Absence Of DNA Synthesis Research Articles

Canavanine-induced fast-sedimenting complexes with Escherichia coli DNA and bacteriophage T4 DNA

Arginine-requiring strains of Escherichia coli grown with l-canavanine formed a heavy canavanyl-protein complex with about 60% of the cell DNA as shown by zonal sedimentation in sucrose-EDTA gradients. DNA, made both during and prior to growth in canavanine, bound to this heavy complex, presumed to include cell membrane. The time course of DNA binding followed the deposition of canavanyl-protein into the complex. Both formation of the complex and canavanine death occurred in the presence or absence of DNA synthesis without apparent coordination with the DNA replication cycle. Various treatments of lysates prior to zonal sedimentation suggested that protein and perhaps lipid components were essential for binding of DNA to the heavy complex. Parental DNA of 32P-labeled bacteriophage T4 was bound to a fast-sedimentating fraction of arginine-starved E. coli dependent upon protein synthesis during infection. Cells pregrown with l-canavanine formed the heavy 32P-T4 DNA complex in the absence of protein synthesis. A canavanine-resistant mutant grown with canavanine formed a heavy protein fraction that did not complex with cellular DNA, and binding of T4 DNA required protein synthesis during infection of canavanine-grown cells. The results suggest that canavanine death of E. coli is caused by the incorporation of canavanine into a heavy membrane-protein that binds DNA abnormally.

Changes of Membrane Proteins and Their Relation to Deoxyribonucleic Acid Synthesis and Cell Division of Escherichia coli

Abstract Escherichia coli K-12 were treated in five ways to alter DNA synthesis, cell division, or both. Their membranes were then examined for changes. The bacteria were grown in the presence of radioactive l-arginine to label the membranes. 14C-Arginine was used for the experimental culture and 3H-arginine for the control, or vice versa. The cells were mixed, and their membranes were isolated and run on acrylamide gels in 0.5% sodium dodecyl sulfate to obtain a spectrum of proteins. Gel fractions were counted for both labels in order to find differences caused by the experimental treatment. Two principal changes were found in the membranes. At a position Y, corresponding to molecular weight 34,000, less protein appeared whenever DNA synthesis was inhibited. At a position X, molecular weight 39,000, extra protein appeared whenever bacterial division was inhibited. Rates of changes of Y and X were examined during thymidine deprivation to stop DNA synthesis, and after restoration of thymidine. Whereas DNA synthesis was rapidly affected, the changes of Y were gradual, suggesting that Y is not responsible for the synthesis of DNA; rather, its slowed rate of formation might be a consequence of arrested DNA synthesis. Changes of X were more immediate, and could possibly be responsible for altered cell division which is only affected after a lag. No significant changes in synthesis of X and Y were noted between cells near the beginning and end of the division cycle, suggesting that these proteins are normally made continuously or are not produced under normal conditions. Pulse chase experiments indicated that X and Y do not have a precursor-product relation. This is also suggested by the kinetic studies. Temperature-sensitive changes of membrane proteins in a previously described temperature-sensitive DNA synthesis mutant were further examined. The mutation appears to influence the formation of Y indirectly by stopping DNA synthesis. But production of X in the absence of DNA synthesis was much less in the mutant than in the wild type, which is compatible with ability of the mutant to divide even in the absence of DNA synthesis.

The expression of two classes of late genes of bacteriophage T4

Two alternative models have been tested which explain why onset of phage DNA synthesis is required in order for the “late” genes of bacteriophage T4 to begin function. One, the cell state model, proposes that the turn-on of late genes requires some state of the infected cell which is reached only after the onset of DNA replication, so that once DNA replication has begun the late genes of any phage DNA molecule, whether or not that particular DNA molecule has replicated, can function. The other, DNA state, model, proposes that the state of newly replicated phage DNA is somehow different from that of unreplicated (parental) DNA, and that only the late genes of replicated DNA can function. The two models were distinguished operationally by means of superinfection rescue experiments: primary infection is carried out with a T4 phage which is mutant in an essential late gene. Late in infection, after its onset, DNA synthesis is shut off, and the infected cells are superinfected with a phage carrying the wild-type allele of the essential late gene which is mutant in the primary phage. If, in the absence of DNA synthesis, that late gene of the superinfecting phage can function (cell state model) to provide the missing product, infective phage will be produced. If it cannot function (DNA state model), no phage will be produced. The results of the superinfection rescue experiments for some late genes are in good agreement with the prediction of the DNA state model in that superinfection produces no rescue. The results of the experiments for other late genes deviate somewhat from that prediction in that here superinfection produces a low level rescue. Additional experiments indicate that the two types of results derive from the existence of two classes of late genes: (1) the true-late genes can function only from replicated but not from parental DNA, and only in the presence of the normal product of the “maturation defective” phage gene 55. (2) The quasilate genes can function to a limited extent from unreplicated parental DNA and in the absence of the gene 55 product. Low-level rescue is observed for the quasilate genes because of their limited ability to act like early genes.

The relationship between the synthesis of DNA and the synthesis of phage lysozyme in Escherichia coli infected by bacteriophage T4

In Escherichia coli cells infected by bacteriophage T4, lysozyme synthesis may occur in the absence of DNA synthesis. The rate of lysozyme synthesis that is observed to occur in the absence of DNA synthesis is a function of the multiplicity of infection. In experiments where the level or the time of initiation of DNA synthesis was affected, the rate of lysozyme synthesis again showed a dependence on the number of phage DNA present in the infected cells.

5-bromodeoxyuridine-induced differentiation of a neuroblastoma.

5-Bromodeoxyuridine induces the differentiation of mouse neuroblastoma C1300 to cells that morphologically resemble mature neurons. The induced differentiation can take place in the absence of DNA synthesis. This suggests that the halogenated pyrimidine need not be incorporated into DNA to alter the phenotype of the cell.

Macrophage turnover in inflamed connective tissue.

It has previously been shown that macrophages in various types of inflamed tissue proliferate, whereas macrophages on subcutaneously inserted glass cover slips showed little such activity. This discrepancy suggested that macrophage proliferation in inflammation might be a complex and variable process. The present investigation indicates that inflammatory macrophages that have entered them mitotic cycle fall into one of three categories; cells newly arrived from the circulation, cells forming a high turn over pool heavily dependent on recruitment from the bone marrow , and cells forming a self-sufficient population largely independent of marrow recruitment. In reactions provoked by subcutaneous insertion of cover slips, uptake of a pulse of [ 3 H]T by perivascular but haematogenous macrophages begins at 4 h but is not evident in macrophages adhering to the cover slip until 2 to 4 days. Contrary to previous belief, DNA synthesis in multinucleate giant cells does occur but not until the cells are about 14 days old. DNA synthesis is frequently synchronous in the nuclei of a given cell. The percentage of connective tissue and cover-slip macrophages undergoing DNA synthesis becomes equal at 4 to 6 days. Selective X-irradiation of the bone marrow or the reaction site showed that the cells remain in the tissues or on the cover slip for only one or two days and that the lesion is totally dependent on daily recruitment from the marrow . Turnover is highest in the first few days of the lesion. The newly arrived macrophages that monopolize these early lesions have the curious property of resisting removal from the granuloma by dissection, whereas established arrivals are teased out with ease. The presence or absence of DNA synthesis in viable macrophages is determined partly by factors within the cell and partly by its environment. This was shown by transferring coverslip macrophages from early lesions to more mature reactions and vice versa and to tissue culture. Early macrophages unable to enter the mitotic cycle retained this disability under all circumstances. Macrophages from older reactions that normally synthesize DNA continued to do so in tissue culture but ceased this activity on transfer to early inflammatory lesions, indicating the inhibitory effect of this particular environment. Peritoneal macrophages that synthesize DNA very poorly in the normal peritoneum and in tissue culture entered the mitotic cycle in large numbers when transferred to inflammatory reactions old enough to have ceased to inhibit DNA synthesis. Similarly, blood monocytes that show even less proliferation than peritoneal cells in vitro , exhibited intense DNA synthesis 2 days after implantation into inflamed subcutaneous tissue. Since small numbers of monocytes gave rise, within a few days, to large numbers of macrophages, an association was established between incorporation of [ 3 H]T into nuclear DNA and net increase in cell numbers. The technique of selective X -irradiation and dissection were applied to various types of dermal and subcutaneous inflammation. In reactions to bovine fibrinogen all DNA-synthesizing macrophages were found to be of the newly arrived type. More unexpectedly in even long-established reactions to B. pertussis vaccine, almost all the macrophages were again of this type, presumably because of the toxicity of the killed organisms. Granulomata due to incomplete Freund adjuvant were similar to the reactions provoked by subcutaneous cover slips in that once the lesion was established most of the macrophages belonged to a high-turnover pool continuously replenished by fresh recruits from the marrow. Carrageenan granulomata revealed the existence of a third type of macrophage population, largely independent of further marrow recruitment. Presumably the low toxicity of carrageenan permits the expression of macrophage longevity which augmented by occasional mitotic activity maintains a self-sufficient population.

Hormonal regulation of cell elongation in the hypocotyl of rootless soybean: an evaluation of the role of DNA synthesis.

A method was developed where soybean seedlings were grown without roots to study the influence of hormones of root origin on shoot growth. Excision of the root resulted in inhibition of apical section growth and DNA synthesis and inhibited elongating section growth. A synthetic cytokinin restored DNA synthesis in the apical section, but did not influence growth in either the apical or elongating sections. Low concentrations of gibberellin with the cytokinin restored growth in the apical section. Gibberellin alone was sufficient to restore growth in the elongating section. An inhibitor of DNA synthesis, 5-fluorodeoxyuridine, inhibited the increase in apical section DNA without inhibiting control or gibberellin-induced growth in the elongating section. Experiments with (14)C-thymidine resulted in no DNA labeling differences in the elongating section under conditions where gibberellin-induced elongation varied from 50% to 73% above controls. It was concluded that gibberellin-induced elongation in soybean hypocotyl occurred in the absence of DNA synthesis. Gibberellin does stimulate DNA synthesis in the apical tissue apart from its effect on cell elongation. Excised soybean hypocotyl elongated maximally at 10(-6)m auxin. At higher auxin concentrations, fresh weight and ethylene production increased, but elongation was reduced. Addition of GA to the higher auxin concentrations resulted in a 50% inhibition in auxin-induced ethylene production and resumption in maximal elongation. Added ethylene inhibited elongation 30% at 2 mul/l. Addition of up to 100 mul/l ethylene did not inhibit elongation with GA present in the incubation medium. Thus GA may counteract ehtylene inhibition of cell elongation in addition to inhibiting ethylene production in auxin-treated tissues.

Nuclear segregation without DNA replication in Bacillus subtilis

When spores of a Bacillus subtilis temperature-sensitive mutant (NV-75) are germinated and grown at 45°, a small amount of either [ 14C]thymine or [ 3H]thymidine is incorporated into acid-insoluble material (DNA). It may be shown by autoradiography that the newly-synthesized material which is located in the center of the small cell emerging from a spore becomes separated into two discrete regions (“nuclei”) as the cell continues to grow. Measurements of the distance between grain clusters in cells of various lengths demonstrates the relationship of cell growth to nuclear separation. By counting the number of grains per cluster it may be shown that nuclear separation procedes in the absence of DNA synthesis. Finally, it was noted that nuclei occupy various positions along the length of the cell when the cells are grown at the restrictive temperature, although a statistical preference for two positions may be demonstrated. It should be noted that there is appreciable heterogeneity in both the time of outgrowth and initiation of DNA synthesis when this mutant is grown under restrictive conditions. After 2 h at 45°, for example, only about 49% of the spores have grains demonstrable by autoradiography which are indicative of DNA synthesis.

The relationship between histone and DNA synthesis in HeLa cells

The relationship between histone and DNA synthesis in HeLa cells has been investigated. Radioactive amino acids were incorporated in histones using synchronized as well as logarithmic cultures treated with inhibitors of the DNA synthesis. It is demonstrated that only a part of the synthesis of all histone fractions in HeLa cells is dependent on concurrent DNA synthesis, whereas all histones are synthesized in varying degrees even in the absence of DNA synthesis, i.e. during the G 1 phase. Different histone fractions of HeLa cells show differences in their degree of dependence on DNA synthesis. On the basis of previously published studies of other organisms, we suggest that the relationship between the synthesis of histones and DNA may be governed by different mechanisms in cells belonging to different physiological and developmental stages.

The splitting of human chromosomes into chromatids in the absence of either DNA or protein synthesis

If cells are irradiated during most of the G 1 portion of the cell cycle, chromosome aberrations are produced. If, however, they are irradiated while in S or G 2, chromatid aberrations are formed. The best estimate of when the transition occurs is late G 1. That is, in both plant and animal cells, the chromosome splits into chromatids before DNA synthesis. This doubling of the chromosome conceivably could be caused by either a synthesis of chromosomal protein prior to S, or by a loosening of a multistranded struture. Experiments were carried out with phytohemagglutinin-stimulated human lymphocytes to test which of the two possible explanations cited above would be the more likely. Phytohemagglutinin-stimulated lymphocytes were cultured in the presence of 2·10 −3 M hydroxyurea and triated thymidine for 40 h. The hydroxyurea was used to inhibit DNA synthesis and to enrich the population of cells at the end of G 1. Cells were then irradiated with 200 R of X-rays and recultured in the absence of tritium. When the cells reached metaphase, unlabeled cells were scored for the types of aberrations induced by the prior irradiation. Chromatid aberrations were found, indicating that in human cells, too, the split occurs in G 1 in the absence of DNA synthesis. Similar experiments in which the protein synthesis inhibitor cycloheximide was added after the cells were in culture for 16 h (before any DNA synthesis had occurred) and kept in until the fortieth hour showed that the transition occurred when protein synthesis was inhibited, too. The experiments indicate that the human somatic chromosome is a multistranded structure that loosens in late G 1 to form functional chromatids.

Studies on Deoxyribonucleic Acid Polymerase from Ehrlich Ascites Tumor Cells: I. ISOLATION AND PURIFICATION OF THE POLYMERASE AND SOME GENERAL PROPERTIES OF THE ENZYME CATALYZED REACTION

Abstract DNA polymerase obtained from Ehrlich ascites tumor cells by hypotonic shock has been purified by a procedure utilizing acid precipitation, fractionation on diethylaminoethyl cellulose, phosphocellulose, hydroxylapatite, and Sephadex G-100, resulting in a purification of the order of 300-fold with respect to the activity of the initial extract. The purified polymerase was free of endonuclease but contained an exonuclease whose activity was proportional to the polymerase activity throughout the purification. Exonuclease activity, as reflected by loss of template, was not evident under conditions of synthesis, but was easily detected in the absence of DNA synthesis. The enzyme was inactivated by heat in the absence of DNA. In the presence of activated DNA, a transitory enhancement of activity was observed upon heating. Sucrose density gradient centrifugation in the presence of native DNA showed poor affinity of the enzyme for native DNA. All four deoxyribonucleoside triphosphates and magnesium ions were required for activity. Optimal activity was found at pH 7.4, with 7 mm magnesium, 50 mm potassium phosphate, and with less than 120 µm eq of template. The properties of the enzyme suggest that it is neither mitochondrial in origin nor chromatin-bound. It is found both in nuclear and in cytoplasmic fractions of cell homogenates.

The process of infection with bacteriophage φX174: XXVI. Transfer of the parental DNA of bacteriophage φX174 into progeny bacteriophage particles

The experiments described in this paper show the following: (1) Parental DNA of bacteriophage φX174 appears as an intact molecule in a newly synthesized protein coat among the progeny phage particles. (2) This transferred parental DNA has to pass through a double-stranded state, the parental replicative form DNA, before it is released into progeny particles. (3) Cytoplasmic parental replicative form DNA rather than the membrane-associated parental replicative form DNA is the precursor for the transferred DNA. (4) Single-stranded φX DNA free of coat protein can be detected in the host cell when the infection has been started in the absence of DNA synthesis. Normally, the parental single-stranded virus DNA is rapidly converted to a double-stranded replicative form DNA. (5) The super-infection exclusion phenomenon in φX-infected cells is probably based on alterations in the host cell surface.

Studies on the fixation and development of cellular transformation by Rous sarcoma virus

The requirement for DNA synthesis in transformation of cells by Rous sarcoma virus (RSV) was confirmed using a nontoxic inhibitor, thymidine (20 m M). Inhibition of DNA synthesis for a 12-hour period immediately after exposure of cells to RSV prevented subsequent transformation. Similar inhibition of DNA synthesis immediately prior to RSV or after a 12-hour delay had little effect on the number of resulting transformed foci. When DNA synthesis was continuously inhibited beginning 12 hours after RSV, morphological transformation developed in the absence of cellular divisions, and transformed loci were comprised of two cells or single cells. Experiments using mitotically synchronized cells demonstrated that both daughter cells of an initially infected cell became transformed. The results demonstrate that there is a fixation period for transformation by RSV requiring DNA synthesis, that the viral genome is duplicated within a single cellular generation and distributed to both cellular progeny at division, and that transformation, once fixed, can develop in the absence of DNA synthesis.

Chromosome replication and cell division in Escherichia coli 15T after growth in the absence of DNA synthesis.

Chromosome Replication and Cell Division in Escherichia coli 15T after Growth in the Absence of DNA Synthesis

X-ray survival and DNA synthesis in Chinese hamster cells. I. The effect of inhibitors added before x-irradiation.

Opinions differ as to the identity of the principal sites of damage in cells killed by X radiation, but certainly DNA has been implicated.'-' Results with synchronized mammalian cells in vitro have established that the X-ray resistance of these cells exhibits periodic changes with age between mitotic divisions,4-9 and therefore depends upon certain cellular processes, as yet unknown. Results in irradiated HeLa cells10 showed an immediate increase in survival with the onset of DNA synthesis whereas irradiated Chinese hamster cells showed a lag.9 Peak survival in irradiated Chinese hamster cells occurred after DNA synthesis had passed its peak rate.9 In addition, in cell lines in which a long period is spent in the G, (preDNA synthesis) phase of the generation cycle, a further X-ray-resistant phase was observed which was apparently unconnected with DNA synthesis.5 It is the purpose of present research in this area to establish the number and biochemical nature of cellular processes which relate to X-ray resistance so that specific theories may be developed concerning the interrelationship of X-ray damage and the over-all biochemistry of the cell. In the process of such an investigation, we might also expect some useful empirical information about modifications of X-ray resistance involving the inhibition of specific cellular processes. The results in this paper further elucidate the relationship between DNA synthesis and X-ray resistance. T'he techniques employed include the use of a specific inhibitor of DNA synthesis, hydroxurea (HU), in Chinese hamster cells while continuing observations of X-ray survival as measured by reproductive integrity. To shed further light on the changes in survival observed with hydroxyurea, I also used another inhibitor of DNA synthesis, excess thymidine (TdR); an inhibitor of protein synthesis, cycloheximide (CH); and an inhibitor of RNA synthesis, actinomycin D (AcD). The results show that changes in survival occur in the absence of DNA synthesis but only in the unbalanced condition produced by the action of DNA inhibitors when used alone. Materials and Methods.-Cells: Cells of Chinese hamster (Cricetulus griseus), V79 line,1 were grown in log phase cultures on 85-mm plastic Petri dishes using EM-15 medium, i.e., HU-15 medium without NCTC 109.12 Cultures were maintained at 37?C in a humid atmosphere of CO2 and air. In log phase, the generation cycle lasted about 10 hr and was subdivided into a DNA synthetic period of 6 hr, G, of 1.5 hr, G2 of 1.5 hr, and mitosis of 0.51.0 hr.6 Synchronous populations of cells at or near division were obtained by harvesting log-phase cells under controlled conditions.6 Tests have been applied to ensure that such populations were ergodic.13 Suspensions of such synchronously dividing cells were inoculated into plastic dishes and were incubated at 37?C. After an appropriate incubationperiod, the cultures were treated with the test drug and were X irradiated at various times thereafter. Control untreated cultures were irradiated and otherwise handled identically. The first cycle often lasted 11.5-12 hr instead of 10 hr mainly because of a lowered temperature in the incubator while sampling or testing. In addition, the cultures spent 10-20 min at room temperature during the delivery of the X rays

Histones of meiosis

The meiotic cells in the anthers of lily and tulip contain a unique histone which is absent or nearly so, from the somatic tissues of these plants. This histone, termed the meiotic histone, is synthesized during the premeiotic histone synthesis, persists through meiosis, microsporogenesis, and pollen maturation. Its concentration decreases during this developmental sequence indicating that it is synthesized but once and conserved thereafter. The histones of the contracted meiotic chromosomes are strikingly similar to the histones of the extended postmeiotic chromosomes. This similarity is interpreted as indicating that if the histones have a role in chromosome contraction, such a role does not require any gross changes in histone composition. During the differentiation of microspores into mature pollen grains, some of the histones decrease and other histones increase in concentration. Some of these changes occur in the absence of DNA synthesis.

Nucleic acid synthesis during insect development—II. Control of DNA synthesis in the Cecropia silkworm and other Saturniid moths

Nucleic acid synthesis during insect development—II. Control of DNA synthesis in the Cecropia silkworm and other Saturniid moths

Susceptibility of HeLa cells in S-phase to inhibition of DNA synthesis by poliovirus infection.

SummaryThe inhibition of DNA synthesis in the 4 to 5 hour interval following infection with poliovirus was the same in untreated HeLa cells as in those whose DNA synthesis had been blocked up to this point by action of FUDR. The viral mechanism which inhibits DNA synthesis can develop in the absence of DNA synthesis. The inhibitory action is directed against cells already in the DNA productive phase rather than to prevent cells from entering this phase.

The metabolism of basic proteins in HeLa cell nuclei.

It has been widely suggested that histones, the basic proteins of the nucleus, are integral units of mammalian chromosomes and that they assist in the regulation of genetic expression.'-3 If so, the metabolism of these proteins might be expected to show a specific temporal relationship to the replication of the chromosomal DNA. In the present study, synchronized cultures of HeLa cells have been used to look for such a relationship. For this purpose three types of experiments were carried out: (1) Direct labeling studies, in which the specific activities of isolated basic nuclear proteins were compared after incorporation of leucine-Cl4 during different intervals in the synchronized cell cycle. (2) Accumulation studies, in which the amounts of the individual basic proteins were determined at different intervals in the cell cycle. (3) Dilution studies, in which cultures were prelabeled with leucine-C'4 and the changes in specific activities of the isolated bands were measured during subsequent synchronized or logarithmic growth in nonradioactive media. The three types of data supplement each other and permit four fundamental conclusions: (1) A fraction of the basic proteins of the nucleus is metabolically unstable and can be renewed in the absence of DNA synthesis. (2) Another fraction of the basic proteins is conserved in a form that was not isolatable during interphase; this form contributes to the bands of extractable, metabolically labile proteins during nuclear replication. (3) The accumulation of basic nuclear proteins is closely coupled to the synthesis of DNA and coincides with the accumulation of the latter. (4) Individual basic nuclear proteins exhibit independent metabolic patterns during the cell cycle. The implications of these findings for the regulation of genetic expression are discussed.

On the transcription of repressed genes in the absence of DNA-synthesis

In four thymine requiring strains ofE. coli the synthesis of induced and repressed β-galactosidase and of repressed alkaline phosphatase have been investigated under thymine-less conditions in different growth media. In agreement with previous studies induced synthesis of β-galactosidase occurred in the absence of DNA-synthesis. When the genes were in the repressed state their expression under conditions of thymine deprivation were not uniform for the strains and conditions used: Enzyme synthesis was either proportional to or less than total protein synthesis. The data do not favour the hypothesis that the expression of repressed genes is dependent on DNA-replication.