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PubMed1987

Computer simulation of T3/T7 phage infection using lag times.

A minimal mechanism is proposed which describes the transcriptional and translational processes for four phage proteins (RNA polymerase, DNase, primase and DNA polymerase) involved in T3/T7 DNA replication. Phage DNA replication is also included. It is shown how lag times may be incorporated into a kinetic mechanism. The distinct three-stage transport of phage DNA into the bacterial host (E. coli) is considered. DNA transport is assumed to be rate-determining for the transcription of class I and II proteins. Transcriptional and translational lag times have been calculated on the basis of available gene mapping of T7 phages. The kinetic behavior of T7 and T3 phage infection is practically identical. The hydrolysis of bacterial DNA by phage DNase (endonculease and exonuclease) as well as the subsequent phosphorylation to the deoxymononucleoside triphosphates are assumed to be rate-determining in phage DNA replication. Good agreement with experiment is obtained in our computer simulations.

Computer Simulation↗

PubMed1978

Chromosome replication during the division cycle in slowly growing, steady-state cultures of three Escherichia coli B/r strains.

The period of DNA synthesis C during the cell cycle was determined over a broad range of generation times in slowly growing, steady-state batch cultures in the exponential phase and in chemostat cultures of three strains of Escherichia coli, strains B/r A, B/r K, and B/r TT, utilizing measurements of average amounts of DNA per cell and cell survival after radioactive decay of 125I incorporated into the DNA of synthesizing cells. At each growth rate, values for cell survival and for C periods were the same within experimental errors for the three strains. The length of the DNA synthesis period increased linearly with generation (doubling) time T of the culture and approached a limiting value of C = 0.36T at very long generation times. In very slowly growing cultures, DNA replication was limited almost entirely to the final third of the cell cycle. D periods, between termination of DNA replication and cell division, were found to be relatively short at all growth rates for each strain. Average amounts of DNA per cell measured in slowly growing cultures of strains B/r A and B/r TT were indistinguishable from results for strain B/r K at the same growth rates. Amounts of DNA per cell calculated from the cell survival values alone are completely consistent with the measured DNA per cell.

Cell Cycle↗

PubMed1980

Inhibitors of RNA synthesis and passage of chick embryo fibroblasts through the G1 period.

Events that are essential for progression through the G1 period begin immediately or shortly after resting chick embryo cells are given fresh medium with serum. The following observations support the contention that the critical events include the production of non-ribosomal RNAs: (1) Addition to the "shift-up" medium of either of two inhibitors of RNA formation, camptothecin or 5, 6-dichloro--1-beta-D-ribofuranosylbenzimidazole, delays the onset of DNA replication by about the length of time the cells are exposed to the drugs. (2) Although entry into the S phase is delayed by the inhibitors, the slopes of the DNA response curves are identical to that of control cultures. (3) Neither drug reduces significantly the rate of overall protein synthesis. Observations (2) and (3) are taken to mean that expansion of the G1 period is not due to cell damage. (4) A third inhibitor of RNA synthesis, cordycepin, also delays passage of stimulated cells through the G1 phase, but, in this case, the length of the delay period is greater than that of the exposure period. (5) A low dose of actinomycin D does not impede movement towards the S phase, even though the synthesis of preribosomal RNA is considerably reduced. The possibility is considered that the essential G1 molecules are mRNAs.

Animals↗

PubMed1982

[Uracil-DNA-glycosylase in the rat: correlation between the enzyme activity and rate of DNA synthesis in different tissues].

Uracil-DNA-glycosylase which releases uracil residues from DNA has been purified from rat liver more than 300-fold. The enzyme has a molecular weight of about 28,0000, Km = 1.7 . 10(-9) M. The content of uracil-DNA-glycosylase in five different tissues of the rat is correlated with the rate of DNA synthesis in the tissues. The enzyme activity in rat liver increases more than 3 times after partial hepatectomy, showing a peak 28-32 hrs following the surgery. The results obtained suggest that uracil-DNA-glycosylase from mammalian cells releases uracil residues from DNA at time intervals close to those of replication.

Animals↗

PubMed1999

Transformation of Candida albicans by electroporation.

In contrast to a variety of other yeasts, Candida albicans has proved difficult to transform with high efficiency. Lithium acetate transformation is fast and simple but provides a very low efficiency of DNA transfer (50-100 transformants/microg DNA), while spheroplast transformation, although more efficient ( approximately 300 transformants/microg integrative DNA and 10(3)-10(4) transformants/microg replicative DNA), is complicated and time-consuming. In this study we applied various yeast transformation techniques to C. albicans and selected an electroporation procedure for further optimization. Transformation efficiencies of up to 300 transformants/microg were obtained for an integrative plasmid and up to 4500 transformants/microg for a CARS-carrying plasmid. This reasonably high transformation efficiency, combined with the ease and speed of electroporation in comparison to alternative techniques, make it the preferred method for transformation of C. albicans.

Blotting, Southern↗

PubMed2005

Cell cycle synchronization of Cupriavidus necator by continuous phasing measured via flow cytometry.

The continuous phasing technique was successfully used to obtain a high degree of cell cycle synchrony in cultures of the model organism Ralstonia eutropha JMP 134 (today reclassified into Cupriavidus necator). The responses of the organism were evaluated with flow cytometric determinations of DNA contents and cell size (by fluorescence and forward scatter measurements, respectively, after staining with the DNA-binding dye 4',6-diamidino-2'-phenylindole, DAPI), and cell concentration, after staining with the nucleic acid binding dye LDS-751. The strain was cultivated on a mineral medium with pyruvic acid sodium salt as the limiting carbon and energy source. Famine conditions, and thus cell dormancy, were achieved in every cycle. The best synchronization, according to the determination of DNA contents, was induced with phasing cycle durations of at least 4 h. The method allows the induction of synchrony for an indefinite period if the medium is exchanged rapidly and precisely. The results show that the time required for a complete cell cycle of Cupriavidus necator JMP 134 is independent of the chosen phasing cycle duration, provided that each process cycle lasts at least 3 h which is much longer than the time needed for a single DNA replication cycle. With shorter cycling periods DNA-synthesis is carried out in an uncoupled manner and only weak cell cycle synchrony can be attained. The results also show that DNA-synthesis can only be undertaken by cells when they have exceeded a critical size.

Bioreactors↗

PubMed1986

Early replicative intermediates of Escherichia coli chromosome isolated from a membrane complex.

The replication origin region of the Escherichia coli chromosome was isolated from an outer membrane fraction. The chromosome was further purified by centrifugation in a cesium chloride gradient. Early replicative intermediates were enriched in the preparation when cytosine-1-beta-arabinofuranoside was added to the culture at the time of initiation of chromosome replication. DNA fragments with an eye structure having two branches of less than 400-500 bp in length were associated with components that were removed by phenol treatment. We conclude that the replication fork usually proceeds counter-clockwise toward the unc operon in the earliest period of replication.

Base Composition↗

PubMed1990

Chromosome site-specific immunohistochemical detection of DNA adducts in N-acetoxy-2-acetylaminofluorene--exposed Chinese hamster ovary cells.

In these studies a polyclonal antiserum elicited against a carcinogen-DNA adduct was used to explore the localization of DNA adducts in metaphase chromosomes of cultured cells. Morphological visualization of the adduct N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF) in Chinese hamster ovary (CHO) cells exposed to the direct-acting carcinogen N-acetoxy-2-acetylaminofluorene (N-Ac-AAF) was accomplished by indirect immunofluorescence with an anti-G-C8-AF antiserum. At the same time the pattern of chromosomal DNA replication was determined by replicative incorporation of bromodeoxyuridine (BrdUrd) and chromosomal staining with anti-BrdUrd. Visualization of DNA in chromosomes was accomplished with Hoechst 33258 dye. When synchronized CHO cells were exposed to N-Ac-AAF for 0.5 h during early S phase, the chromosomal pattern of dG-C8-AF adduct formation was not random. Metaphase chromosome spreads from cells exposed to N-Ac-AAF in different experiments contained certain chromosome regions that had a consistently high adduct concentration. The regions of high DNA damage corresponded to the regions active in DNA synthesis when BrdUrd and the carcinogen were given simultaneously in early S phase. In addition, the patterns of high adduct concentration and replicative synthesis shifted when the carcinogen and BrdUrd were given simultaneously during late S phase. Thus, the stage of cell cycle in which adducts are induced is an important factor in the specific location of the highest concentrations of this type of DNA lesion.

2-Acetylaminofluorene↗

PubMed1994

SV40 large T antigen functions at two distinct steps in virion assembly.

The SV40 large T antigen mutant 5002 has two amino acid substitutions (L19-F; P28-S) and is defective for productive viral infection as demonstrated by its small plaques that arise very late and by a 100-fold reduced yield of infectious progeny. 5002 replicates viral DNA at the same time postinfection as wild-type SV40, and the production of progeny DNA molecules is only marginally reduced. Furthermore, the viral capsid proteins accumulate to near normal levels following infection with 5002. In this manuscript we report evidence that 5002 infection is blocked at a specific stage of viral assembly. The SV40 viral assembly pathway involves conversion of 75S chromatin complexes to 240S virions. Unlike mutants within the T antigen host range (HR) domain, that are also defective for viral assembly and accumulate 75S particles (Spence and Pipas, 1994), 5002 particles are blocked as 150S previrions containing viral DNA and capsid proteins. We have previously shown that 5002 and HR mutants cooperate to produce viable progeny in trans complementation tests. Thus, by two criteria, SV40 large T antigen encodes two distinct activities that function at different steps in virion assembly.

Antigens, Polyomavirus Transforming↗

PubMed1996

Mammalian DNA repair responses and genomic instability.

A cell responds to damage to its DNA in one of three ways: by tolerating the damage, by repairing the damage or by undergoing apoptosis. The latter two responses represent defenses against genomic instability and tumorigenesis resulting from unrepaired damage. There are multiple DNA repair pathways to cope with a variety of damage reflecting the importance of DNA repair in maintaining both cell viability and genomic stability. These include base excision repair, mismatch repair, double-strand break repair and nucleotide excision repair. Several signal transduction pathways are activated by DNA damage resulting in cell-cycle arrest. Cell-cycle arrest increases the time available for DNA repair before DNA replication and mutation fixation. Recently, there has been tremendous progress in our understanding of the molecular components repair processes and to examine recently observed interactions between DNA repair, signal transduction pathways and other cellular processes such as cell-cycle control, transcription, replication and recombination.

Animals↗

PubMed2006

Recombinant alpha2(IV)NC1 domain of type IV collagen is an effective regulator of retinal capillary endothelial cell proliferation and inhibits pre-retinal neovascularisation.

BACKGROUND: A recombinant form of the alpha2(IV)NC1 domain of type IV collagen has been shown to have potent anti-angiogenic activity although this peptide has not been studied in the context of proliferative retinopathies. In the current investigation we examined the potential for alpha2(IV)NC1 to regulate retinal microvascular endothelial cell function using a range of in vitro and in vivo assay systems. MATERIALS AND METHODS: alpha2(IV)NC1 at concentrations between 0.1 and 1 mug/ml was added to retinal microvascular endothelial cells (RMECs) followed by assessment of cell attachment, proliferation and survival. This agent was also tested within a novel in vitro three-dimensional retinal angiogenesis assay and the number of angiogenic sprouts quantified. alpha2(IV)NC1 was also delivered intra-vitreally to mice with oxygen-induced proliferative retinopathy (OIR) and neovascularisation evaluated in comparison with vehicle-treated controls. RESULTS: RMECs treated with alpha2(IV)NC1 (0.1, 0.5 and 1 microg/ml) showed delayed attachment at 3 h post-seeding, although this deficit had been restored at the 6-h time point. BrdU assay of DNA replication revealed that confluent RMECs treated with alpha2(IV)NC1 showed no measurable response in comparison with vehicle-treated controls. By contrast, proliferation of sub-confluent RMECs was significantly reduced by alpha2(IV)NC1 at 0.5 microg/ml (P<0.01). alpha2(IV)NC1 also induced apoptosis in RMECs and inhibited angiogenesis of pre-existing retinal vascular networks in vitro (P<0.001). Intra-vitreal injection of alpha2(IV)NC1 in the OIR model significantly inhibited pre-retinal neovascularisation compared with vehicle-treated controls (P<0.001). CONCLUSION: alpha2(IV)NC1 inhibits angiogenesis in the retinal microvasculature. This recombinant protein has potential for the treatment of neovascularisation in proliferative retinopathies.

Animals↗

PubMed1979

Specificity and functions of guanine methylase of Shigella sonnei DDVI phage.

DNA methylase methylating adenine with formation of 6-methylaminopurine has been identified in Shigella sonnei 1188 cells which are the natural host of DDVI phage. At the same time, in DNA of DDVI phage replicating both in Sh. sonnei 1188 cells and in Escherichia coli B cells 7-methylguanine was found as the only minor base in amounts of 0.25 and 0.27 mol per 100 mol of nucleotides, respectively. The extract of the infected cells was found to contain both kinds of DNA methylases: virus-specific guanine methylase and cellular adenine methylase. The lack of 6-methylaminopurine in DNA of this phage is explained by reversible inhibition of the cell enzyme in the infected cells. The amount of methyl groups transferred by DDVI-specific methylase on DNA does not depend on the species of the infected cells and is similar in the case of unmodified SD phage DNA and DNA of T2 phage methylated by E. coli B enzyme. Guanine methylase has been shown to be a DDVI-induced modification enzyme and to protect against restriction of B-type. It methylates double-stranded DNAs only and is inhibited by S-adenosylhomocysteine.

Bacteriophages↗

PubMed1985

Poly ADP-ribosylation--a cellular emergency reaction?

We propose that the activation of poly(ADP-ribose) synthetase by DNA damage serves to decrease rapidly and transiently the cellular level of NAD (by production therefrom of poly ADP-ribose). The result is a slow-down of energy-requiring reactions, in particular of replicative DNA synthesis giving cells more time to repair the damage. We do not attribute any specific role to poly ADP-ribosylated proteins in this reaction beyond their action as acceptors for poly ADP-ribose.

DNA↗

PubMed1979

Effect of cell growth rate and dose fractionation on chemically-induced ouabain-resistant mutations in Chinese hamster V79 cells.

Chinese hamster V79 cells were grown in medium containing either 10% or 2% FCS during the expression time following exposure to MNNG. The lower serum concentration was used to reduce the rate of cell replication, thereby allowing more time for DNA repair prior to "fixation" of the mutagenic lesion. In addition, fractionated and continuous exposures to MNNG and MAM, respectively, were carried out to determine their effect on the number of induced ouabain-resistant mutants. The results indicated that lowering the rate of cell growth effectively reduces the mutation frequency at low, but not at high doses of MNNG. Fractionated doses of MNNG result in a potentiation of their mutagenic effects compared to single doses. Also, continuous exposures to MAM result in an exponential increase in the mutation frequency. Collectively, these results suggest the importance of a repair process in Chinese hamster V79 cells which is dependent upon cell growth rate and the dose of the mutagen for its effectiveness.

Animals↗

PubMed2000

Proliferation is necessary for both repair and mutation in transgenic mouse cells.

Proliferating cells are often presumed to be more mutable than quiescent cells because they have less time to repair DNA damage before DNA replication. Direct tests of this hypothesis have been confounded by the need for cell division before a mutation can be detected. We have avoided this problem by showing that the Big Blue mouse cell line permits the dynamic quantification of both lesions and mutations in the complete absence of cell division. These cells carry the bacterial lacI gene in a lambda shuttle vector. Mutant plaques recovered by in vitro packaging of the mouse DNA can arise from mutations sustained either in mouse cells or in the bacteria. The proportion of mutant phage contained within a mutant plaque can distinguish these two types of mutation. Mutations formed in mouse cells yield >90% mutant phage because both DNA strands are mutant. On the other hand, mutations formed in the bacteria from adducted DNA yield </=50% mutant phage, because one of the DNA strands is wild type. Immediately after exposure to a test mutagen, ethylnitrosourea, all induced mutations were formed in the bacteria, but after approximately one cell division, the reverse was true and all mutations arose in the mouse cells. Only one-fifth as many mutations were recovered from quiescent cells and all arose in the bacteria, showing that the mouse cells made no mutations in the absence of proliferation. Surprisingly, the mouse cells did not repair any of the premutagenic damage during 4 days of quiescence. When these quiescent cells were induced to proliferate, however, both repair and mutation fixation ensued.

Animals↗

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↗