EVIDENCE FOR AN ESSENTIALLY CONSTANT DURATION OF DNA SYNTHESIS IN RENEWING EPITHELIA OF THE ADULT MOUSE

Tritiated thymidine autoradiography has been applied to several renewing epithelial tissues of the adult mouse in order to determine (a) the average time required for DNA synthesis; and (b) the temporal relationship of the synthesis period to the progenitor cycles of these populations. The average duration of DNA synthesis has been computed from curves describing the rates of appearance and disappearance of labeled metaphase figures in epithelia of colon, ileum, duodenum, esophagus, and oral cavity, in both normal and colchicine-treated animals. In general, application of colchicine does not significantly influence the derived values for DNA synthesis duration. The DNA synthetic time is remarkably similar in the tissues examined, despite wide differences in the times required for completion of the progenitor cycle (and for tissue renewal). Synthesis of DNA in these epithelial cells of the mouse requires approximately 7 hours. Agreement between this value and those derived by other investigators for mammalian cells in vivo and in vitro indicates that DNA synthetic time may be a temporal constant, of considerable potential utility to studies of cell proliferation. The advantages and shortcomings of this experimental approach to problems of cell population kinetics in vivo are discussed.     

CELL DIVISION AND DNA SYNTHESIS IN TETRAHYMENA PYRIFORMIS DEPRIVED OF ESSENTIAL AMINO ACIDS

The question of amino acid requirements for DNA synthesis and cell division has been studied in Tetrahymena pyriformis by depriving cells of histidine and tryptophan at defined stages in the interdivision interval. Deprivation any time before DNA synthesis does not prevent the initiation of such synthesis but completely inhibits the following division and limits the increase in DNA, as measured microspectrophotometrically, to 20 per cent. H³-thymidine added to the medium is not incorporated during the 20 per cent increase. Deprivation after DNA synthesis is initiated does not prevent the continuation (to completion) of DNA synthesis, and cell division ensues. H³-thymidine added to the medium under these conditions is incorporated into macronuclear DNA. The data indicate that some amino acid-dependent event occurs, about the time of the beginning of the DNA synthesis period, which is not essential for initiation of DNA synthesis but which is essential for the maintenance of synthesis once it has begun. These results are further discussed in terms of enzymes required to convert thymidine (and possibly the other three deoxyribonucleosides) to the immediate precursor of DNA synthesis.     

MACROMOLECULAR SYNTHESES RELATED TO THE REPRODUCTIVE CYST OF TETRAHYMENA PATULA

Macromolecular syntheses in encysted Tetrahymena patula were studied using Feulgen fluorescence cytophotometry, autoradiography, and inhibitors of RNA and protein synthesis. Cycloheximide significantly depressed protein synthesis and D-actinomycin effectively blocked RNA synthesis. Under these conditions, the cells within the cyst were unable to divide. Both cytophotometric measurements and autoradiographic data with tritiated thymidine show that DNA synthesis does not occur during the encystment divisions. Excysted cells placed in nutrient broth medium showed a prolonged generation time after the first cell growth cycle, and by the third generation the mean DNA content per cell was almost triple that of starved excysted cells. These findings indicate that (a) the encystment divisions require RNA and protein synthesis, which are apparently effected through turnover, (b) the encystment division cycles occur in the absence of DNA synthesis, and (c) excysted cells placed in culture medium may go through more than one DNA replication per cell cycle.     

AN ANALYSIS OF HETEROCHROMATIN IN MAIZE ROOT TIPS

The B chromosomes of maize are condensed in appearance during interphase and are relatively inert genetically; therefore they fulfill the definition of heterochromatin. This heterochromatin was studied in root meristem cells by radioautography following administration of tritiated thymidine and cytidine, and was found to behave in a characteristic way, i.e. it showed asynchronous DNA synthesis and very low, if any, RNA synthesis. A cytochemical comparison of normal maize nuclei with nuclei from isogenic maize stock containing approximately 15–20 B-chromosomes in addition to the normal complement has revealed the following: (a) the DNA and histone contents are greater in nuclei with B chromosomes; (b) the proportion of DNA to histone is identical with that of nuclei containing only normal chromosomes; (c) the amount of nonhistone protein in proportion to DNA in interphase is less in nuclei with B chromosomes than in normal nuclei. In condensed B chromosomes the ratio of nonhistone protein to DNA is similar to that in other condensed chromatin, such as metaphase chromosomes and degenerating nuclei. The B chromosomes appear to have no effect on nucleolar RNA and protein. Replication of B chromosomes is precisely controlled and is comparable to that of the ordinary chromosomes not only in synthesis for mitosis but also in formation of polyploid nuclei of root cap and protoxylem cells.     

EVIDENCE FOR A TEMPORAL INCOMPATIBILITY BETWEEN DNA REPLICATION AND DIVISION DURING THE CELL CYCLE OF TETRAHYMENA

The mechanism of coordination between DNA replication and cell division was studied in Tetrahymena pyriformis GL-C by manipulation of the timing of these events with heat shocks and inhibition of DNA synthesis. Preliminary experiments showed that the inhibitor combination methotrexate and uridine (M + U) was an effective inhibitor of DNA synthesis. Inhibition of the progression of DNA synthesis with M + U in exponentially growing cells, in which one S period usually occurs between two successive divisions, or in heat-shocked cells, when successive S periods are known to occur between divisions, resulted in the complete suppression of the following division. In further experiments in which the division activities were reassociated with the DNA synthetic cycle by premature termination of the heat-shock treatment, it was shown that (a) the completion of one S period during the treatment was sufficient for cell division, (b) the beginning of division events suppressed the initiation of further S periods, and (c) if further S periods were initiated while the heat-shock treatment was continued, division preparations could not begin until the necessary portion of the S period was completed, even though DNA had previously been duplicated. It was concluded that a temporal incompatibility exists between DNA synthesis and division which may reflect a coupling mechanism which insures their coordination during the normal cell cycle.     

AN ANALYSIS OF DNA LOSS AND SYNTHESIS IN THE RAT ADRENAL MEDULLA NUCLEI UPON COLD STIMULATION

The peculiar changes previously observed in DNA content of rat adrenal medulla cell nuclei upon intermittent cold exposure (15 hr at +4°C followed by 9 hr at room temperature) have been further studied with the aid of Feulgen histophotometry and H₃-thymidine radioautography. The amount of DNA decreases progressively with increasing length of cold exposure until 300 hr (-32%). Later a rapid change takes place, whereby DNA content per nucleus returns to values which are slightly, but consistently lower than normal. At termination of a period of cumulative exposure to cold, an analysis of a whole-day experimental cycle shows that the DNA decrease is due to loss of DNA during cold exposure and that DNA synthesis occurs upon return to room temperature. The balance between these two processes can be divided into three stages: (a) loss of DNA up to 300 hr of cumulative cold exposure; (b) marked increase in DNA by 350 hr; (c) oscillation around zero or slightly negative at 400 hr and beyond. These variations are due to: (1) the extension of DNA synthesis into the period of cold exposure as clearly demonstrated by radioautography (stage b), and (2) a later still greater DNA loss (stage c) which partly offsets the increased synthesis. A complex pattern of adaptation of the adrenal medulla cells, as regards DNA content, to the repetitive cold stimulus is thus demonstrated.     

The requirements for conjugal DNA synthesis in the donor strain during flac transfer.

Although neither rifampicin nor spectinomycin had any effect on the frequency of Flac transfer by a sensitive donor, rifampicin but not spectinomycin prevented donor conjugal DNA synthesis as measured in matings between a dnaB donor and a tdk recipient. An untranslated RNA species is therefore probably required for this synthesis, although transfer took place even in its absence. Donor conjugal DNA synthesis was abolished in a dnaE donor, showing that DNA polymerase III is responsible for this process; again, plasmid DNA transfer was not affected.Flac mutants lacking the F pilus gave neither donor conjugal DNA synthesis nor plasmid DNA transfer, probably because they could not receive a “mating signal” to activate the transfer process. The products of traI and traM were also required both for donor conjugal DNA synthesis and for physical transfer of plasmid DNA, probably being involved in the conversion of covalently closed circular plasmid DNA into the open circular form that is the substrate for the independent although normally simultaneous synthesis and transfer steps. In contrast, donor conjugal DNA synthesis took place at a normal rate in both piliated traG and traN mutants, and at a reduced rate in traD mutants, although in no case was there physical transfer of plasmid DNA. These gene products are therefore required for DNA transfer to the recipient, and in addition, the absence of the traD product may hinder DNA synthesis.Based upon these results, a scheme for the processing of DNA during conjugation is presented.     

The DNA Replication Factor RFC1 Is Required for Interference-Sensitive Meiotic Crossovers in Arabidopsis thaliana

During meiotic recombination, induced double-strand breaks (DSBs) are processed into crossovers (COs) and non-COs (NCO); the former are required for proper chromosome segregation and fertility. DNA synthesis is essential in current models of meiotic recombination pathways and includes only leading strand DNA synthesis, but few genes crucial for DNA synthesis have been tested genetically for their functions in meiosis. Furthermore, lagging strand synthesis has been assumed to be unnecessary. Here we show that the Arabidopsis thaliana DNA REPLICATION FACTOR C1 (RFC1) important for lagging strand synthesis is necessary for fertility, meiotic bivalent formation, and homolog segregation. Loss of meiotic RFC1 function caused abnormal meiotic chromosome association and other cytological defects; genetic analyses with other meiotic mutations indicate that RFC1 acts in the MSH4-dependent interference-sensitive pathway for CO formation. In a rfc1 mutant, residual pollen viability is MUS81-dependent and COs exhibit essentially no interference, indicating that these COs form via the MUS81-dependent interference-insensitive pathway. We hypothesize that lagging strand DNA synthesis is important for the formation of double Holliday junctions, but not alternative recombination intermediates. That RFC1 is found in divergent eukaryotes suggests a previously unrecognized and highly conserved role for DNA synthesis in discriminating between recombination pathways.     

Untersuchungen über die Regulation der DNS-Synthese während des Aktivitätswechsels vonAgrostemma githago-Samen

The relationships between DNA synthesis and germination capacity ofAgrostemma seeds have been studied. Protein synthesis and RNA synthesis are activated at the very beginning of imbibition, whereas DNA synthesis starts in the second part of the imbibition phase. Agrostemma seeds inhibited by higher temperature (30° C), or aged seeds with a low germination capacity are characterized by a remarkably reduced protein synthesis. DNA synthesis is also reduced. The inhibition of protein-synthesis ofAgrostemma embryos fed with cycloheximid or actinomycin D causes a depression of DNA synthesis. These results indicate that the initiation of DNA synthesis of imbibingAgrostemma seeds depends on the synthesis of special proteins. Abscisic acid inhibits growth as well as DNA synthesis of isolatedAgrostemma embryos. Mitomycin inhibits germination and DNA synthesis to the same extent. Dormant seeds with an undiminished intensity of protein synthesis also show a reduced incorporation of³H-thymidine in DNA. We suggest that DNA synthesis of imbibed seeds, which is a necessary prerequisite for the radicle protrusion, is involved in the mechanism of afterripening of theAgrostemma seeds.     

DNA synthesis in pretreated and ultraviolet-irradiated cells ofEscherichia coli B/rHcr + andHcr −

DNA synthesis after the ultraviolet irradiation was followed in the excision proficient strainEscherichia coli B/rHcr ⁺, in which the ability to excise thymin dimers was suppressed by a preirradiation inhibition of DNA and protein syntheses and in the excision deficient strainEscherichia coli B/rHcr ^?. Synthesis of pulse-labeled DNA, its stability and semiconservative DNA synthesis were compared in both strains. It was found that cells of theHcr ⁺ strain restore semiconservative DNA synthesis and the pulselabeled DNA appears stable, in spite of the fact that dimers are not excised under these conditions. On the other hand, cells of theHcr ^? strain are unable to restore semiconservative DNA synthesis and the pulselabeled DNA is degraded. As the repair by the excision of dimers under the used experimental conditions may be excluded in both strains, it is possible to assume that activity of enzymes included in theHcr ⁺ marker is prerequisite for restoring the DNA synthesizing system in theHcr ⁺ strain.     

Properties of ts Cl mouse L cells which exhibit temperature-sensitive DNA synthesis.

ts Cl mouse L cells are temperature-sensitive (ts) in DNA synthesis. The protein involved undergoes inactivation at 38.5 °C, with an apparent half-life of 3–4 h. A variety of experimental approaches yield data indicating that the ts Cl gene product acts directly during the DNA-synthesis period, probably late during the duplication of chromosomal DNA. The specificity of the ts lesion is reflected in the fact that replication of mitochondrial DNA is unaffected for many hours after nuclear DNA synthesis is almost totally inhibited. Temperature inactivation is not due to degradation or to loss of template capacity of preformed DNA. ts Cl cells are able to enter a DNA-synthesis phase at the higher temperature, as indicated by radioautographic experiments and by studies in which cells, blocked at the permissive temperature (34 °C) in a pre-DNA synthesis phase by isoleucine deprivation, are subsequently incubated at 38.5 °C. Cells arrested early in DNA synthesis by hydroxyurea treatment at 34 °C continue such synthesis for a short interval after up-shift to 38.5 °C. However, they are then unable to complete the S phase in progress nor can they proceed into cell division. The kinetics of DNA synthesis in cells incubated at 38.5 °C and back-shifted to 34 °C are compatible with the model that the ts Cl locus encodes an S phase function.     

INCORPORATION OF H3-THYMIDINE INTO CHLOROPLAST DNA OF MARINE ALGAE

The chloroplasts of three genera of marine algae, Dictyota, Padina, and Bryopsis, were labeled with tritiated-thymidine for various time periods during culture in "Erd-Schreiber's" solution. Autoradiographs were prepared from both smeared and sectioned material. They revealed that almost all of the radioactivity was in the cytoplasm and associated with the chloroplasts, as detected in the overlying silver halide crystals. Deoxyribonuclease, ribonuclease, and hot trichloracetic acid treatments indicated that the loss of radioactivity corresponded to the removal of DNA and not RNA. Quantitative studies of silver grain distribution suggested that the radioactivity of the labeled DNA originated from the edge of the pyrenoids on either side in the longitudinal direction of Bryopsis chloroplasts. Nuclei did not incorporate H³-thymidine even though cells were dividing rapidly in the three genera examined. It is postulated that the enzyme, thymidine kinase, is absent as a coding sequence of nuclear DNA in algae, but is present in chloroplast DNA. When the chloroplasts of Dictyota and Padina in various stages of division were scored for labeling, there appeared to be a DNA synthesis period, analogous to S period in cell division. This chloroplast-labeling period occurred just previous to fission. Many of the criteria seem to have been satisfied to establish the self-reproducing and semi-autonomous nature of chloroplasts, especially when combined with the chemical, genetic, and morphological evidence.     

Radiation-stimulated DNA synthesis in cultured mammalian cells

A type of DNA synthesis in mammalian cells that is stimulated by ultraviolet light has been studied by means of radioautography and density gradient centrifugation. The characteristics of this synthesis are: (a) it is not semiconservative; (b) it is enhanced by the presence of 5-bromodeoxyuridine in the DNA molecule; (c) the degree of stimulation is dose dependent; (d) there is less variability in the rate of incorporation of H³-thymidine during this synthesis than during normal DNA synthesis; (e) it occurs in cells that are not in the normal DNA synthesis phase (G₁ and G₂ cells). This kind of synthesis has been found in cultured cell lines from five different species; however, in some strains, the presence of bromouracil in the DNA is required before it can be demonstrated by radioautography.     

THE DISTRIBUTION OF NEWLY SYNTHESIZED DNA IN MITOTIC DIVISION

The chromosomes of Crepis capillaris were labelled with thymidine-2-C¹⁴ in their DNA fraction. Quantitative analysis of the distribution of newly synthesized DNA in postmetaphase stages of the division following the period of label incorporation led to the conclusion that the new DNA is not necessarily equally distributed by the mitotic process and that, therefore, chromosome duplication does not involve the equal partition of parental DNA. The implications of these findings with respect to DNA duplication are discussed. An attempt is made to translate the pattern of new DNA distribution into a probable number of units of synthesis per chromosome.     

CHARACTERIZATION OF LYMPHOCYTE TRANSFORMATION INDUCED BY ZINC IONS

Lymphocyte cultures from all normal human adults are stimulated by zinc ions to increase DNA and RNA synthesis and undergo blast transformation. Optimal stimulation occurs at 0.1 mM Zn⁺⁺. Examination of the effects of other divalent cations reveals that 0.01 mM Hg⁺⁺ also stimulates lymphocyte DNA synthesis. Ca⁺⁺ and Mg⁺⁺ do not affect DNA synthesis in this culture system, while Mn⁺⁺, Co⁺⁺, Cd⁺⁺, Cu⁺⁺, and Ni⁺⁺ at concentrations of 10^-7–10^-3 M are inhibitory. DNA and RNA synthesis and blast transformation begin to increase after cultures are incubated for 2–3 days with Zn⁺⁺ and these processes reach a maximum rate after 6 days. The increase in Zn⁺⁺-stimulated lymphocyte DNA synthesis is prevented by rendering cells incapable of DNA-dependent RNA synthesis with actinomycin D or by blocking protein synthesis with cycloheximide or puromycin. Zn⁺⁺-stimulated DNA synthesis is also partially inhibited by 5'-AMP and chloramphenicol. Zn⁺⁺ must be present for the entire 6-day culture period to produce maximum stimulation of DNA synthesis. In contrast to its ability to independently stimulate DNA synthesis, 0.1 mM Zn⁺⁺ inhibits DNA synthesis in phytohemagglutinin-stimulated lymphocytes and L1210 lymphoblasts.     

VITAMIN B12 AND THE MACROMOLECULAR COMPOSITION OF EUGLENA : III. Effect of Cycloheximide on the Recovery from B12-Induced Unbalanced Growth

When cycloheximide is added to (B12)-deficient cultures before or after replenishment of the cells with B₁₂, reversion of these cells is inhibited. This inhibition is not caused by interference of the inhibitor in the uptake of B₁₂ as measured by division kinetics. Cycloheximide does not inhibit the initial increase in the rate of DNA synthesis caused by B₁₂ replenishment, but within 30–45 min the rate decreases and DNA synthesis ceases. Cycloheximide added to replenished deficient cells after completion of DNA duplication inhibits cell division. The total cellular protein and RNA in replenished cells treated with cycloheximide does not change. B₁₂ added to deficient cells does not stimulate the incorporation of [¹⁴C]leucine into protein during resumption and completion of DNA duplication. However, there is a large increase in [¹⁴C]leucine incorporation into the protein of these cells soon after completion of DNA duplication and before resumption of cell division. The addition of cycloheximide to B₁₂-replenished or to nonreplenished deficient cells rapidly inhibits the incorporation. We suggest that the addition of B₁₂ accelerates the rate of DNA synthesis in the deficient cells and that possibly no new protein synthesis is required except for mitosis. However, protein synthesis is needed for continuous DNA synthesis.     

The role of DNA polymerases in DNA repair in Escherichia coli: DNA polymerase I-dependent repair following chemical alkylation of permeabilized bacteria.

Many mutagens and carcinogens damage DNA and elicit repair synthesis in cells. In the present study we report that alkylation of the DNA of Escherichia coli that have been made permeable to nucleotides by toluene treatment results in the expression of a DNA polymerase I-directed repair synthesis. The advantage of the system described here is that it permits measurement of only DNA polymerase I-directed repair synthesis and serves as a simple, rapid method for determining the ability of a given chemical to elicit “excision-repair” in bacteria.DNA ligation is intentionally prevented in our system by addition of the inhibitor nicotinamide mononucleotide. In the absence of DNA ligase activity, nick translation is extensive and an “exaggerated” repair synthesis occurs. This amplification of repair synthesis is unique for DNA polymerase I since it is not observed in mutant cells deficient in this polymerase. DNA ligase apparently controls the extent of nucleotide replacement by this repair enzyme through its ability to rejoin “nicks” thereby terminating the DNA elongation process.The nitrosoamides N-methyl-N-nitrosourea and N-ethyl-N-nitrosourea, as well as the nitrosoamidines N-methyl-N′-nitro-N-nitrosoguanidine and N-ethyl-N′-nitro-N-nitrosoguanidine, elicit DNA polymerase I-directed repair synthesis. Methyl methanesulphonate is especially potent in this regard, while its ethyl derivative, ethyl methanesulphonate, is a poor inducer of DNA polymerase I activity in permeabilized cells.