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At least 793 records · Page 44Linked to original sources
PubMed1980

The effect of cytosine arabinoside on the synthesis of rapidly labeled RNA during DNA replicating and non-DNA replicating periods of the cell cycle.

The effects of various concentrations of cytosine arabinoside (Ara-C) on the rates of DNA and RNA synthesis were investigated during the peak of DNA synthesis, using a naturally synchronized culture of Aedes aegypti (mosquito) cells. During this stage of the cell cycle, the synthesis of both DNA and RNA was found to be progressively inhibited with increasing concentrations of Ara-C. When the same concentrations of Ara-C were added to the culture at a time period when no DNA was being synthesized, it was found that the synthesis of RNA was not inhibited. Rapidly labeled polysomal RNA species were isolated from cultured cells in which DNA was being synthesized. The synthesis of these RNA species is inhibited completely in the presence of a concentration of Ara-C which inhibits 90% of DNA synthesis. It is suggested that during periods of DNA replication, Ara-C is a selective inhibitor of the synthesis of rapidly labeled RNA species or of the processing of these RNA species from the nucleus to the polysomes.

Aedes↗
PubMed1982

Organization of the newly replicated chromatin in the vicinity of the replication fork.

Ehrlich ascites tumour cells were pulse-labelled with [3H]thymidine for 1 min or were treated with cycloheximide and labelled with [3H]thymidine for 45 min. The kinetics of digestion with micrococcal nuclease of both pulse-labelled and cycloheximide chromatins showed that they exhibited increased susceptibility towards the enzyme. At the same time their release from the nucleus was retarded and this was interpreted to mean that, unlike the bulk of chromatin, they were tightly bound to a fixed nuclear structure. When subjected to an equilibrium metrizamide-triethanolamine density gradient centrifugation both pulse-labelled and cycloheximide chromatins banded at higher density than control chromatin, which was an indication of their higher protein to DNA ratio. After a mild trypsinization, eliminating H1 and the nonhistone proteins, the pulse-labelled chromatin sedimented to the same density as control chromatin, and the cycloheximide chromatin sedimented to a density which was intermediate between those of control chromatin and free DNA. This result showed that the newly replicated chromatin had the same, and the cycloheximide chromatin half the amount of core histones present in control chromatin.

Animals↗
PubMed1982

Self-replication with errors. A model for polynucleotide replication.

A model for polynucleotide replication is presented and analyzed by means of perturbation theory. Two basic assumptions allow handling of sequences up to a chain length of v approximately 80 explicitly: point mutations are restricted to a two-digit model and individual sequences are subsumed into mutant classes. Perturbation theory is in excellent agreement with the exact results for long enough sequences (v greater than 20).

DNA Replication↗
PubMedDate not supplied

The replication origin of pSC101: the nucleotide sequence and replication functions of the ori region.

The nucleotide sequence of a 770-bp ori region of plasmid pSC101 is presented. The sequence shows homologies to some parts of Escherichia coli oriC and phage G4 ori. Several other features are an 80-bp A + T-rich region overlapping a part of the region homologous to oriC, three direct repeats of an 18-bp sequence adjacent to the A + T-rich region, a typical promoter sequence just upstream of the longest open reading frame (ORF) and a long inverted repeat sequence overlapping the putative promoter region. Analysis of successive deletions by BAL31 exonuclease demonstrated that one of the regions homologous to oriC along with the A + T-rich region are essential for autonomous replication of the plasmid. The three 18-bp repeats are responsible for incompatibility phenotype. The region containing the promoter-like sequence is required for expression of a trans-acting function.

Base Sequence↗
PubMed1983

Methylation of replicating and post-replicated mouse L-cell DNA.

We have introduced [alpha-32P]dGTP into permeabilized cells and measured the degree of methylation at CpG sites by nearest-neighbor analysis. This method reveals a lag of approximately 1 min between DNA synthesis and the modification event. When methylation is inhibited by the addition of S-adenosyl-L-homocysteine in the presence of continued DNA synthesis, the resulting hemimethylated sites are methylated immediately after the release of inhibition. The results suggest that the methylase activity in the cell allows immediate methylation but conditions at the replication fork bring about a short delay in the onset of the modification reaction.

Animals↗
PubMed1998

Processing of an HIV replication intermediate by the human DNA replication enzyme FEN1.

The role of human FEN1 (flap endonuclease-1), an RTH1 (RAD two homolog-1) class nuclease, in the replication of human immunodeficiency virus (HIV) type 1 has been examined using model substrates. FEN1 is able to endonucleolytically cleave a primer annealed to a template, but with a 5'-unannealed tail. The HIV (+)-strand is synthesized as two discontinuous segments, with the upstream segment displacing the downstream segment to form a central (+)-strand overlap. Given a substrate with the exact HIV nucleotide sequence, FEN1 was able to remove the overlap. After extension of the upstream primer with DNA polymerase epsilon, human DNA ligase I was able to complete the continuous double strand as would occur for an integrated provirus. FEN1 may represent a target for new therapeutic interventions.

Base Sequence↗
PubMed1994

Emergence of early region 1-containing replication-competent adenovirus in stocks of replication-defective adenovirus recombinants (delta E1 + delta E3) during multiple passages in 293 cells.

Early region 1 (E1)-deleted human adenovirus (AV) recombinants have been shown to be powerful tools of gene transfer in vivo and in vitro and are considered for application in human gene therapy. We could detect increasing titers of E1-containing adenovirus in two independent E1 + E3-deleted recombinant AV stocks during multiple passages in 293 cells, most likely due to a recombinant event with the host cell genome. We show the deleterious effects of this E1-containing, mostly replication-competent AV subpopulation in vivo and compare different screening methods of AV stocks for its detection. These considerations are important for the safety of human gene therapy trials.

Adenovirus E1 Proteins↗
PubMed1974

Replication of colicinogenic factor E 1 DNA: evidence for a discontinuous replication mechanism.

The mechanism of Col E 1 DNA replication was investigated in a plasmolysed cell system prepared from chloramphenicoltreated E. coli JC 411 (Col E 1). After pulse-labelling with (3)H-dTTP a considerable fraction of the newly synthesized DNA was recovered as single-stranded fragments. Upon alkali denaturation the pulse label was found in DNA chains sedimenting slower than unit length Col E 1 strands with a prominent peak at 5 S. During a chase with unlabeled precursors the label is transferred nearly completely into supercoiled Col E 1 DNA. DNA ligase appears to be required for the joining of the 5 S pieces since in the absence of NAD an accumulation of short fragments is observed.

Bacterial Proteins↗
PubMed1982

Design and characterization of N2-arylaminopurines which selectively inhibit replicative DNA synthesis and replication-specific DNA polymerases: guanine derivatives active on mammalian DNA polymerase alpha and bacterial DNA polymerase III.

The 2-amino substituted derivatives of guanine, N2-(p-n-butylphenyl)guanine (BuPG) and N2-(3',4'-trimethylenephenyl) guanine (TMPG), were synthesized and found to selectively inhibit, respectively, HeLa cell DNA polymerase alpha (po1 alpha) and B. subtilis DNA polymerase III (po1 III). Both purines, like their corresponding uracil analogs, BuAu and TMAU (2,9), were specifically competitive with dGTP in their inhibitory action on their target polymerases. BuPG, the pol alpha-specific purine, was also toxic for HeLa cells in vivo, selectively inhibiting DNA synthesis. These N2-substituted purines, in contrast to the 6-substituted uracils, provide a structural basis for the synthesis of nucleosides and nucleotides with considerable potential as probes for the analysis of the structure of specific replicative DNA polymerases and their function in cellular DNA metabolism.

Bacillus subtilis↗
PubMed1997

Co-packaging of non-vector RNAs generates replication-defective retroviral vector particles: a novel approach for blocking retrovirus replication.

A Moloney murine leukemia virus (MoMuLV)-derived packaging retroviral vector, pUCMoTN-PR3, was previously developed in which the packaging (psi) signal was cloned within the 5'-long terminal repeat (LTR) U3-r and U5 sequences. The MoTN-PR3 vector particles released from a transfected packaging cell line contain RNAs with r-psi-U5 sequences at the 5'-end and U3-r sequences at the 3'-end. Upon infection, these vector particles can efficiently transduce the neomycin phosphotransferase (neo) gene to the target cells. The structure of the proviral DNA synthesized in these cells was shown to contain modified 5'- and 3'-LTRs with U3-r-psi-U5 sequences, indicating that this vector can undergo reverse transcription and integration. Analysis of psi signal-containing RNAs revealed that in addition to vector RNA transcribed from the MoMuLV 5'-LTR promoter, readthrough neo RNA transcribed from the internal herpes simplex virus (HSV) thymidine kinase (tk) promoter and cellular RNAs transcribed from the MoMuLV 3'-LTR promoter are produced. Of these, the downstream cellular RNAs are also packaged within the vector particles. These vector particles containing the vector and non-vector RNAs carrying the MoMuLV psi signal are non-infectious. It is proposed that intracellular expression of packageable non-viral RNAs may represent an effective strategy for inhibiting animal and plant virus replication.

3T3 Cells↗
PubMed1996

Simultaneous measurement of unscheduled and replicating DNA synthesis by means of a new cell culture insert DNA retention method: rapid induction of replicating DNA synthesis in response to genotoxic carcinogens.

In order to measure simultaneously replicating DNA synthesis (RDS) and unscheduled DNA synthesis (UDS) in rat hepatocytes responding to exposure to carcinogens, a new method, namely the "cell culture insert DNA retention (CDR)" method, was developed. All CDR procedures for cell culture, digestion of cytoplasm and retention of DNA were performed on membranes attached to cell culture containers. Four subgroups of primary cultures of hepatocytes prepared from rats were exposed to a genotoxic or non-genotoxic carcinogen with or without 10 mM hydroxyurea and incubated for 4 h with 10 microCi/ml [3H]thymidine. The membranes were then processed for both liquid scintillation and autoradiography. Among seven tested chemicals, three genotoxic agents, 3,2'-dimethyl-4-aminobiphenyl, 2-acetylaminofluorene and diethylnitrosamine, and two non-genotoxic carcinogens, nafenopin and phenobarbital, induced RDS within 4 h after the exposure, indicating that these carcinogenic agents induce cell proliferation is non-proliferating rat hepatocytes prior to the emergence of genotoxic changes. Several indices were devised to characterize the genotoxicity of the tested chemicals. The induction patterns obtained showed a wide variation in the individual characteristics of carcinogen-induced genotoxicity and mitogenicity in the early phase of initiation. This is the first report of simultaneous measurement, by using a combination of autoradiography and liquid scintillation, of UDS and RDS induced in rat hepatocytes. The described CDR approach will be useful for risk assessment and characterization of carcinogenic and tumor-promoting agents.

2-Acetylaminofluorene↗
PubMed1975

Replication of colicinogenic factor E1 DNA in plasmolysed Escherichia coli cells. Coupling of DNA replication and RNA synthesis.

Plasmolysed chloramphenicol-treated Escherichia coli cells carrying the colicinogenic factor E1 utilize deoxynucleoside triphosphates for the semi-conservative synthesis of Col E1 DNA. Col E1 DNA replication in plasmolysed cells can be dissociated into two temporally separated processes: (a) a rifampicin-sensitive RNA synthesis, which is stimulated by adenosine 3':5'-monophosphate (cyclic AMP) and requires all four ribonucleoside triphosphates and (b) an ATP-dependent DNA synthesis, which is inhibited by arabinosylnucleoside triphosphates and sulfhydryl-blocking reagents. Thes two processes exhibit different sensitivities to inhibition by polyamines and actinomycin D.

Adenosine Triphosphate↗
PubMed1973

Temperature-sensitive mutants for the replication of plasmids in Escherichia coli: requirement for deoxyribonucleic acid polymerase I in the replication of the plasmid ColE 1 .

An Escherichia coli mutant (polA1), defective in deoxyribonucleic acid (DNA) polymerase I, (EC 2.7.7.7) is unable to maintain colicinogenic factor E1 (ColE1), whereas several sex factor plasmids are maintained normally in this strain. polA1 mutant strains containing these sex factor plasmids do not exhibit a readily detectable plasmid-induced polymerase activity. A series of E. coli mutants that are temperature sensitive for ColE1 maintenance, but able to maintain other plasmids, were isolated and shown to fall into two phenotypic groups. Mutants in one group are defective specifically in ColE1 maintenance at 43 C, but exhibit normal DNA polymerase I activity. Mutations in the second group map in the polA gene of E. coli, and bacteria carrying these mutations are sensitive to methylmethanesulfonate (MMS). Revertants that were selected either for MMS resistance or the ability to maintain ColE1 were normal for both properties. The DNA polymerase I enzyme of two of these mutants shows a pronounced temperature sensitivity when compared to the wild-type enzyme. An examination of the role of DNA polymerase I in ColE1 maintenance indicates that it is essential for normal replication of the plasmid. In addition, the presence of a functional DNA polymerase I in both the donor and recipient cell is required for the ColV-promoted conjugal transfer of ColE1 and establishment of the plasmid in the recipient cell.

Centrifugation, Density Gradient↗
PubMed2002

Replication-dependent recruitment of the beta-subunit of DNA polymerase III from cytosolic spaces to replication forks in Escherichia coli.

The beta-subunit of DNA polymerase III is located as one or two condensed clusters within the nucleoid-occupied space in exponentially growing cells of Escherichia coli. When chromosome replication is terminated after incubation at nonpermissive temperature in a temperature-sensitive dnaC mutant, the beta-subunit is located in the cytosolic spaces of the cell poles.

Bacterial Outer Membrane Proteins↗
PubMed1973

Herpes simplex virus and human cytomegalovirus replication in WI-38 cells. I. Sequence of viral replication.

A comparison, under standardized conditions, of herpes simplex virus (HSV) and human cytomegalovirus (CMV) revealed differences in viral morphology, in the timing of their infectious cycles, and in several morphological events during those cycles. Structural distinctions between the two viruses included the coating of unenveloped cytoplasmic CMV capsids, but not those of HSV, and a variation in the structure of their cores. Since the two cycles were carried out in the same host cell strain under conditions of one-step growth (input multiplicity = 10 PFU/cell), it was possible to construct time scales locating the major events of each cycle. Comparison of the two showed that HSV replicated and released progeny within 8 h postinfection, whereas CMV required 4 days. These results correlated well with those of concurrent plaque assays. Within the longer CMV cycle, most of the major events appeared retarded to a similar degree, and no obvious limiting step in particle production could be identified. Distinctions between the two cycles included the following: condensation of the chromatin in HSV- but not CMV-infected cells; the greater tendency of HSV to produce membrane alterations; and the appearance of cytoplasmic dense bodies in CMV- but not HSV-infected cells. Identification of these differences even under identical conditions of culture and infection strongly implies that they result from intrinsic differences in the nature of the viruses, and are not caused by variations in experimental conditions.

Cell Line↗
PubMed1984

[DNA replication in mammalian cells exposed to physical, chemical or biological factors. II. DNA-complex and nucleoid recovery and DNA replication after gamma irradiation].

In the irradiated mammalian cells the repair of DNA-membrane complexes (DMC) and nucleoid structure was shown to proceed more quickly than the restoration of DNA synthesis. It is suggested that the repair of DNA-membrane complexes and nucleoid structure is necessary but not sufficient for normalization of DNA replication.

Animals↗
PubMed1978

[DNA replication in HeLa cells after gamma irradiation. II. The periodicity of DNA replication in the process of giant cell formation].

HeLa G-63 cells irradiated by 5 krads of 6 degrees Co gamma-rays 1.5--2 hours after mitosis (G1-phase) were incubated in growth medium during 9 days. The number of proliferating and eliminated cells, the content of DNA per cell nucleus, and kinetics of the labeled cell fraction upon 3H-TdR continuous incubation were studied. It has been concluded that: during the giant cell growth the nuclear DNA is replicated periodically without following mitoses; 90% of cells are involved in such a cycle; the duration of the period of DNA synthesis in giant cells is the same as that in non-irradiated cells; the duration of the gap between two successive S-periods in giant cells is 2--6 times as long as the sum of G2+M+G1 periods in the normal non-irradiated cells.

Cell Division↗