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PubMed1979

Nucleosome-like structural subunits of intranuclear parental adenovirus type 2 DNA.

The intranuclear structure of parental adenovirus 2 DNA was studied using digestion with micrococcal nuclease as a probe. When cultures were infected with 32P-labeled virions, at a multiplicity of 3,000 particles per cell, 14 to 21% of parental DNA penetrated the cell and reached the nucleus. Of this parental DNA, 60% could be solubilized by extensive digestion with micrococcal nuclease. The nuclease-resistant fraction contained viral deoxyribonucleoprotein monomers and oligomers. These nucleosome-like structures contained DNA fragments which are integral multiples of a unit-length DNA of approximately 185 base pairs. The monomeric DNA is similar in length to the unit-length DNA contained in cellular nucleosomes. However, the viral oligomers are slightly smaller than their cellular counterparts. DNA-DNA hybridization demonstrated that all segments of the viral genome, including those expressed as mRNA only at late times, are represented in the nucleosomal viral DNA. The amount of early intranuclear viral chromatin was proportional to multiplicity of infection up to multiplicities of 4,000 particles per cell. However, viral transcriptional activity did not increase in direct proportion to the amount of viral chromatin. Maximum accumulation of intranuclear viral chromatin was achieved by 3 h after infection. The intranuclear parental viral chromatin remained resistant to nuclease digestion even at late times in infection, after viral DNA replication had begun.

Adenoviruses, Human↗
PubMed1997

Readthrough activation of early adenovirus E1b gene transcription.

In cells productively infected with adenovirus type 5, transcription is not terminated between the E1a gene and the adjacent downstream E1b gene. Insertion of the mouse beta(maj)-globin transcription termination sequence (GGT) into the E1a coding region dramatically reduces early, but not late, E1b expression (E. Falck-Pedersen, J. Logan, T. Shenk, and J. E. Darnell, Jr., Cell 40:897-905, 1985). In the study described herein, we showed that base substitution mutations in the globin DNA that specifically relieved transcription termination also restored early E1b promoter activity in cis, establishing that maximal early E1b expression requires readthrough transcription originating from the adjacent upstream gene. To identify potential targets of readthrough activation, a series of recombinant viruses with double mutations was constructed. Each double-mutant virus strain had the transcription termination sequences in the first exon of E1a and a deletion within the transcription control region of E1b. Early E1b expression from the double-mutant strains was more defective than that from strains containing either mutation alone, indicating that the deleted regions (positions -362 to -35) are not the target for readthrough activation. Two findings suggested that a cis-dominant property of early viral templates is important for readthrough activation. First, the early E1b defect caused by the GGT insertion was not complemented in trans by factors present in late-infected cells. Second, restoration of E1b transcription at late times occurred concurrently with viral DNA replication. Readthrough activation may help convert virion DNA into a transcriptionally competent template prior to DNA replication and late transcription.

Adenovirus E1A Proteins↗
PubMed2003

Transit of rat uterine stromal cells through G1 phase of the cell cycle requires temporal and cell-specific hormone-dependent changes on cell cycle regulators.

Progesterone pretreatment increases the number of synchronously proliferating stromal cells in the ovariectomized rat uterus, but estrogen is necessary to stimulate reentry into the cell cycle. To investigate the mechanisms underlying differential hormone actions, sexually mature ovariectomized rats were injected with progesterone (2 mg) for three consecutive days. Estradiol 17-beta (0.6 microg) was administered to initiate cell proliferation. Uterine samples were collected at timed intervals. Cell entry into DNA replication was monitored by injecting 5-bromo-2'-deoxyuridine (1 mg/100 g body weight) 2 h before necropsy. Demicolchicine (400 microg) was injected 30 min before necropsy to assess transit into M phase. Temporal progress through G1 was determined by spatial changes in cyclin D1/D3 proteins. Total cyclin D1/D3 protein and mRNA was measured by Western and Northern blotting. Estrogen increased the number of 5-bromo-2'-deoxyuridine-positive stromal cells (P < 0.05), compared with the number in rats treated with progesterone alone. An increase (P < 0.05) in the number of M-phase cells occurred at 12 h post estrogen. There was no evidence for epithelial cell proliferation in response to steroid treatments. Cyclin D1/D3 mRNA was expressed in the uteri of ovariectomized and hormone treated rats. The D-type cyclin proteins, however, were not evident in stromal cells without estrogen treatment. Progesterone pretreatment inhibited estrogen-dependent epithelial cell proliferation while redirecting D-type cyclin expression to the uterine stroma. Stromal cell transit through G1 required nongenomic steroid-dependent action on signal transduction pathways that control the nuclear localization and cell type-specific expression of the D-type cyclin proteins.

Animals↗
PubMed1995

BCR-ABL-mediated inhibition of apoptosis with delay of G2/M transition after DNA damage: a mechanism of resistance to multiple anticancer agents.

A critical determinant of the efficacy of antineoplastic therapy is the response of malignant cells to DNA damage induced by anticancer agents. The p53 tumor-suppressor gene is a critical component of two distinct cellular responses to DNA damage, the induction of a reversible arrest at the G1/S cell cycle checkpoint, and the activation of apoptosis, a genetic program of autonomous cell death. Expression of the BCR-ABL chimeric gene produced by a balanced translocation in chronic myeloid leukemia, confers resistance to multiple genotoxic anticancer agents. BCR-ABL expression inhibits the apoptotic response to DNA damage without altering either the p53-dependent WAF1/CIP1-mediated G1 arrest or DNA repair. BCR-ABL-mediated inhibition of DNA damage-induced apoptosis is associated with a prolongation of cell cycle arrest at the G2/M restriction point; the delay of G2/M transition may allow time to repair and complete DNA replication and chromosomal segregation, thereby preventing a mitotic catastrophe. The inherent resistance of human cancers to genotoxic agents may result not only by the loss or inactivation of the wild-type p53 gene, but also by genetic alterations such as BCR-ABL that can delay G2/M transition after DNA damage.

Antineoplastic Agents↗
PubMed1969

Simian virus 40 deoxyribonucleic acid replication. I. Effect of cycloheximide on the replication of SV40 deoxyribonucleic acid in monkey kidney cells and in heterokaryons of SV40-transformed and susceptible cells.

Infectious deoxyribonucleic acid (DNA) was extracted from green monkey kidney (CV-1) cultures at various times after the cultures were infected with simian virus 40 (SV40) at input multiplicities of 0.01 and 0.1 plaque-forming unit (PFU) per cell. A pronounced decrease in infectious DNA was observed from 3 to 16 hr after virus infection, suggesting that structurally altered intracellular forms may have been generated early in infection. Evidence is also presented that SV40 DNA synthesis requires concurrent protein synthesis. DNA replication was studied in the presence and absence of cycloheximide in: (i) SV40-infected and uninfected cultures of CV-1 cells; (ii) cultures synchronized with 1-beta-d-arabinofuranosylcytosine (ara-C) for 24 to 30 hr prior to the addition of cycloheximide; and (iii) in heterokaryons of SV40-transformed hamster and susceptible monkey kidney cells. DNA synthesis was determined by pulse-labeling the cultures with (3)H-thymidine at various times from 24 to 46 hr after infection. In addition, the total infectious SV40 DNA was measured. Addition of cycloheximide, even after early proteins had been induced, grossly inhibited both SV40 and cellular DNA syntheses. The activities of thymidine kinase, DNA polymerase, deoxycytidylate deaminase, and thymidylate kinase were measured; these enzyme activities remained high for at least 9 hr in the presence of cycloheximide. SV40 DNA prelabeled with (3)H-thymidine before the addition of cycloheximide was also relatively stable during the time required for cycloheximide to inhibit further DNA replication.

Animals↗
PubMed1982

Characteristics of enzymatic DNA methylation in cultured cells of human and hamster origin, and the effect of DNA replication inhibition.

In mammalian cells, inhibitors of DNA replication have been shown to induce chromosomal aberrations, cell death and changes in gene control. Inhibition of DNA synthesis has been reported to induce hypermethylation of mammalian DNA (enzymatic postsynthetic formation of 5-methylcytosine). These 5-methylcytosines in mammalian DNA have variously been suggested to be important in gene control, DNA repair, and control of DNA replication. In establishing the normal characteristics of enzymatic DNA methylation, we have demonstrated that, in asynchronously growing cells of both human and hamster origin, some cytosine methylation is delayed for several hours after strand synthesis and that this delayed methylation is completed before the DNA strand acts as a template for DNA replication in the next S-phase. Further, in testing whether the deleterious effects on mammalian cells of DNA synthesis inhibitors might be mediated via changes in enzymatic DNA methylation, we have found, contrary to some previous findings, no evidence for any change in the level of DNA methylation in DNA strands synthesized during 6 h of treatment of cells of human origin with high concentrations of four different inhibitors of DNA replication or during the 4 h following the 6 h treatment. Almost totally blocking DNA replication had no effect on the small amount of delayed methylation of DNA strands not involved in semi-conservative replication during the time of the experiment. This lack of effect on DNA methylation was obtained when the labelling medium contained normal, undialysed serum. In contrast, if dialysed serum was used in the labelling medium in order to maximize L-[Me-3H]methionine utilization, highly variable, totally irreproducible patterns of apparent DNA hypermethylation were obtained.

5-Methylcytosine↗
PubMed2002

Systemic infection and limited replication of SHIV vaccine virus in brains of macaques inoculated intracerebrally with infectious viral DNA.

SHIV deleted in two accessory genes, DeltavpuDeltanef SHIV(PPC), functioned well as a vaccine against later challenge with highly pathogenic SHIV(KU), and it was able to reach the brain after oral inoculation of live virus. In this study, the proviral genome cloned into a plasmid was inoculated as DNA intracerebrally and spread systemically. Few regions of the brain had detectable proviral DNA by real-time PCR. Two measures of virus replication, detection of viral mRNA expression and circular proviral DNA, were negative for those brain regions, with the exception of the infection site in the right parietal lobe, whereas lymphoid tissues were positive by both measures. Histopathological analyses of all the sampled brain and spinal cord regions did not reveal any abnormalities. Despite intracerebral inoculation of the viral DNA, the brain was not targeted for high levels of virus replication.

Animals↗
PubMed1986

Chromatin assembly during SV40 DNA replication in vitro.

A cytosol extract from human 293 cells supports efficient replication of SV40 origin-containing plasmid DNA in the presence of the SV40 T antigen. Addition of a nuclear extract from the same cells promotes negative supercoiling of the replicated DNA but not the bulk of the unreplicated DNA. The level of superhelicity is affected by the concentrations of T antigen and nuclear extract factors and by the time of addition of the nuclear extract. The replicated DNA in isolated DNA-protein complexes resists relaxation by purified HeLa cell topoisomerase I. Micrococcal nuclease digestion, sucrose gradient sedimentation, and electron microscopy demonstrate that the negative supercoils result from assembly of the replicating DNA into a chromatin structure. These results suggest that, during DNA replication, the core histones can be assembled on both sides of the replication fork by an active, replication-linked mechanism that does not require a template of preexisting nucleosomes.

Antigens, Polyomavirus Transforming↗
PubMed2005

Human cytomegalovirus inactivates the G0/G1-APC/C ubiquitin ligase by Cdh1 dissociation.

The anaphase promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase that targets regulators of the cell division cycle for degradation by the 26S proteasome. Discovered as a key regulator of mitosis, the APC/C has more recently been recognized to also play a limiting role in the control of G(0) maintenance, G(1)/S-transition and DNA-replication. Human cytomegalovirus (HCMV) has been shown to interfere with cell cycle regulation at different levels. It can induce an S phase-prone proliferation program in quiescent cells but at the same time this virus directly inhibits competitive cellular DNA replication. Here we show, that human cytomegalovirus (HCMV) inactivates the G(0)/G(1) APC/C rapidly after infection of quiescent fibroblasts, resulting in the untimely stabilization of APC/C substrates. APC/C inactivation is caused by the dissociation of its positive regulator, Cdh1. Surprisingly, this dissociation is independent from known Cdh1 inhibitors, Emi1 and Cyclin A, suggesting that APC/C-Cdh1 inhibition by HCMV is directly caused by a viral protein or an intermediate cellular factor distinct from Emi1 and Cyclin A. Thus, upon infection of quiescent cells HCMV not only activates the E2F-dependent G(1)/S transcription program but also facilitates protein accumulation of APC/C substrates by rapid Cdh1 dissociation.

Anaphase-Promoting Complex-Cyclosome↗
PubMed1987

Herpesvirus simiae (B virus): replication of the virus and identification of viral polypeptides in infected cells.

The events and products of replication of Herpesvirus simiae (B virus) in Vero cells were studied. The time course of the synthetic events of DNA replication and protein synthesis were found to be similar to the processes of the herpes simplex viruses and SA 8. Infectious progeny virus were detected by 4 hours post infection and were first found extracellularly between 6 and 8 hours post infection (PI). As in the case of SA 8, all cell lines tested were permissive for lytic infection by B virus. Analyses of B virus-infected cells by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) revealed approximately 50 infected cell polypeptides (ICP) ranging in molecular weight from about 26,000 to 239,000 daltons. The kinetics of synthesis of the ICPs were also identified. At least nine glucosamine-containing glycopeptides were noted ranging from 133,000 to 29,000 daltons.

Animals↗
PubMed1986

Free and viral chromosome-bound simian virus 40 T antigen: changes in reactivity of specific antigenic determinants during lytic infection.

Simian virus 40 (SV40) large T antigen (TAg), both free and bound to mature 70S and replicating 90S SV40 chromosomes, was prepared from lytically infected cells. The relative reactivity of the different TAg-containing fractions toward 10 monoclonal antibodies directed against three different regions in SV40 TAg and toward an antibody against the p53 protein was measured. The results for free TAg indicated that all of the determinants in both the amino-terminal (0.65 to 0.62 map units) and carboxy-terminal (0.28 to 0.17 map units) regions were highly reactive, whereas all five determinants located between 0.43 and 0.28 map units in the midregion of TAg were poorly reactive. For TAg bound to replicating chromosomes, all but one of the antibodies specific for TAg were highly reactive. Thus, antigenic sites in the middle of TAg, the region important for nucleotide binding and ATP hydrolysis (an activity required for viral DNA replication), were more accessible in TAg-replicating DNA complexes. As replicating molecules matured into 70S chromosomes, three or more determinants at different locations in TAg bound to chromatin became two- to fivefold less reactive, indicating other changes in TAg structure. Overall, at least nine different antigenic determinants in the TAg molecule were identified. Anti-p53 was reactive with about 10% of the free TAg and the same amount of SV40 chromosomes of all ages, suggesting that p53-TAg complexes are not preferentially associated with either replicating or mature viral chromosomes. When the reactivity of both mature and replicating labeled SV40 chromosomes with polyclonal tumor anti-T was measured as a function of time after purification, TAg bound to mature chromosomes appeared to dissociate about fourfold faster than that bound to replicating chromosomes. The relative amount of TAg in various subcellular fractions was measured by an enzyme-linked immunosorbent assay. Approximately 1.3% of the total TAg was estimated to be associated with SV40 chromosomes in infected cells. Based on the relative amounts of TAg and viral DNA in the 70S and 90S fractions, replicating chromosome-TAg complexes were estimated to bind 4.8 times more TAg per DNA molecule, on the average, than mature chromosome-TAg complexes. Together, these results are consistent with major differences in TAg structure when free and associated with replicating and nonreplicating SV40 chromosomes.

Animals↗
PubMed1986

Conversion of replicative intermediates in human DNA-repair defective cells.

We have examined the conversion of intermediates of DNA replication in normal human skin fibroblasts and fibroblasts isolated from patients with genetic diseases caused by putative DNA repair defects. Experiments were performed in non-transformed, unchallenged cells using alkaline sucrose sedimentation analysis to demonstrate precursor low molecular weight (LMW) DNA molecules which converted into high molecular weight (HMW) DNA with time. Analyses of conversion of replicative intermediates were conducted in cells from patients with ataxia telangiectasia (AT), Fanconi anemia (FA), Bloom syndrome (BS), Cockayne syndrome (CS) and xeroderma pigmentosum (XP). Our studies show that conversion of replicative intermediates occurs in all cell strains examined. However, XP cells (complementation groups A and E) show evidence of abnormalities in the conversion of LMW replicative intermediates, with the most dramatic alterations shown by cells from complementation group A.

Ataxia Telangiectasia↗
PubMedDate not supplied

Post-antibiotic effects of ofloxacin on Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, and Streptococcus pyogenes.

A viable counting technique was used to determine the post-antibiotic effect (PAE) of ofloxacin against four bacterial species, treated with either once of four times the minimum inhibitory concentration for 1 or 3 h. Similar to the results obtained previously with ciprofloxacin, ofloxacin gave PAE values with Escherichia coli, Staphylococcus aureus, and Streptococcus pyogenes. Cell division of Klebsiella pneumoniae was inhibited on removal of ofloxacin, but no clear PAE was demonstrated with this species because once replication recommenced, the mean generation times of drug-treated cultures were much shorter than those of untreated controls. Therefore, although the results obtained with ciprofloxacin and ofloxacin imply a consistency of PAE for 4-quinolones within a species, the response to DNA damage induced by 4-quinolones is multifaceted and species dependent. 4-quinolones inhibit both DNA replication and cell division, whilst at the same time stimulating DNA repair pathways. Thus, in some cases PAEs result from an increased post-treatment lag phase which may be followed by nearly normal multiplication, whereas in other cases a long lag may be followed by abnormally rapid cell division, with the generation times of treated cultures being shorter than those of corresponding drug-free controls. The PAE of a drug-induced lag may thus be masked by rapid cell division once growth resumes.

Colony Count, Microbial↗
PubMed1989

Changes of Escherichia coli cell cycle parameters during fast growth and throughout growth with limiting amounts of thymine.

It is generally accepted that during fast growth of Escherichia coli, the time (D) between the end of a round of DNA replication and cell division is constant. This concept is not consistent with the fact that average cell mass of a culture is an exponential function of the growth rate, if it is also accepted that average cell mass per origin of DNA replication (Mi) changes with growth rate and negative exponential cell age distribution is taken into account. Data obtained from cell composition analysis of E. coli OV-2 have shown that not only Mi but also D varied with growth rate at generation times (tau) between 54 and 30 min. E. coli OV-2 is a thymine auxotroph in which the replication time (C) can be lengthened, without inducing changes in tau, by growth with limiting amounts of thymine. This property has been used to study the relationship between cell size and division from cell composition measurements during growth with different amounts of thymine. When C increased, average cell mass at the end of a round of DNA replication also increased while D decreased, but only the time lapse (d) between the end of a replication round and cell constriction initiation appeared to be affected because the constriction period remained fairly constant. We propose that the rate at which cells proceed to constriction initiation from the end of replication is regulated by cell mass at this event, big cells having shorter d times than small cells.

Cell Cycle↗
PubMed1990

Replication of the dihydrofolate reductase genes on double minute chromosomes in a murine cell line.

The purpose of this study is to determine the kinetics of the replication of intrachromosomal versus extrachromosomal amplified dihydrofolate reductase (DHFR) genes. Previous studies reported that the DHFR gene, when carried intrachromosomally on a homogeneously staining region, replicates (as a unit) within the first 2 h of the S phase of the cell cycle. We wished to determine if the extrachromosomal location of the amplified genes carried on double minute chromosomes effects the timing of their replication. Equilibrium cesium chloride ultracentrifugation was used to separate newly replicated (BUdR-labeled) DNA from bulk DNA in a synchronized cell population. Hybridization with the cDNA for the DHFR gene allowed us to determine the period of time within the cell cycle in which the DHFR DNA sequences were replicated. We found that, in contrast to intrachromosomal dihydrofolate reductase genes that uniformly replicate as a unit at the beginning of the S phase of the cell cycle, dihydrofolate reductase genes carried on double minute chromosomes (DMs) replicate throughout the S phase of the cell cycle. These results suggest that control of replication of extrachromosomal DNA sequences may differ from intrachromosomal sequences.

Animals↗
PubMed2002

Global and gene-specific methylation patterns in cancer: aspects of tumor biology and clinical potential.

Heritable alterations of DNA that do not affect the base pair sequence itself but nevertheless regulate the predetermined activity of genes are referred to as epigenetic. Epigenetic mechanisms comprise diverse phenomena including stable feedback loops, nuclear compartmentalization, differential replication timing, heritable chromatin structures, and, foremost, DNA cytosine methylation (1-3). DNA cytosine methylation has recently gained major attention in the field of basic molecular biology as well as in studies of human diseases including cancer. Changes in DNA methylation patterns in human malignancies have been shown to contribute to carcinogenesis in multiple ways. Both hypo- and hypermethylation events have been described in various neoplasias leading to chromosomal instability and transcriptional gene silencing. DNA methylation research has entered the clinical arena and methylation patterns have become a major focus of clinicians seeking novel prognostic factors and therapeutic targets. The following minireview covers aspects of the basic molecular biology of DNA methylation and summarizes its importance in human cancers.

Apoptosis↗
PubMed1980

Cell cycle phase specificity of hydroxyurea and its effects on the cell kinetics in embryonic spinal cord.

Pregnant mice (NMRI) received an intravenous injection of 500 mg/kg of hydroxyurea (HU) on day 10 of gestation. The animals were killed 1, 1.5, 2, 2.5, 4, 6, 8 or 10 h after application of the drug and the embryos removed. Forty-five min. prior to sacrifice, the animals were given an intraperitoneal injection of 5 microCi/g 3H-thymidine (specific activity 5 Ci/mmol). From autoradiographs of the embryos the percentage of mitoses, necroses and labelled cells of the spinal cord was determined at the various time intervals. It was observed that HU inhibited DNA-synthesis in all S-phase cells for more than 4 hours. All cells damaged by HU did not start DNA-synthesis again and became necrotic. Only cells entering the S-phase after the influence of HU could replicate their DNA in a normal manner. It is assumed that HU possibly blocks cells in the G2-phase and in the mitosis for a certain period of time.

Animals↗
PubMed2001

Characterization of the bacteriophage phi29-encoded protein p16.7: a membrane protein involved in phage DNA replication.

An early expressed operon, located at the right end of the linear bacteriophage phi29 genome, contains open reading frame (ORF)16.7, whose deduced protein sequence of 130 amino acids is conserved in phi29-related phages. Here, we show that this ORF actually encodes a protein, p16.7, which is abundantly and early expressed after infection. p16.7 is a membrane protein, and the N-terminally located transmembrane-spanning domain is required for its membrane localization. The variant p16.7A, in which the N-terminal membrane anchor was replaced by a histidine-tag, was purified and characterized. Purified p16.7A was shown to form dimers in solution. To study the in vivo role of p16.7, a phi29 mutant containing a suppressible mutation in gene 16.7 was constructed. In vivo phage DNA replication was affected in the absence of p16.7, especially at early infection times. Based on the results, the putative role of p16.7 in in vivo phi29 DNA replication is discussed.

Amino Acid Sequence↗