribonuclease inhibitor prevented the action of released cell nucleases; and protoplasts cultivated with actinomycin D did not synthesize RNA. First RNA synthesis was evident 18 h after Petunia hybrida protoplasts had been put into culture. An increase of RNA above a critical level was required for cells to be able to initiate DNA replication from G1, termed G1B. G2 nuclei had an RNA:DNA ratio similar to that of G1 nuclei.     

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.     

Nutritional Requirements for Polyploid Mitoses in Cultured Pea Root Segments

Diploid and polyploid mitoses could be stimulated in excised segments of the mature region of pea roots grown on a sterile culture medium. Diploid mitoses were observed in segments cultured on water alone for 72 hours. Their frequency was increased by the presence of salts, sucrose, vitamins, and any two or all three of the following: an amino mixture, auxins, and kinetin. Polyploid mitoses were observed 72 hours after the beginning of the culture period in segments cultured on salts, sucrose, vitamins, auxins, and kinetin. Polyploid mitoses required the presence of auxins and kinetin in the culture medium. Their frequency was not affected by the presence of a reduced nitrogen source. Light treatments had no effect on the frequency of diploid or polyploid mitoses. Diploid mitoses were first observed about 24 hours after the beginning of the culture and their frequency increased thereafter. Experiments with colchicine showed that diploid cells were entering mitosis for the first time as late as 60 hours after the beginning of the culture. Polyploid mitoses showed a long lag time when compared with diploid mitoses. They began at about 60 hours and their frequency increased thereafter. Experiments with colchicine showed that polyploid cells were entering mitosis for the first time as late as 84 hours after the beginning of the culture. The presence of kinetin in the medium was not required during the first 24 hours in culture for the appearance of polyploid mitoses at 74 hours. However, the presence of kinetin was required after 24 hours. Auxin was required at some time during the first 24 hours of the culture and its continuous presence may be required for the stimulation of polyploid mitoses.     

contribution to the study of nuclear total proteins and DNA during the mitotic cycle in fibroblasts cultivated in vitro and in Ehrlich ascites cells

Summary The dry weight and total protein content of nuclei has been measured by interferometry in living or fixed cells cultivated in vitro (freshly prepared chick, mouse or rat embryo fibroblasts) and in fixed Ehrlich ascites tumor cells of the mouse growing in vivo. The DNA content was estimated by cytophotometry after Feulgen reaction in the same nuclei. The dry weight of nucleoli in fibroblasts and the dry weight and DNA content of chromosomes in dividing fibroblasts and Ehrlich tumor cells have also been measured.During the interphase in fibroblasts, the dry weight of the living nucleus and the nuclear total protein content as measured in fixed cells doubles during the preparation for mitosis, as the DNA content does. In chick and mammal fibroblasts and within the limits of accuracy of our measurements, the synthesis curves for nuclear proteins and DNA do not seem to be necessarily identical.In our fibroblasts, the nucleolar total dry weight per nucleus doubles during the interphase (nucleolar preparation for mitosis); it increases in proportion to the nuclear total protein content, even in polyploid nuclei.During the mitosis, the chromosomes contain all the DNA of the nucleus but some nuclear proteins (non chromosomal proteins) seem to move into the cytoplasm during the mitosis and return into the nucleus at the post-telophase.According to our observations, Ehrlich ascites mouse tumor cells are near-tetraploid as far as the number of chromosomes, nuclear total protein content and DNA content are concerned. During the preparation for mitosis, these amounts double but no necessary close time relation seems to link these premitotic syntheses. Prom this point of view, our results show no clear-cut differences between these tumor cells and the fibroblasts. Except the polyploidy, the behaviour of nuclear proteins and DNA during mitosis in the tumor cells is the same as that observed in our fibroblasts.The effects of various antimitotic agents on rat fibroblasts cultivated in vitro have also been studied with our cytochemical methods. Our measurements of nuclear protein, DNA and nucleolar material content have been made in cells in which mitosis was prevented by alkylating agents, beryllium sulphate, RNase or neutral DNase. The effects of colchicine on these cellular parameters have also been studied.     

Electron-microscope observations of mitosis and cytokinesis in multinucleate protoplasts of soybean.

Multinucleate soybean protoplasts produced by spontaneous fusion during enzyme digestion of the cell wall initiated cell division after approximately 40 h in culture. The structure of these protoplasts during mitosis and cytokinesis was studied with both light and electron microscopes. Most nuclei did not fuse but divided synchronously. Interphase nuclei was commonly connected by short narrow nuclear bridges. At prophase and metaphase the nuclei appeared typical of those in most higher plants; technical difficulties prevented an adequate examination of protoplasts at anaphase. Telophase was characterized by cytokinesis involving phragmoplast and cell plate formation; however, complete partitioning of the cytoplasm by cell plants was not observed. Numerous coated vesicles were present near to or continuous with the cell plate and plasmalemma. The presence of a few dividing protoplasts with at least double the normal chromosome number suggests that some nuclear fusion occurred prior to mitosis. Very little cell wall material was detected at the margin of the dividing protoplasts.     

Transformation of protoplasted yeast cells is directly associated with cell fusion.

The frequency of cell fusion during transformation of yeast protoplasts with various yeast plasmids with a chromosome replicon (YRp or YCp) or 2 mu DNA (YEp) was estimated by two methods. In one method, a mixture of protoplasts of two haploid strains with identical mating type and complementary auxotrophic nuclear markers with or without cytoplasmic markers was transformed. When the number of various phenotypic classes of transformants for the nuclear markers was analyzed by equations derived from binominal distribution theory, the frequency of nuclear fusion among the transformants was 42 to 100% in transformations with the YRp or YCp plasmids and 28 to 39% with the YEp plasmids. In another method, a haploid bearing the sir mutation, which allows a diploid (or polyploid) homozygous for the MAT (mating type) locus to sporulate by the expression of the silent mating-type loci HML and HMR, was transformed with the plasmids. Sporulation ability was found in 43 to 95% of the transformants with the YRp or YCp plasmids, and 26 to 31% of the YEp transformants. When cytoplasmic mixing was included with the nuclear fusion, 96 to 100% of the transformants were found to be cell fusants. Based upon these observations, we concluded that transformation of yeast protoplasts is directly associated with cell fusion.     

Cell-specific endopolyploidy in developing Artemia

Summary Cells in developing Artemia franciscana SFB demonstrated tissue-specific differences in DNA content, as determined by fluorescence intensity of bisbenzimide-stained nuclei and by nuclear area. The general epidermis comprised proliferating diploid (2C) cells. The setal cells had 4C–8C DNA content and did not divide during the first two instars. Salt gland cells were polyploid (>8C) and also did not undergo mitosis. Neural cells in the brain were diploid and were replicating. Cells in the thorax region of the gut had a 4C–8C DNA content and were proliferating. The muscle cells in the cephalic appendages contained 2C non-replicating nuclei. Only diploid epidermal cells were involved in segment morphogenesis. There was no difference in number of chromosomes (n=42) in the epidermal cells and the gut cells, indicating that the tissue-specific endopolyploidy was due to endoreduplication.     

Experession and integration of genes introduced into highly synchronized plant protoplasts

Summary Chimaeric genes containing the chloramphenicol acetyltransferase (CAT) coding sequence were introduced into protoplasts of suspension-cultured tobacco cells using improved conditions of electroporation (Okada et al. 1986). CAT activity became detectable in the protoplasts within 3 h, was maximal during a period of 18–36 h after electroporation, and then declined gradually. Alpha-amanitin added to the medium abolished the transient expression of the CAT gene. The closed circular form of input DNA was as effective as the linear form for the transient expression. The suspension culture was treated with aphidicolin, and S, G₂, M and G₁ phases were identified in the highly synchronized cell cycle obtained by releasing the cells from the inhibition of DNA synthesis. When a chimacric CAT gene was introduced into M phase protoplasts prepared from the synchronized culture, the transient expression of the CAT gene was 3–4 times higher than when it was introduced into protoplasts of other cell cycle phases. The frequency of stable transformation with a chimaeric neomycin phosphotransferase II gene was studied using the same system. G-418-resistant transformants were obtained from M phase protoplasts at frequencies 2–8 times those obtained from protoplasts at other cell cycle phases. The results indicate that the absence of the nuclear membrane in mitotic cells favours delivery to the nucleus of exogenous DNA introduced into the cytoplasm.     

Delinking of S phase and cytokinesis in the protozoan parasite Entamoeba histolytica

The alternation of DNA replication in S phase and chromosome segregation in M phase is a hallmark in the cell cycle of most well-studied eukaryotes and ensures that the progeny do not have more than the normal complement of genes and chromosomes. An exception to this rule has been described in cancer cells that occasionally become polyploid as a result of failure to restrain S phase despite the failure to undergo complete mitosis. Here, we describe the cell division cycle of the human pathogen, Entamoeba histolytica, which routinely accumulates polyploid cells. We have studied DNA synthesis in freshly subcultured cells and show that, unlike most eukaryotes, Entamoeba cells reduplicate their genome several times before cell division occurs. Furthermore, polyploidy may occur without nuclear division so that single nuclei may contain 1-10 times or more genome contents. Multinucleated cells may also accumulate several genome contents in each nuclei of one cell. Thus, checkpoints that normally prevent DNA reduplication until after cytokinesis in most eukaryotes are not observed in E. histolytica.     

Novel alterations in CDK1/cyclin B1 kinase complex formation occur during the acquisition of a polyploid DNA content.

The pathways that regulate the S-phase events associated with the control of DNA replication are poorly understood. The bone marrow megakaryocytes are unique in that they leave the diploid (2C) state to differentiate, synthesizing 4 to 64 times the normal DNA content within a single nucleus, a process known as endomitosis. Human erythroleukemia (HEL) cells model this process, becoming polyploid during phorbol diester-induced megakaryocyte differentiation. The mitotic arrest occurring in these polyploid cells involves novel alterations in the cdk1/cyclin B1 complex: a marked reduction in cdk1 protein levels, and an elevated and sustained expression of cyclin B1. Endomitotic cells thus lack cdk1/cyclin B1-associated H1-histone kinase activity. Constitutive over-expression of cdk1 in endomitotic cells failed to re-initiate normal mitotic events even though cdk1 was present in a 10-fold excess. This was due to an inability of cyclin-B1 to physically associate with cdk1. Nonetheless, endomitotic cyclin B1 possesses immunoprecipitable H1-histone kinase activity, and specifically translocates to the nucleus. We conclude that mitosis is abrogated during endomitosis due to the absence of cdk1 and the failure to form M-phase promoting factor, resulting in a disassociation of mitosis from the completion of S-phase. Further studies on cyclin and its interacting proteins should be informative in understanding endomitosis and cell cycle control.     

Cell wall regeneration and cell division in isolated tobacco mesophyll protoplasts

Summary Protoplasts were isolated from palisade tissue of tobacco leaves by treatment with pectinase and cellulase under aseptic conditions, and were cultured in a synthetic liquid medium. Calcofluor, a fluorescent brightener, was found to be an excellent stain for plant cell walls and was used to demonstrate regeneration of cell walls in these protoplasts. The cultured protoplasts regenerated cell walls by the 3rd day of culture, giving rise to spherical cells. The majority of the protoplasts regenerating cell walls underwent mitosis and cell division. The cycle of mitosis and cell division was repeated 2–3 times during 2 weeks of culture. Some of the nutritional conditions affecting division in the cultured protoplasts were studied.     

Structural and ultrastructural analysis of Solanum lycopersicoides protoplasts during diploid plant regeneration

Light, fluorescence and electron microscopy were used to analyse the structural properties of protoplasts obtained from established suspension culture of Solanum lycopersicoides Dun, composed of meristematic cell aggregates. Four types of protoplasts were distinguished immediately after isolation: (1) mononuclear; (2) polynuclear, (3) anuclear and (4) homogeneous protoplasts. Only mononuclear protoplasts were capable of complete cell wall regeneration and mitotic division. Other types of protoplasts were eliminated during culture. Three phases were distinguished in the developing protoplast culture: (1) the elimination phase during which protoplasts damaged during isolation underwent complete degradation; (2) a phase of intense division during which both mitotic cell division and amitotic nuclear division took place; and (3) a stabilization phase leading to the formation of suspension culture. The cell suspension culture obtained from protoplasts was capable of regenerating diploid plants.