Specific nuclear elimination in polyploid plasmodia of the slime mold Physarum polycephalum

In growing plasmodia of the myxomycete Physarum polycephalum (G2-phase), three distinct classes of nuclei with a relative DNA content of 1x, 2x, and 4x are observed in the presumed haploid strain CL. The 2x and 4x species comprise up to 35% and 5% of the nuclei. Quantitative cytofluorometric studies of nuclei isolated in either G2- or S-phase or after FUDR treatment (G1 arrest) show that the three nuclear populations undergo a synchronous mitotic cycle and that the relative DNA content of the nuclear fractions in G-2 phase reflects the 2c, 4c, and 8c state. The heterogeneity of the nuclear population does, however, seem to be restricted to the growth phase. During a starvation period of 4 days that always preceeds sporulation (and also meiosis), the 4c nuclear population is reduced to 7%, 8c nuclei are no longer detected. These results suggest that a mechanism exists in Physarum for the selective detection and elimination of polyploid nuclei.     

Nuclear DNA content and chromosome numbers in the myxomycete Physarum polycephalum

An improved method is described for making chromosome spreads of the plasmodium of the myxomycete, Physarum polycephalum. It consists of isolating metaphase nuclei, spreading the chromosomes with hot lactic acid, and staining with acetic-orcein.Most sublines derived from the Backus Wis 1 sclerotium had about 1 pg of DNA per nucleus, and had nuclei with 50 and 75 chromosomes in both the growing and sporulating plasmodium. Mature spores contained 0.6 pg of DNA, and hatching amoebae had 20–25 chromosomes and 0.6 pg of DNA. Plasmodia of the homothallic Colonia strain had a nuclear DNA content of about 1 pg, and had 35–40 chromosomes during growth and sporulation. Polyploid plasmodial sublines were found which had 1.5 and 3 times the normal DNA content and chromosome number. The polyploid sublines had the same plasmodial protein:DNA and RNA:DNA ratios as normal cultures. DNA content of nuclei varied directly with nuclear surface area. Ploidy was determined by the parent amoebae and therefore can serve as a genetic marker.A simple technique is given for completing the life cycle of P. polycephalum axenically. Germinating spores are plated without bacteria on one-tenth strength semidefined plasmodial growth medium, containing 2% agar. Plasmodia are visible in 2–4 days.     

Post-meiotic DNA synthesis in nocodazole-blocked nuclei during conjugation of Tetrahymena thermophila : Induction of polyploidy in the micronucleus

The formation of single, polyploid micronuclei was induced during conjugation of Tetrahymena thermophila with Nocodazole (ND) according to Kaczanowski et al. The increase in DNA content in these nuclei was measured cytophotometrically in conjugating pairs continuously exposed to the drug. ND-treated micronuclei ('restitution nuclei') undergo two complete rounds of DNA replication and enter the third at the time of pair separation. The DNA content of these micronuclei in late pairs was in the range 16-30C (mean 20C). This amount was similar to the sum of the DNA content of all post-meiotic products during normal conjugation at about the same stage. Thus the increase in DNA content in 'restitution' nuclei reflects some intrinsic ability of nuclei in pairing cells to replicate DNA independently of nuclear division.     

FEULGEN MICROSPECTROPHOTOMETRIC STUDIES OF PANDORINA MORUM AND OTHER VOLVOCALES (CHLOROPHYCEAE)12

The nuclear DNA content during normal vegetative growth and division has been examined in three species of Volvocales, Chlamydomonas reinhardtii Dangeard, Pandorina morum Bory, and Volvox carteri f. nagariensis Iyengar. The results are consistent with the nuclear cycle reported in the literature for Eudorina. Nuclear DNA content does not increase during the prolonged cell growth phase. At the time of colony formation, nuclear DNA doubles, the nucleus divides, and this alternation continues until the final 2ⁿ complement of progeny nuclei is formed. The 4- and 8-nucleate stages of dividing gonidia of V. carteri have a nuclear DNA content in the same range as the somatic cells; they are not polyploid or polytene. Four normal clones of Pandorina, having 2, 5 or 12 chromosomes, all had similar amounts of DNA per nucleus, suggesting that the species has a nuclear genome of fairly constant size rather than consisting of many strains representing a polyploid series. One unique clone, a hybrid with double the chromosome number of either its parents, had twice as much DNA as the normal clones. The Feulgen spectrophotometric method is sufficiently sensitive to detect 2-fold differences in DNA content at the level of 2 × 10^?13 g of DNA /nucleus, and its use avoids the complications associated with the presence of organelle DNA.     

Isolation of a stimulatory factor for nuclear DNA replication

Aqueous extracts of isolated nuclei and intact plasmodia of Physarum contain a heat-stable stimulator of nuclear DNA replication. The stimulatory factor is present throughout the mitotic cycle, and its activity is unaffected by prior exposure of plasmodia to cycloheximide. The stimulatory substance has been partially purified by heat treatment, precipitation with ethanol, chromatography on DEAE cellulose, and gel filtration. The purified material contains both carbohydrate and protein, and exhibits a molecular weight of about 30 000. The active substance increases the rate and overall extent of DNA replication in S-phase nuclei, but does not trigger the initiation of DNA synthesis in nuclei isolated from G2-phase plasmodia. The stimulatory material contains little or no deoxyribonuclease or DNA polymerase activity, and it does not affect DNA polymerase activity assayed using a purified DNA template.     

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.     

Both Nucleolar Organizers Are Replicated in Dipteran Polyploid Tissues: A Study at the Level of Individual Nuclei

Working with the Dipteran Calliphora erythrocephala, we have tested the hypothesis that only one nucleolar organizer region (NO) is replicated during polyploidization. NO replication was examined in two very different highly polyploid nuclear types: salivary gland nuclei and nurse cell nuclei. Two strains of the organism containing NO regions with highly diagnostic nontranscribed spacer (NTS) polymorphisms were prepared and reciprocal single pair-matings between members of the strains were performed. The representation of the two distinguishable NOs in diploid and polyploid DNAs of individual F1 progeny from each cross was then examined. DNA from a total polyploid nuclear DNA preparation and from individual polyploid nuclei of both tissue types was analyzed. Our results show conclusively that both genomic NOs are replicated in individual polyploid nuclei of both types. Further, evidence for variation in the relative replication of cistrons from the two NOs by individual nuclei was obtained. The cistron types present in the NOs of both strains showed differential replication upon polyploidization. In general, the patterns of differential cistron replication seen in salivary gland and nurse cell nuclei were similar.     

Nuclear division cycle in germinating conidia of Neurospora crassa.

A temperature-sensitive mutant has been shown to be blocked at a specific point in the nuclear division cycle: just before the initiation of DNA synthesis at the time when the spindle pole bodies have duplicated but not separated. The metabolic activities of conidia of this mutant strain at the nonpermissive temperature have led us to conclude that the nuclei in a population of dormant conidia are arrested at various points in the nuclear division cycle. This conclusion is substantiated by the activities of conidia in the presence of the inhibitory drugs cycloheximide and hydroxyurea. In each inhibitory situation we observed that some, but not all, of the conidia were able to accomplish DNA synthesis and/or nuclear division.     

Spatial and temporal distribution of a Drosophila melanogaster embryonic variable nuclear antigen

A polyclonal antibody has been prepared that specifically recognises a nuclear protein antigen in Drosophila embryos. During development, the antigen appears initially to be uniformly distributed but by nuclear division cycle 10 is seen to accumulate in nuclei in a manner suggesting that it is destroyed or becomes modified upon transition from S- to M phase of the nuclear division cycle. This conclusion is supported by the observed disappearance of the antigen from the postblastoderm nuclei in a manner that reflects the pattern of the first asynchronous postblastoderm cell division and persistence in the polyploid nuclei of the amnioserosa that do not undergo further cell or nuclear division. In Western blot experiments, the antibody detects specifically a 105 kDa nuclear protein that probably corresponds to the antigen detected in embryos by immunocytochemical means.     

Formation of a micronuclei-like structure induced by colchicine treatment in Saccharomyces cerevisiae

Minute nuclei named “smaller nuclei” were generated when the cells of Saccharomyces cerevisiae were treated with colchicine. The formation of “smaller nuclei” seemed to be related to nuclear division because those nuclei were only produced under conditions suitable for nuclear division. The fact that the average DNA content of “smaller nuclei” was almost one tenth of that of the isolated normal diploid nuclei showed that the “smaller nuclei” are not condensed nuclei but aneuploid nuclei like micronuclei in animal cells. It appeared therefore likely that a micronuclei-like structure could be produced by colchicine treatment in S. cerevisiae.     

Development of muscle fibre nuclei in holometabolous insect larvae

The growth and development of the nuclear population from intersegmental dorsal longitudinal muscles (IDLM) of Bombyx mori and Pieris brassicae during larval development were studied. Using whole muscle mounts, examined with an image analysis system, we observed increases in muscle size but a constant number of fibres and concomitant increases in the number and size of fibre nuclei. The gradual growth of the nuclei throughout larval life, corresponding very probably to an increase in their DNA content, is unequal and results in heterogeneity of the nuclear population. This suggests periodic incorporation of myoblasts into the larval muscles; the nuclei arising from these myoblasts would therefore undergo endomitotic processes and grow polyploid. The possibility of somatic reduction divisions of the polyploid muscle nuclei is discussed.     

A comparative karyological and cytophotometric study of normal and wounded stem tissues ofLathyrus odoratus L.

Wounding of stems ofLathyrus odoratus induced increased mitoses with polyploid in vascular and pith tissues, but not in cortex. Cell division area extended 200 μm from the wound edge. These facts confirm the previous observations inPisum sativum. DNA content of normal stem nuclei was high in vascular and pith tissues, but not in cortex.     

DNA replication in Physarum polycephalum: characterization of DNA replication products made in vivo in the presence of cycloheximide in strains sensitive and resistant to cycloheximide.

Synchronous plasmodia of cycloheximide-sensitive and cycloheximide-resistant strains of Physarum polycephalum were labelled with 3[H]-deoxyadenosine in pulse and pulse-chase experiments in presence and absence of cycloheximide. The replication products were studied with alkaline sucrose gradient sedimentation analysis. We show that the action of cycloheximide on DNA replication in Physarum is mediated through the ribosome, since the ribosomally located resistance also makes the plasmodial DNA replication refractile to the action of cycloheximide. Cycloheximide caused inhibition of three stages in DNA replication in the wild type: first, the formation of primary replication units ("Okazaki" size fragments), secondly, the ligation of primary units into secondary ("Replicon" size) units and thirdly, the ligation of secondary units into mature DNA.     

Analysis of cell ploidy in histological sections of mouse tissues by DNA-DNA in situ hybridization with digoxigenin-labelled probes

Summary DNA-DNA in situ hybridization, with two digoxigenin-labelled, chromosome-specific DNA probes, was used to determine the number of copies of a given chromosome in interphase nuclei and so identify putatively polyploid nuclei in histological sections of several mouse tissues. One hybridization site per diploid genome was expected for tissues with hemizygous markers: male mice hybridized with a Y chromosome probe (pY353/B) or hemizygous transgenic mice hybridized with a -globin probe (pM02). Nuclei with more than one hybridization site were considered putative polyploids. Three groups of experiments were undertaken: (1) evaluation of the method, using mouse liver sections; (2) studies of tissues already known to contain polyploid nuclei, and (3) studies that resulted in the discovery that the mouse ovary contains polyploid nuclei. First, control studies showed that the ability to detect the target DNA sequences was affected by section thickness. Studies of nuclear ploidy in the developing mouse liver revealed a pattern similar to that established by previous studies using DNA content as a criterion for ploidy. At birth, only about 5% of the liver nuclei were polyploid; this increased to 10–15% by 10–20 days and was followed by a sharp increase in the frequency of tetraploid nuclei between 20 and 40 days (to about 35%) and a more gradual increase in higher order polyploid nuclei. Secondly, this technique was used to confirm that polyploid (mostly tetraploid) nuclei were present in the bladder epithelium, heart, uterine decidua and placental trophoblast. Higher order polyploidy was seen in large bone marrow cells (megakaryocytes) but not in the even larger trophoblast giant cells of the placenta, thus confirming previous claims that these cells are polytene rather than polyploid. Thirdly, putatively tetraploid nuclei were found in the ovarian follicle and corpus luteum. As far as we are aware, this is the first time polyploid nuclei have been reported for the mouse ovary.     

Cytological studies on degenerative nuclei at pollen development ofCarex ciliato-marginata

Chromosomes in degenerative and functional nuclei ofCarex ciliato-marginata Nakai were investigated during meiotic and primary pollen nuclear division. The nuclear DNA content of these nuclei was also measured using Feulgen microspectrophotometry. At metaphase of the primary pollen nuclear division, the chromosomes of degenerative nuclei were the same length as those of the functional nucleus, but only half their width. The functional nucleus divided into two, each of which moved to a pole, but the degenerative nuclei did not divide. The nuclear DNA content of the degenerative nucleus was half that of the functional nucleus and equal to that of one of the tetrads of a meiotic division. It is concluded that DNA replication was carried out in only one nucleus of the tetrad and that the other three nuclei were composed of unreplicated chromosomes at metaphase of the primary pollen nuclear division.     

SIMULTANEOUS SYNTHESIS OF HISTONE AND DNA IN SYNCHRONOUSLY DIVIDING TETRAHYMENA PYRIFORMIS

Histone and DNA syntheses have been studied in synchronously dividing Tetrahymena pyriformis GL. During the heat treatment necessary to synchronize cultures of this amicronucleate protozoan, the DNA content of the already polyploid macronucleus increases. When the cells begin synchronous division, their DNA content is reduced in a stepwise process which is closely paralleled by reduction of macronuclear histone content. During cell division, the contents of DNA and histone decrease by slightly more than twofold, and in the subsequent S phase, DNA and histone increase simultaneously to 85% of the values expected if all chromosomes were to double. The first step in the process of reduction of DNA and histone contents is their decrease in excess of twofold, and this is accomplished by removal of extrusion bodies from the nuclei of dividing cells. The second step is a mechanism which allows, in effect, only 70% of the chromatin in the average nucleus to duplicate. Such partial duplication suggests that both histone and DNA syntheses in synchronous Tetrahymena depend upon a regulatory mechanism, the mediating elements of which are localized in only certain chromosomes.     

Formation and division of binucleated cells in kidney cell cultures of microtus agrestis

Summary Epithelial kidney cell cultures of Microtus agrestis contain 10 to 25% binucleated cells. Observations of living cells under the phase contrast microscope showed that binucleated cells can arise by nuclear mitosis without cytoplasmic division. When binucleated cells divide the two nuclei are highly synchronized as they enter mitosis. In mitosis the chromosomes of both nuclei combine to a common metaphase plate leading to polyploid cells. In one case a tripolar spindle was seen after formation of a metaphase by the chromosomes of the two nuclei of a binucleated cell. This tripolar mitosis resulted in one binucleated and one mononucleated cell. The DNA-content (Feulgen photometry) and the distribution of heterochromatic bodies of the nuclei were corresponding to a tetraploid, a triploid and a haploid chromosome set. This suggests the possibility of somatic segregation of complete haploid sets.Supported by the Deutsche Forschungsgemeinschaft.     

The nuclear cycle of the myxomyceteEchinostelium minutum: II. Cytophotometric analysis of the nuclear DNA content of the sporangial phase

Nuclei of uncleaved sporangia ofEchinostelium minutum isolate D-3 1971 have a mean Feulgen-DNA content of 1.84. This value does not differ significantly from that recorded for the nuclei of the amoebal and plasmodial phases. No nuclei were found with a DNA content twice that of the nuclei in the amoebae, plasmodia, or uncleaved sporangia. This observation appears to rule out the possibility of zygotic meiosis. A single endosporic nuclear division occurs during the maturation which follows spore cleavage. The mean DNA content of the metaphases measured was 1.99. No metaphases were observed with a DNA content that would be indicative of a second division of meiosis. Also, no 4:2:1 reduction in DNA content was observed. These data confirm our previous hypothesis that this isolate ofE. minutum normally lacks a sexual phase. The single nuclear division in the maturing spore is followed by one of the division products becoming euchromatic and undergoing a single round of DNA replication, while the other division product becomes very pycnotic, eventually degenerating. The mean nuclear DNA values for the euchromatic and pycnotic nuclei are 1.84 and 0.88, respectively. Mature spores normally become uninucleate and have a nuclear DNA content which is equivalent to that of the amoebal and plasmodial phases.     

Arrest of Micronuclear DNA Replication during Genomic Exclusion in Tetrahymena Produces Haploid Strains

Diploid cells of Tetrahymena thermophila were crossed to strain A*V, whose micronucleus is defective, to induce the unilateral transfer of gametic nuclei from the diploid cells to the A*V cells (round I of genomic exclusion). These haploid nuclei presumably undergo one endomitotic cycle and then become diploid with a G1 (2C) DNA content. However, further DNA replication from 2C to 4C was transiently arrested until the pairs separated. When endomitosis was blocked by treatment with cycloheximide during 6-8 hours of conjugation, the exconjugants of round I of genomic exclusion remained haploid. Competence for diploidization is apparently limited to some period of time after nuclear transfer. Blocking of diploidization during round I of genomic exclusion can be used as an efficient way to induce haploid strains in Tetrahymena.