Mitochondrial DNA synthesis in synchronous cultures of the yeast,Saccharomyces cerevisiae

The synthesis of mitochondrial DNA (mtDNA) has been investigated by three independent methods of analysis during consecutive synchronous cell cycles in the yeast, Saccharomyces cerevisiae. The rates of pulse-label incorporation indicate maximal [³H]adenine uptake into mtDNA at the time of nuclear DNA synthesis. In contrast, the relative concentrations of mtDNA as determined by both the ratio of mtDNA to total cellular DNA and by the kinetics of isotope dilution analysis were found to increase continuously during synchronous growth. We conclude that whereas nuclear DNA replicates discontinuously during the cell cycle, mitochondrial DNA is synthesized continuously during this time. The discontinuous pattern of pulse-label incorporation into mtDNA is not considered to reflect its true mode of replication during the cell cycle.     

THE CYTOPLASMIC CONTROL OF NUCLEAR ACTIVITY IN ANIMAL DEVELOPMENT

1.This article reviews the occurrence, mechanism, and functional significance of the cytoplasmic regulation of nuclear activity during cell differentiation and especially during early animal development. 2.Nuclei from brain, and from other kinds of adult cell normally inactive in DNA synthesis, are rapidly induced to commence DNA synthesis by components or properties of intact egg cytoplasm. The components of egg cytoplasm which induce DNA synthesis are not species-specific and they are likely to include DNA polymerase. It is known that DNA polymerase exists in egg cytoplasm before it becomes associated with nuclei in which it is effective. The induction of DNA synthesis in brain nuclei by living egg cytoplasm is always preceded by a pronounced nuclear swelling, a dispersion of chromosomes or chromatin, and the entry of cytoplasmic protein into the nucleus. 3.RNA synthesis can be experimentally induced or repressed by living cytoplasm. The cytoplasm of unfertilized and fertilized eggs appears to contain components which can reversibly and independently repress the synthesis of ribosomal RNA, transfer RNA, and heterogeneous RNA. RNA synthesis can be induced by introducing nuclei inactive in this respect into the cytoplasm of cells very active in RNA synthesis. The induction and repression of RNA synthesis is preceded by a marked swelling of the nucleus and the dispersion of its chromosome material. 4.The cytoplasmic control of chromosome condensation before division has been demonstrated by introducing sperm or adult brain nuclei into the cytoplasm of oocytes undergoing meiotic maturation. 5.The evidence that regional differences in the composition of eggs and other cells are associated with changes in nuclear and gene activity is reviewed in Section 111. While it is certain that these regional differences are of great importance in cell differentiation, evidence that they have a direct effect on nuclear activity has been obtained in a few instances only. In some species it has been shown that the cytoplasmic components related to germ-cell differentiation include RNA and, frequently, granules. 6.It is concluded that whenever nuclei are introduced experimentally into the cytoplasm of another cell, they very quickly assume, in nearly every respect, the nuclear activity characteristic of the host cell. In many instances, altered function has been demonstrated in nuclei which subsequently support normal development. The induced nuclear changes are therefore regarded as normal and it is believed that they are achieved through the same mechanism as that by which the host cell nucleus originally came to function in its characteristic way. Examples are cited to show that changes in gene activity very frequently arise immediately after mitosis. The changes induced experimentally in transplanted nuclei resemble in very many respects those undergone by nuclei which are naturally reconstituted after mitosis, and it is argued that the two processes are functionally equivalent, It is suggested that during telophase of mitosis, chromosomes are reprogrammed in respect of potential gene activity by association with cytoplasmic proteins. Inter-phase nuclei seem not to show changes of gene activity except when they undergo a pronounced enlargement after entering a new cytoplasmic environment.     

The effect of sodium butyrate on Tipula iridescent virus synthesis in insect suspension cell cultures

The effect of sodium butyrate on Tipula iridescent virus (TIV) synthesis in suspension-cultured cells of Estigmene acrea was investigated. Sodium butyrate reduces viral-induced cell fusion but this is reversible with the removal of butyrate. At 7 mM sodium butyrate, TIV replicates in cells within 8 hr, but does not replicate in this time with 10–20 mm butyrate in the cell medium; cells so treated contain large vesicles with inoculum. Upon removal of the inhibitor, TIV replication appears normal, but large inoculum vesicles can still be found in the cytoplasm, and many infected cells have highly condensed chromatin in their nuclei. Sodium butyrate causes a lag of at least 2 hr in viral DNA synthesis as detected by [³H]thymidine incorporation into viroplasmic centres and at 7 mm butyrate viral DNA synthesis is reduced by 50–60%. In comparison, butyrate at 7 and 10 mm concentration does not inhibit host DNA synthesis, but at 15 and 20 mm, nuclear DNA synthesis is markedly reduced.     

Vaccinia virus infection of HeLa cells. I. Synthesis of vaccinia DNA in host cell nuclei.

The replication of vaccinia virus is thought to take place exclusively in the cytoplasm of host cells. However, using DNA-DNA hybridization techniques, it can be shown that a significant fraction of the synthesis of vaccinia DNA takes place in the nucleus as well as the cytoplasm. The (3H) thymiding pulse-labeled vaccinia DNA synthesized in the nucleus reaches a maximum at about 3 h after infection, corresponding to the time of maximal DNA synthesis in infected cells. At this time host DNA synthesis drops to about 25% of the rate of the uninfected cells. Even with short labeling times (2 min) the nucleus is found to contain 60% of the incorporated (3H)thymidine, much of which is in vaccinia DNA. Prior inhibition of host nuclear DNA synthesis with mitomycin C, followed by removal of the antibiotic causes a subsequent inhibition of vaccinia DNA synthesis and complete suppression of mature virus. Purified nuclei, isolated from vaccinia-infected cells, also synthesize vaccinia DNA in vitro. Over 90% of the DNA synthesized in vitro by isolated nuclei contain vaccinia-specific sequences.     

A study of the change in DNA synthesis of S phase cells treated with N-methyl-N-nitrosourethane: a study using Amoeba proteus as a single cell model.

The present experiments using Amoeba proteus as a single cell model show that DNA synthesis continues during and after exposure of S phase cell to N-methyl-N'-nitrosourethane (MNU). At sublethal dose levels which caused long division delays, division and growth abnormalities and mutations, the amount of [3h] thymidine ([3h]Tdr) incorporated was decreased by 20-30%; at dose levels which killed all S phase cells it was inhibited by up to 90%. There was a direct correlation between the dose of MNU used and the degree of inhibition of [3H]Tdr incorporated. The effect was rapid, mainly taking place within 20 min of treatment. Amoeba heterokaryons (HKs) were used to examine the rate of DNA synthesis of treated and untreated nuclei in the same cytoplasm, i.e. where the nuclei would have the same [h]tdr intake, the same thymidine kinase (TK) activity and the same endogenous precursor pools. Direct comparison of the nuclear DNA synthetic activity in this way revealed less difference between treated and untreated nuclei than comparisons made using the nuclear grain counts from treated and untreated amoebae. This suggested that much of the decrease in [3H]Tdr incorporation by MNU-treated S phase cells was due to a change in the cytoplasm and/or the cell membrane, rather than to nuclear damage. Thus MNU-treated nuclei were able to synthesize DNA at a near normal rate when they could draw on the resources of untreated cytoplasm, while the rate of DNA synthesis of control nuclei decreased when they occupied cytoplasm which had been exposed to high doses of MNU. These studies suggest that nuclear sites of damage were only involved when lethal doses of MNU had been used.     

Analysis of the initial stages of plant protoplast development using 33258 Hoechst: reactivation of the cell cycle

A microfluorimetric procedure, employing the fluorescent stain 33258 Hoechst, has been developed for the investigation of the process of DNA synthesis during the initial stages of culture of tobacco ( N. tabacum cv. Xanthi) leaf protoplasts.
In this system, the freshly-isolated protoplasts exhibited a unimodal distribution of nuclear DNA content characteristic of the diploid state. The almost immediate onset of DNA synthesis during culture resulted in a doubling of nuclear DNA levels prior to the first mitoses. Although the majority of the protoplasts subsequently entered into synchronous mitosis and cell division, a proportion of the remainder developed into large polyploid cells. Upon further culture, the polyploid cells became subdivided into clusters of small diploid cells. Measurement of total cell protein and cell volumes during culture indicated that a relationship existed between these parameters and the initiation of mitosis. The significance of these observations is discussed.     

Cytoplasmic DNA synthesis in rhinovirus type 14-inoculated KB cells.

Rhinovirus type 14 (RV14) incuced a transient statistically significant stimulation in synthesis of DNA which appeared between 0 and 3 h post-inoculation in the cytoplasm of high density monolayer cultures of KB cells. Newly synthesized DNA was measured by incorporation of [3H] thymidine into acid-insoluble DNAase-sensitive material and the cytoplasmic location established by cell fractionation and electron microscope radioautographic methods. A minimum of 10 plaque-forming units per cell of RV14 was required to stimulate DNA synthesis which did not occur above 34.5 degrees C, a temperature optimal for virus replication. Cytoplasmic DNA taken from RV14-infected or control cells could be differentiated from the bulk of cell (nuclear) DNA by several criteria, including: (1) RV14 induction of synthesis; (2) lower buoyant density and greater heterogeneity in CsCl and ethidium bromide/CsCl gradients; and (3) a different kinetic complexity upon reannealing. The Cot 1/2 value of cytoplasmic DNA, calclated as 50--100 from reassociation profiles, was about 10-fold less complex than the Cot 1/2 value of nuclear DNA (800-1000). These data rule out the possibility that cytoplasmic DNA arises by random breakage of nuclear DNA during cell disruption and extraction and are compatible with the hypothesis that inoculation of KB cells with RV14 results in stimulation of synthesis of a specific class of cell DNA which is detected in the cytoplasm.     

LIMITED DNA SYNTHESIS IN THE ABSENCE OF PROTEIN SYNTHESIS IN PHYSARUM POLYCEPHALUM

Actidione (cycloheximide), an antibiotic inhibitor of protein synthesis, blocked the incorporation of leucine and lysine during the S phase of Physarum polycephalum. Actidione added during the early prophase period in which mitosis is blocked totally inhibited the initiation of DNA synthesis. Actidione treatment in late prophase, which permitted mitosis in the absence of protein synthesis, permitted initiation of a round of DNA replication making up between 20 and 30% of the unreplicated nuclear DNA. Actidione treatment during the S phase permitted a round of replication similar to the effect at the beginning of S. The DNA synthesized in the presence of actidione was replicated semiconservatively and was stable through at least the mitosis following antibiotic removal. Experiments in which fluorodeoxyuridine inhibition was followed by thymidine reversal in the presence of actidione suggest that the early rounds of DNA replication must be completed before later rounds are initiated.     

Enzyme regulation during the synchronous nuclear cycle of Physarum polycephalum: Effects of methotrexate and hydroxyurea on thymidine kinase and deoxycytidine kinase

Specific enzyme activities of thymidine kinase (TK) and deoxy-cytidine kinase (dCK) increase sharply at the onset of synchronous mitosis in macroplasmodia of Physarum polycephalum. They reach a maximum in early S-phase (Physarum lacks a G1 period) and decline to a minimum in late G2. Partial inhibition of DNA synthesis with methotrexate (MTX) or hydroxyurea (HU) retards the onset of the next mitosis and provokes a superinduction of both enzymes, with dCK responding stronger than TK. The temporal pattern observed suggests that the drugs interfere with the postmitotic down-regulation of enzyme expression, possibly due to alterations of the chromatin structure. Moderate inhibition of DNA synthesis still permits the appearance of (delayed) mitoses associated with peaks of enzyme activity at elevated levels. On the other hand, stronger inhibition completely suppresses the onset of mitosis and keeps the enzyme activities at an elevated level without further oscillations. The timing mechanism of periodic enzyme induction therefore appears to be functionally linked to the mitotic signal and does not persist under a stringent DNA block.     

The H3.3K27M oncohistone affects replication stress outcome and provokes genomic instability in pediatric glioma

While comprehensive molecular profiling of histone H3.3 mutant pediatric high-grade glioma has revealed extensive dysregulation of the chromatin landscape, the exact mechanisms driving tumor formation remain poorly understood. Since H3.3 mutant gliomas also exhibit high levels of copy number alterations, we set out to address if the H3.3K27M oncohistone leads to destabilization of the genome. Hereto, we established a cell culture model allowing inducible H3.3K27M expression and observed an increase in mitotic abnormalities. We also found enhanced interaction of DNA replication factors with H3.3K27M during mitosis, indicating replication defects. Further functional analyses revealed increased genomic instability upon replication stress, as represented by mitotic bulky and ultrafine DNA bridges. This co-occurred with suboptimal 53BP1 nuclear body formation after mitosis in vitro, and in human glioma. Finally, we observed a decrease in ultrafine DNA bridges following deletion of the K27M mutant H3F3A allele in primary high-grade glioma cells. Together, our data uncover a role for H3.3 in DNA replication under stress conditions that is altered by the K27M mutation, promoting genomic instability and potentially glioma development.     

DNA SYNTHESIS AND MITOSIS IN ERYTHROPOIETIC CELLS

Studies of newt (Triturus or Diemictylus viridescens) erythropoietic cells showed that DNA synthesis and mitosis normally occur throughout most of the developmental process. Mitotic divisions were found in all immature precursor stages from the proerythroblast to the highly hemoglobinized reticulocyte. Mitoses were absent in mature erythrocytes. Radioautographic examination of thymidine-³H incorporation into DNA revealed that all erythroid cells except the mature erythrocyte were labeled. Microphotometric measurements of Feulgen-stained smears showed that all immature stages were undergoing DNA synthesis whereas the mature erythrocyte was inactive. The results obtained from three independent methods clearly demonstrate that (a) no loss of DNA or of chromosomes occurs during erythrocytic development and (b) highly hemoglobinized and, therefore, well-differentiated cells normally do undergo DNA synthesis and mitosis.     

SYNCHRONIZATION OF MITOCHONDRIAL DNA SYNTHESIS IN CHINESE HAMSTER CELLS (LINE CHO) DEPRIVED OF ISOLEUCINE

Mitochondrial DNA (mit-DNA) synthesis was compared in suspension cultures of Chinese hamster cells (line CHO) whose cell cycle events had been synchronized by isoleucine deprivation or mitotic selection. At hourly intervals during cell cycle progression, synchronized cells were exposed to tritiated thymidine ([³H]TdR), homogenized, and nuclei and mitochondria isolated by differential centrifugation. Mit-DNA and nuclear DNA were isolated and incorporation of radioisotope measured as counts per minute ([³H]TdR) per microgram DNA. Mit-DNA synthesis in cells synchronized by mitotic selection began after 4 h and continued for approximately 9 h. This time-course pattern resembled that of nuclear DNA synthesis. In contrast, mit-DNA synthesis in cells synchronized by isoleucine deprivation did not begin until 9–12 h after addition of isoleucine and virtually all [³H]TdR was incorporated during a 3-h interval. We have concluded from these results that mit-DNA synthesis is inhibited in CHO cells which are arrested in G₁ because of isoleucine deprivation and that addition of isoleucine stimulates synchronous synthesis of mit-DNA. We believe this method of synchronizing mit-DNA synthesis may be of value in studies of factors which regulate synthesis of mit-DNA.     

Cytochemical studies on the protamine-type protein transition in sperm nuclei after fertilization and the early embryonic histones of Urechis caupo.

Cytochemical staining characteristics of nuclear histones during postfertilization maturation division and various early embryonic stages in Urechis have been studied. The transition of protamine-type protein to adult histones in the sperm nucleus is accomplished by 15 min after entrance into the egg cytoplasm. Newly synthesized egg proteins migrate into enlarging male and female pronuclei after this transition, followed by pronuclear DNA synthesis and fusion. The shift from protamine-type protein to adult histones, which occurs in the absence of RNA synthesis during the postfertilization maturation division of the egg, may be one of the processes involved in the normal structural reorganization of chromosomes. Such a reorganization is likely to be a prerequisite for chromosome replication and mitosis. No qualitative differences are detected in the stainability of histones of unfertilized eggs and embryos at the cleavage and later stages of development.     

Stimulation of albumin synthesis in mouse hepatoma cells by Bt2cAMP: cell cycle specificity

Addition of 1 mm dibutyryl cyclic AMP (Bt₂cAMP) to cultures of mouse hepatoma cells, Hepa, specifically stimulates the synthesis of serum proteins including albumin. This stimulation is accompanied by an inhibition of cell proliferation. We have investigated these phenomena in synchronous cultures of Hepa. Proliferation of Hepa was arrested by isoleucine starvation. Synchronous growth was initiated by addition of complete growth medium or complete growth medium supplemented with 1 mm Bt₂cAMP. S phase and mitosis were estimated by determinations of [³H]thymidine incorporation and by cell numbers. The rate of albumin synthesis relative to total protein synthesis was measured by pulse labeling cultures for 30 min with [³H]leucine and comparing amounts of immunoprecipitable label with trichloroacetic acid-precipitable label. Treatment of synchronous cultures with Bt₂cAMP did not alter the duration of S phase or the onset of mitosis. The relative rate of albumin synthesis in Bt₂cAMP-treated culture began increasing after mitosis. The timing of the Bt₂cAMP stimulation of albumin synthesis was further investigated by adding Bt₂cAMP to cultures of Hepa at various times after the initiation of synchronous growth. The relative rate of albumin synthesis was then measured at a fixed postmitotic time. An increased relative rate of albumin synthesis was observed only in cultures exposed to Bt₂cAMP before or during S phase. Thus the postmitotic increase in the synthesis of albumin requires the presence of Bt₂cAMP during S phase.     

A Sericesthis iridescent virus infection of the hemocytes of the waxmoth,Galleria mellonella (Lepidoptera)

The cytopathology of the Seriscethis iridescent virus (SIV) infection of Galleria hemocytes was studied by phase-contrast microscopy, [³H]thymidine autoradiography, and fluorescent antibody and acridine orange staining. Of the five hemocyte classes only the oenocytoid was not infected. Infected plasmatocytes and adipohemocytes undergo nuclear and cytoplasmic hypertrophy accompanied by nuclear DNA synthesis. The timing and identity of nuclear and cytoplasmic DNA synthesis are discussed.     

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on stimulated DNA synthesis in the liver and kidney of the rat

The administration of the toxic agent 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin) to rats at 0, 24 or 72 h before 70% partial hepatectomy had no significant effect on DNA synthesis, measured by [³H]thymidine incorporation 24 h after the operation, in the remaining liver lobes. However, dioxin depressed DNA synthesis in the folate-stimulated rat kidney when given from 0–9 days before the folic acid. A similar effect was observed using inorganic lead to stimulate kidney mitosis. This effect does not appear to be due to direct interaction of dioxin with DNA, inhibition of the protein synthesis required for DNA synthesis, alteration of the time course of events in the mitotic cycle of the kidney or the depression of food intake of the rats caused by dosage with dioxin. Experiments with methylcholanthrene suggest that levels of drug-metabolising enzymes in the liver or kidney might possibly have an indirect effect on folate-stimulated kidney mitosis.