The replication time of the Escherichia coli K12 chromosome as a function of cell doubling time.
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Cell Division↗
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Adenovirus early region 4 and viral DNA synthesis.
Mutants of human adenovirus type 5 (Ad5) lacking early region 4 (E4) display a complex phenotype that includes a delay in the onset of viral DNA replication in low-multiplicity infections. Studies of viral DNA replication in vitro have not revealed a requirement for E4 products in DNA synthesis and, for most E4 mutants, defects in DNA replication are not apparent at high multiplicities of infection. The effects of E4 mutations on DNA replication therefore may reflect a role for E4 in the regulation of replication rather than in the process of DNA synthesis. The E4 mutant H5d/1014 carries two deletion mutations that together destroy all E4 open reading frames (ORFs) except ORF 4. Immunoprecipitation measurements of the level of the ORF 4 product confirm that H5d/1014 accumulates the ORF 4 product in somewhat larger amounts than wild-type Ad5. H5d/1014 is profoundly defective in viral DNA replication at a multiplicity of infection (50 PFU/cell) and time (24 hr after infection) that permit mutants lacking all seven E4 products to accumulate approximately normal amounts of DNA. In contrast, H5d/1019, a derivative of H5d/1014 in which the expression of ORF 4 is prevented by a mutation in the ORF 4 ATG initiator codon, produces DNA normally under these conditions. The product of ORF 4 therefore is necessary for the inhibition of viral DNA replication in H5d/1014-infected cells. H5d/1014 also inhibits, in trans, the synthesis of viral DNA by other E4 mutants that lack both E4 ORFs 3 and 6. Viruses that possess either of those ORFs are not subject to inhibition, indicating that the ORF3 and 6 products antagonize the effect of ORF 4. These observations are consistent with a regulatory role for the E4 ORF 3, 4, and 6 products in viral DNA replication in adenovirus-infected cells.
Adenoviruses, Human↗
Structure-activity relationships of L-dioxolane uracil nucleosides as anti-Epstein Barr virus agents.
A series of 1,3-dioxolanyluracil analogues was prepared from the dioxolane intermediates 2, and their anti-Epstein Barr virus (anti-EBV) activities were determined. The potency of L-dioxolane uracil nucleosides against EBV replication is dependent on the substituents at the 5-position in the following decreasing order: I > Br > Cl > CH3 > CF3 > F. The most active and selective analogue was the iodo derivative (L-I-OddU) with an EC50 value of 0.03 microM and an EC90 value of 0.16 microM. There was no cytotoxicity or depletion of mitochondrial DNA in cells after exposure to L-I-OddU at 50 microM. The action against EBV replication in H1 cells is time-dependent, and EBV DNA in cells treated with L-I-OddU could rebound to pretreatment levels once the drug was removed. In view of the potent antiviral activity plus favorable toxicity profiles, L-I-OddU may be potentially useful for the treatment of EBV-related infectious diseases as well as for delaying the onset or decreasing the incidence of EBV-associated cancers.
Antiviral Agents↗
Mutants of the Rous sarcoma virus reverse transcriptase gene are nondefective in early replication events.
The functional domains of the avian retrovirus polymerase gene are at least tripartite in nature. Three enzymatic domains exist; the RNase H and DNA polymerase activities are located on the alpha subunit while the DNA endonuclease is located on the pp32 moiety. Virus mutants possessing deletions in the pp32 region demonstrated that this region encodes function(s) essential for replication of the virus while separate point mutations generated near the NH2 terminus of pp32 resulted in decreased replication and cell transformation. Molecular analysis of various steps in the virus replication cycle demonstrated that the synthesis of linear viral DNA, transport of viral DNA to the nucleus, and its subsequent circularization and integration into cellular DNA are apparently not affected in these point mutants. However, the synthesis of viral RNA from the integrated provirus of these point mutants appears less than that observed in wild type virus-infected cells. What role the mutated pp32 protein might have on viral transcription is discussed.
Animals↗
The control of timing and spatial organization during Caulobacter cell differentiation.
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Bacterial Proteins↗
The spreading of X inactivation into autosomal material of an x;autosome translocation: evidence for a difference between autosomal and X-chromosomal DNA.
X inactivation involves initiation, propagation, and maintenance of genetic inactivation. Studies of replication timing in X;autosome translocations have suggested that X inactivation may spread into adjacent autosomal DNA. To examine the inactivation of autosomal material at the molecular level, we assessed the transcriptional activity of X-linked and autosomal loci spanning an inactive translocation in a phenotypically normal female with a karyotype of 46,X,der(X)t(X;4)(q22;q24). Since 4q duplications usually manifest dysmorphic features and severe growth and mental retardation, the normal phenotype of this individual suggested the spreading of X inactivation throughout the autosomal material. Consistent with this model, reverse transcription-PCR analysis of 20 transcribed sequences spanning 4q24-qter revealed that three known genes and 11 expressed sequence tags (ESTs) were not expressed in a somatic-cell hybrid that carries the translocation chromosome. However, three ESTs and three known genes were expressed from the t(X;4) chromosome and thus "escaped" X inactivation. This direct assay of expression demonstrated that the spreading of inactivation from the adjoining X chromosome was incomplete and noncontiguous. These findings are broadly consistent with the existence of genes known to escape inactivation on normal inactive X chromosomes. However, the fact that a high proportion (30%) of tested autosomal genes escaped inactivation may indicate that autosomal material lacks X chromosome-specific features that are associated with the spreading and/or maintenance of inactivation.
Adult↗
Growth of replicating DNA chain in adult and old rat spleen.
The rate of growth of the DNA chain length was found to decrease in rat spleen with aging. This can be substantiated by the slowing down of transformation of old spleen 3H-labeled DNA with low molecular weight to that with high molecular weight as labeling time increases. At the same time, the intensity of the DNA total synthesis is higher in old spleens than in adult ones. This may result from an increase of mitotic activity in rodent spleen with aging. In its turn, the increased mitotic activity may represent an adaptive reaction responsible for maintaining the number of immunocompetent cells which tend to decrease with aging. The slowing down of the newly formed DNA chain growth may lead to the decreased rate of stabilization of the secondary structure of replicating DNA in old spleen.
Aging↗
Coupling of mitosis to the completion of S phase through Cdc34-mediated degradation of Wee1.
The dependence of mitosis on the completion of the period of DNA replication in the cell cycle [synthesis (S) phase] ensures that chromosome segregation occurs only after the genome has been fully duplicated. A key negative regulator of mitosis, the protein kinase Wee1, was degraded in a Cdc34-dependent fashion in Xenopus egg extracts. This proteolysis event was required for a timely entrance into mitosis and was inhibited when DNA replication was blocked. Therefore, the DNA replication checkpoint can prevent mitosis by suppressing the proteolysis of Wee1 during S phase.
Anaphase-Promoting Complex-Cyclosome↗
Interaction of gametes with exogenous genes: possible opportunities for incorporation into embryonic genome.
The mechanism of incorporation of foreign DNA into newly fertilized eggs is poorly understood. It is not known with certainty if S phase DNA replication is required or if integration could occur at other times of the cell cycle that involve DNA strand breaks, such as chromatin rearrangements. We have investigated DNA strand breaks in mouse eggs and zygotes with a sensitive terminal uridine nucleotide end labeling (TUNEL) assay. Greater than 90% of all polar bodies and metaphase II chromosomes in freshly ovulated mouse eggs are TUNEL-assay positive. Approximately one-third of zygotes assayed 6 hr after fertilization contain at least one TUNEL assay positive pro-nucleus and/or decondensing sperm head. These results indicate that early embryonic DNA contains multiple transient DNA breaks that could play a role in the incorporation of foreign DNA.
Animals↗
Host DNA replication forks are not preferred targets for bacteriophage Mu transposition.
Bacteriophage Mu DNA integration in Escherichia coli strains infected after alignment of chromosomal replication was analyzed by a sandwich hybridization assay. The results indicated that Mu integrated into chromosomal segments at various distances from oriC with similar kinetics. In an extension of these studies, various Hfr strains were infected after alignment of chromosomal replication, and Mu transposition was shut down early after infection. The positions of integrated Mu copies were inferred from the transfer kinetics of Mu to an F- strain. Our analysis indicated that the location of Mu DNA in the host chromosome was not dependent on the positions of host replication forks at the time of infection. However, the procedure for aligning chromosomal replication affected DNA transfer by various Hfr strains differently, and this effect could account for prior results suggesting preferential integration of Mu at host replication forks.
Amino Acids↗
Variations of DNA polymerase-alpha and -beta during prolonged stimulation of human lymphocytes.
Stimulation of human lymphocytes with phytohemagglutinin is known to induce an increase in overall DNA polymerase activity (DNA nucleotidyltransferase; deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase, EC 2.7.7.7). Previous work [Pedrali Noy, G., Dalprà, L. Pedrini, A. M., Ciarrocchi, G., Giulotto, E., Nuzzo, F. & Falaschi, A. (1974) Nucleic Acids Res. 1, 1183] has shown that two subsequent waves of induction of DNA polymerase can be observed in this system; a first wave occurs in parallel with the increase in DNA replication rate; a second one occurs when the DNA synthesis rate is returned to minimal levels; the second peak is parallel to a maximum in DNA ligase and DNase levels. In the present work we have measured the levels of the DNA polymerases-alpha and -beta in phytohemagglutinin-stimulated lymphocytes during a 12-day period; both enzymes are present at detectable levels at time zero; in correspondence to the peak of DNA synthesis rate (between the fourth and fifth day) a peak of DNA polymerase-alpha is observed, increasing by a factor of approximately 20-fold over the zero time value; subsequently, the level of DNA polymerase-alpha decreases in parallel with DNA synthesis rate. The DNA polymerase-beta is also increased in correspondence to the peak in DNA synthesis rate, but reaches its maximum at later times, between the eighth and tenth day of incubation. The capacity of stimulated lymphocytes to perform repair synthesis following UV damage was measured in the same cells used for the enzyme activity determinations; this capacity also shows two maxima: a first one correlated with the peak in DNA replication rate, and a second one correlated with the peak of DNA polymerase-beta. These data suggest a certain tendency to the specialization of functions in human cell DNA polymerases; the alpha-enzyme seems mainly correlated with DNA replication, whereas the beta-enzyme seems more correlated with the ability of the cell to perform repair type synthesis.
DNA Nucleotidyltransferases↗
Cell cycle timing and developmental checkpoints in Caulobacter crescentus.
Development in Caulobacter reflects a level of complexity once thought only to exist in eukaryotic cells. The cell cycle and development are not isolated from each other, but are interdependent processes. Checkpoints are in place to ensure that both cell cycle and developmental processes are completed accurately before the next stage is initiated. The timing of these processes is regulated by signal transduction networks that integrate signals from DNA replication, cell division and development. These signal transduction networks achieve precise timing of the cell cycle and development by regulating temporal gene expression, and protein activity by dynamic spatial localization within the cell and timed proteolysis.
Caulobacter crescentus↗
Escherichia coli DNA distributions measured by flow cytometry and compared with theoretical computer simulations.
A computer simulation routine has been made to calculate the DNA distributions of exponentially growing cultures of Escherichia coli. Calculations were based on a previously published model (S. Cooper and C.E. Helmstetter, J. Mol. Biol. 31:519-540, 1968). Simulated distributions were compared with experimental DNA distributions (histograms) recorded by flow cytometry. Cell cycle parameters were determined by varying the parameters to find the best fit of theoretical to experimental histograms. A culture of E. coli B/r A with a doubling time of 27 min was found to have a DNA replication period (C) of 43 min and an average postreplication period (D) of 22 to 23 min. Similar cell cycle parameters were found for a 60-min B/r A culture. Initiations of DNA replication at multiple origins in one and the same cell were shown to be essentially synchronous. A slowly growing B/r A culture (doubling time, 5.5 h) had an average prereplication period (B) of 2.3 h; C = 2.4 h and D = 0.8 h. It was concluded the the C period has a constant duration of 43 min (at 37 degrees C) at fast growth rates (doubling times, less than 1 h) but increases at slow growth rates. Thus, our results obtained with unperturbed exponential cultures in steady state support the model of Cooper and Helmstetter which was based on data obtained with synchronized cells.
Cell Division↗
Acyclovir: discovery, mechanism of action, and selectivity.
The reasons for acyclovir's activity and selectivity in cells infected with HSV or VZV may be summarized as follows: 1. Activation by a HSV- or VZV-specified TK. 2. Greater sensitivity of viral DNA polymerase than of the cellular polymerases to ACV-TP. 3. Inactivation of the viral DNA polymerase, but not the cellular polymerases, by ACV-TP. 4. Chain termination of viral DNA by incorporation of ACV-MP. For the Epstein-Barr virus, which is also sensitive to acyclovir, there is no selective activation in infected cells [Colby et al., 1981], but the viral polymerase can be inhibited by very low levels of ACV-TP [Datta et al., 1980]. For HCMV, the activation of acyclovir is very poor but the viral polymerase is also more sensitive to ACV-TP than the cellular polymerases. One of the important contributions of acyclovir was the demonstration for the first time that a compound could prevent the DNA replication of a DNA virus at concentrations far below those that affect cellular DNA synthesis. As we all know, in the past 15 years there has been a complete rejuvenation of antiviral chemotherapy. I think it is very fortunate that we changed our outlook on the possibility of making potent and selective antiviral agents in time so that, when the AIDS epidemic came along, we did not feel completely at a loss on ways to attack viral disease.
Acyclovir↗
Filamentous bacterial viruses. V. Asymmetric replication of fd duplex deoxyribonucleic acid.
Short pulses (30 sec at 32 C) of (3)H-thymidine were found primarily in the viral strands of replicating fd deoxyribonucleic acid (DNA), even at a time when most DNA being synthesized was duplex DNA. Much of the labeled viral strand DNA was longer than unit length, but some was shorter than unit length. Most of the corresponding complementary-strand DNA was recovered in closed supercoiled duplex molecules, even for short pulses; the remainder of the complementary-strand DNA was found in replicative intermediates in pieces shorter than unit length. Some of the viral strands in open replicating DNA lacked a corresponding complementary strand.
Centrifugation, Density Gradient↗
[The effect of am umuC mutation on the induction of an SOS response in E. coli cella under the action of UV and gamma irradiation].
The kinetics of the SOS induction in E. coli cells of wild type and deficient in umuC gene exposed to UV and gamma-rays were analysed. In the presence of UmuC protein SOS induction was 3-5.5 times lower and delayed for about 30 minutes after both UV and gamma rays. It was shown that decrease of the SOS induction in wild type cells irradiated by UV was due to more effective elimination of the photolesions from DNA by excision repair system. UmuCD-dependent inhibition of DNA replication was discussed as a possible mechanism allowing additional time for error-free repair.
Bacterial Proteins↗
Replication timing: histone genes replicate during early S phase in cleavage-stage embryos of sea urchin.
Newly synthesized DNA was separated from the bulk of the DNA by pulse-labeling with BUdR and centrifugation in an alkaline CsCl buoyant density gradient. The content of histone gene in the newly synthesized DNA was determined by DNA dot hybridization. The gene contents in DNA replicated during the early half of S phase and during the whole S phase were compared. Results showed that histone genes were replicated during the first half of the S phase in embryos in the early cleavage stage.
Animals↗
A microplate version of the DNA-synthesis inhibition test for rapid detection of DNA-alteration potentials.
A microplate version of the DNA-synthesis inhibition test (DIT) for fast detection of DNA-alteration potentials has been developed. The DIT is based on the concept that DNA damage causes inhibition of DNA synthesis that becomes detectable some time after replicating cells have been in contact with genotoxic agents. In this test procedure human tissue culture cells (HeLa S3), prelabeled with [14C]thymidine, arfe exposed for 90 min to the substances in question. After the cells are rinsed, they are allowed to recover for 2 1/2 h in fresh culture medium, thereby unspecific interactions interfering with DNA replication are practically eliminated. Next, [3H]thymidine is added for 30 min, and then the cells are harvested and thoroughly rinsed. Finally, incorporated radioactivity is determined by liquid scintillation counting for measurement of the 3H/14C ratio. This allows for the evaluation of DNA synthesis during the 3H-labeling period and of the extent of genotoxic damage. This microplate version of the DIT can be carried out fully automated in a laboratory workstation. The test is compared to other tests for genotoxicity. Its advantages are discussed.
Autoanalysis↗