DNA polymerase alpha, beta and gamma activities in ultraviolet irradiated CV-1 monkey cells.

To determine the possible role of DNA polymerase alpha, beta and gamma during the repair period following ultraviolet (lambda max : 254 nm) irradiation of monkey CV-1 cells, we measured the three enzymatic activities by using specific tests, either in crude extracts or after fractionation by sucrose gradient (5--20%) centrifugation at high salt concentration. When compared to the unirradiated control, we could not detect any significant variation in the levels of activity of DNA polymerases alpha, beta and gamma at any time (0, 12 to 48 h) after ultraviolet irradiation of the cells with doses ranging from 9 to 52.5 J.m-2.     

Comparative sequence and expression analyses of African green monkey (Cercopithecus aethiops) TNPO3 from CV-1 cells

Transportin 3 (TNPO3 or TRN-SR2) is a key host cellular factor involved in the early steps of several lentiviral replications. In the present study, we cloned the TNPO3 gene from CV-1 cells of African green monkey (AGM) using a homologue based cloning technology, analyzed the sequence, and evaluated the cellular expression of the proteins by western blotting and immunostaining assays. DNA sequencing of TNPO3 showed homologies of 99 % with human, rhesus monkey, chimpanzee, and baboon; the predicted protein sequence differed in only one amino acid (leucine in place of methionine). The deduced sequence revealed that AGM is phylogenetically related to human, chimpanzee, rhesus monkey, orangutan and baboon rather than bovine, rate and mouse. Western blot analysis demonstrated immunoreactive proteins in both the cytoplasmic and nuclear fractions. A similar expression pattern was observed in human and baby hamster cells. The specific detection of TNPO3 was also confirmed in the cytoplasm and nucleus by immunostaining. The present findings conclusively demonstrate that AGM-TNPO3 is genetically and physiologically almost identical with that of humans and could be a good candidate for HIV and AGM research as well as an ideal system for a TNPO3 vaccine trial.     

Kinetics of DNA and histone messenger RNA synthesis in CV-1 monkey kidney cells infected with simian virus 40.

Histone messenger RNA has been identified in CV-1 monkey kidney cells and its synthesis during the simian virus 40 (SV40) productive cycle has been correlated with the synthesis of cellular DNA and viral DNA. In cultures of CV-1 cells that have reached confluence, infection with SV40/5 (a high-yield clone of SV40) promotes an increase in the rate of cellular DNA synthesis followed by a decline. During this decline the rate of viral DNA synthesis continues to rise and eventually surpasses that of cellular DNA.The synthesis of histone mRNA rises concomitantly with the increase in the synthesis of cellular DNA. This occurs in a fashion similar to that observed when confluent CV-1 cultures are stimulated by the addition of fresh serum to the growth medium. However, whereas in cells stimulated with serum the synthesis of histone mRNA closely parallels that of cellular DNA, in cells infected with SV40, histone mRNA synthesis continues at a high rate even after the decline of cellular DNA synthesis. The rate of histone mRNA synthesis thus appears to he coupled to the total (cellular plus viral) DNA synthesis and not to the synthesis of the host DNA alone. The high rate of synthesis of the F1 histone at late times after infection suggests that histone genes are transcribed co-ordinately.     

Identification and characterization of an adeno-associated virus integration site in CV-1 cells from the African green monkey

Adeno-associated virus (AAV) is a classification given to a group of nonpathogenic, single-stranded DNA viruses known to reside latently in primates. During latency in humans, AAV type 2 (AAV2) preferentially integrates at a site on chromosome 19q13.3ter by targeting a sequence composed of an AAV Rep binding element (RBE), a spacer, and a nicking site. Here, we report the DNA sequence of an African green monkey AAV integration site isolated from CV-1 cells. Overall, it has 98% homology to the analogous human site, including identical spacer and nicking sequences. However, the simian RBE is expanded, having five perfect directly repeated GAGC tetramers. We carried out a number of in vitro and in vivo assays to determine the effect of this expanded RBE sequence on the Rep-RBE interaction and AAV targeted integration. Using electromobility shift assays it was demonstrated that AAV4 Rep68 bound the expanded RBE with a sixfold-greater affinity than the human RBE. To determine the basis for the affinity increase, DNase I protection and methylation interference (MI) assays were performed. Comparison of footprints on both the human and simian RBEs revealed nearly identical protection; however, MI analysis suggested greater interaction with the guanine nucleotides of the expanded RBE, thus providing a biochemical basis for the increased binding activity. In vivo, integration targeted to the simian RBE was demonstrated by PCR analysis of latently infected Cos-7 cells. Interestingly, the frequency of site-specific integration was twofold greater in Cos-7 cells than in HeLa cells. Overall, these experiments establish that the simian RBE, identified in CV-1 cells, functions analogously to the human RBE and provide further evidence for a developing model that proposes individual roles for the RBE and the spacer and nicking site elements.     

Restricted repair of aflatoxin B1 induced damage in alpha DNA of monkey cells

We have investigated the processing of adducts formed by covalent binding of aflatoxin B1 (AFB1) to DNA in confluent cultures of African green monkey cells. Repair synthesis elicited by AFB1 adducts was deficient in alpha DNA sequences compared to that in bulk DNA, although the initial levels of modification were the same for these DNAs. The removal of the primary initial adduct, AFB1-N7-Guanine, was deficient in alpha DNA and the kinetics of its loss resembled those previously reported for removal from total DNA in xeroderma pigmentosum cells of complementation group A. Spontaneous loss of the AFB1 moiety or the concomitant loss of the guanine to yield an apurinic site account for these results. The formation of the more chemically stable secondary product, AFB1-triamino-Pyrimidine, occurred more rapidly and to a greater extent in alpha DNA than in bulk DNA, probably because of slower removal of the primary product. The excision repair patch size for AFB1 adducts in alpha DNA was only 10 nucleotides compared to 20 nucleotides for repair of AFB1 adducts in bulk DNA. Irradiation of cells with low doses of UV prior to or immediately after treatment with AFB1 increased the rate and extent of removal of AFB1 adducts from alpha DNA to the levels found in the bulk DNA, indicating that the formation of pyrimidine dimers or their repair may alter the chromatin structure of alpha DNA sufficiently to facilitate its repair.     

DNA replication during a serum-induced S phase in primate CV-1 cells

We have investigated components of DNA replication in a serum-induced S phase of primate CV-1 cells. Using DNA fiber autoradiography, we found a relative decrease in the frequency of initiation events in mid-S compared with early and late S phase. The other components of DNA replication measured by autoradiography—synchrony of initiation events, size of replication units, incidence of bidirectional replication, and the rate of replication fork movement—remained constant through S phase. When fork movement was measured by density gradient analysis of BUdR- and [³H]-thymidine-substituted DNA, it was also found to remain constant. These results show that most components of DNA replication are invariable through a serum-induced S phase. The changes in initiation frequency support the view that it may be critical in the regulation of ongoing replication.     

Spin trapping of precursors of thymine damage in X-irradiated DNA

A spin-trapping method combined with ESR spectroscopy was utilized to obtain evidence for the presence of precursor radicals leading to damage in X-irradiated DNA. Two technical improvements were introduced to the conventional spin-trapping method to make possible its application to large molecules such as DNA: prior to X irradiation, sonolysis of aqueous DNA solution by 19.5-kHz ultrasound was made to get a highly concentrated DNA solution and to lower the viscosity of the solution; after precursor radicals in X-irradiated DNA were trapped by a spin-trapping reagent, the DNA was digested to oligonucleotides by DNase I to get an ESR spectrum with a well-resolved hyperfine structure. Thus, it was recognized that the ESR spectrum obtained after X irradiation of the aqueous solution containing DNA and the nitroso spin-trapping reagent 2-methyl-2-nitrosopropane consisted of at least three sets of signals in the DNA. Identification of free radicals was made by comparing the spectrum with that of thymidine, which was precisely examined by a spin-trapping method combining two kinds of spin traps (nitroso and nitrone compounds) with liquid chromatography. As a result, all the signals were identified as the spin adducts of radicals produced at the thymine base moiety of DNA. The 5-hydroxy-5,6-dihydrothymin-6-yl radical was identified as a precursor of 5,6-dihydroxy-5,6-dihydrothymine (thymine glycol), the 6-hydroxy-5,6-dihydrothymin-5-yl radical as a precursor of 6-hydroxy-5,6-dihydrothymine, and the 5-methyleneuracil radical as a precursor of 5-(hydroxymethyl) uracil.     

Lack of enhanced mutation of UV- and gamma-irradiated herpes virus in UV-irradiated CV-1 monkey cells

The frequency of forward mutation of unirradiated, UV-irradiated or gamma-irradiated herpes virus was determined after infecting UV-irradiated or unirradiated CV-1 monkey kidney cells, to investigate the correlation between UV-enhanced reactivation (UVER) and mutagenesis. UV-irradiation to cells had no effect on mutation frequency of irradiated virus even in the conditions in which UVER was maximally expressed for the survival of UV-irradiated virus.     

Mutation of potassium permanganate- and hydrogen peroxide-treated plasmid pZ189 replicating in CV-1 monkey kidney cells

We examined the effect of the oxidation of plasmid pZ189 by KMnO4, which does not produce free radicals, and H2O2/Fe(2+)-diethylenetriaminepentaacetic acid (DTPA), which does, on the mutation frequency of pZ189 transfected into monkey kidney CV-1 cells. Treatment with 1.5 mM KMnO4 increased the content of certain modified bases, principally Thy and Cyt modified at C-5 and C-6, by up to 300-fold, as measured by GC/MS; however, the mutation frequency increased only 5-fold above background. 1.0 mM H2O2/0.1 mM Fe(2+)-DTPA treatment, which increased the mutation frequency 10-fold above background, increased the content of certain modified bases by up to 4-fold. Sequence analysis revealed both deletions and point mutations, with a predominance of C-G substitutions, among H2O2/Fe(2+)-DTPA-associated mutations. These data suggest that KMnO4-modified DNA is only weakly mutagenic in DNA replicating in mammalian nuclei, despite substantial production of Thy glycol and other base modifications, whereas H2O2/Fe(2+)-DTPA-modified DNA is more mutagenic. H2O2/Fe(2+)-DTPA generated mutations occur predominantly at C-G base pairs.     

The sequence specificity of bleomycin-induced DNA damage in intact cells

Bleomycin causes lesions to DNA in intact cells and in purified DNA under appropriate conditions. Using a middle repetitive DNA sequence called alpha-DNA as a target sequence, we have compared the sequence specificity of bleomycin-induced DNA cleavage in intact human cells and in purified human DNA. Bleomycin induces numerous cleavage sites in alpha-DNA which vary widely in intensity and give rise to a complex pattern of bands on a DNA sequencing gel. Unexpectedly, the intensity and position of bleomycin-induced DNA cleavage sites are very similar in intact human cells and in purified human DNA.     

Biochemical and immunofluorescence analysis of the constitutively expressed HSP27 stress protein in monkey CV-1 cells

The α-crystallin-related stress protein HSP27, which promotes cellular resistance to different types of stress, is constitutively expressed during the growth of several primate tissue culture cells. Here, we report an analysis of the cellular localization of this protein in CV-1 monkey cells. Following cell lysis and fractionation in the absence of detergent about 2 5 % of the cellular content of HSP27 was recovered in the particu late fractions while the remaining of this protein was in the soluble cytoplasmic fraction. This association of HSP27 with particulate fractions was no more observed when cells were lysed in the presence of non-ionic detergent or when cells were pretreated with drugs, such as monensin and colcemid, that disrupt cytoskeletal architecture. Immunofluorescence analysis revealed that HSP27 is concentrated in a polarized perinuclear zone of CV-1 cells from where microtubules radiate. The particular locale of HSP27 was investigated in cells exposed to drugs or treatments, such as monensin, colcemid, cold stess and serum starvation, that disrupt the cellular architecture of microtubules. A correlation was observed between HSP27 cellular locale and microtubules integrity. Our results suggest a possible interaction of a fraction of HSP27 with cytoplasmic organelles or structures, different from the Golgi apparatus, whose distribution depends upon the organization of microtubules.     

The formation of photoreactivable damage by direct excitation of DNA in X-irradiated E. coli cells

The role of the direct excitation process in the formation of photoreactivable damage (pyrimidine dimers) in E. coli WP2 hcr-exr- cells has been studied. The pyrimidine dimers were detected by photoreactivation following anoxic irradiation by X-rays (220 kVp). The dose modifying factor (DMF) is 1.28 +/- 0.09. A biophysical model is used for a theoretical examination of the importance of the direct excitation process in the formation of photoreactivable damage and the experimental data are consistent with this model.     

Cell-cycle-dependent repair of damage in alpha and bulk DNA of monkey cells

Excision repair of bulky chemical adducts in alpha DNA of confluent cultures of African green monkey cells has previously been shown to be deficient relative to that in the overall genome. We have compared the removal of adducts produced by treatment with aflatoxin B1 (AFB1) and N-acetoxy-2-acetylamino-fluorene (NA-AAF) from alpha DNA sequences in synchronized and exponentially growing cultures of monkey cells. Proficient removal of AFB1 adducts in alpha DNA was observed in exponentially growing cultures. However, as the cultures approached confluence, adduct removal from alpha DNA became deficient. Cells synchronized by subculturing confluent cultures exhibited proficient removal of adducts from both alpha and bulk DNA when treated in early G1 or late S/G2 while those cells treated in early S phase did not remove adducts from either alpha or bulk DNA. We conclude that the accessibility of chemical adducts to repair in alpha chromatin is influenced by the growth state and the cell cycle stage.     

DNA damage and DNA sequence retrieval from ancient tissues.

Gas chromatography/mass spectrometry (GC/MS) was used to determine the amounts of eight oxidative base modifications in DNA extracted from 11 specimens of bones and soft tissues, ranging in age from 40 to >50 000 years. Among the compounds assayed hydantoin derivatives of pyrimidines were quantitatively dominant. From five of the specimens endogenous ancient DNA sequences could be amplified by PCR. The DNA from these specimens contained substantially lower amounts of hydantoins than the six specimens from which no DNA could be amplified. Other types of damage, e.g. oxidation products of purines, did not correlate with the inability to retrieve DNA sequences. Furthermore, all samples with low amounts of damage and from which DNA could be amplified stemmed from regions where low temperatures have prevailed throughout the burial period of the specimens.     

Differential repair of DNA damage in specific nucleotide sequences in monkey cells.

An immunological method was developed that isolates DNA fragments containing bromouracil in repair patches from unrepaired DNA using a monoclonal antibody that recognizes bromouracil. Cultured monkey cells were exposed to either UV light or the activated carcinogen aflatoxin B1 and excision repair of damage in DNA fragments containing the integrated and transcribed E. coli gpt gene was compared to that in the genome overall. A more rapid repair, of both UV and AFB1 damage was observed in the DNA fragments containing the E. coli gpt genes. The more efficient repair of UV damage was not due to a difference in the initial level of pyrimidine dimers as determined with a specific UV endonuclease. Consistent with previous observations using different methodology, repair of UV damage in the alpha sequences was found to occur at the same rate as that in the genome overall, while repair of AFB1 damage was deficient in alpha DNA. The preferential repair of damage in the gpt gene may be related to the functional state of the sequence and/or to alterations produced in the chromatin conformation by the integration of plasmid sequences carrying the gene.     

Enhancement of DNA-mediated gene transfer by high-Mr carrier DNA in synchronized CV-1 cells.

Synchronized CV-1 cells were transfected with SV40 (simian virus 40) DNA-calcium phosphate co-precipitates. In the presence of carrier DNA, the transfection efficiency of SV40 DNA was decreased 5-fold in S-phase cells and was increased 4-fold in preparations of mitotically enriched cells as compared with asynchronous controls. No difference was observed when carrier DNA was omitted, when cells had progressed through S-phase and into G2-phase, or when the infectivity of cells to intact SV40 virus was tested. These results highlight the importance of cell-cycle-dependent factors on DNA-mediated gene transfer.     

Identification of DNA polymerase delta in CV-1 cells: studies implicating both DNA polymerase delta and DNA polymerase alpha in DNA replication

DNA polymerases delta and alpha were purified from CV-1 cells, and their sensitivities to the inhibitors aphidicolin, (p-n-butylphenyl)deoxyguanosine triphosphate (BuPdGTP), and monoclonal antibodies directed against DNA polymerase alpha were determined. The effects of these inhibitors on DNA replication in permeabilized CV-1 cells were studied to investigate the potential roles of polymerases delta and alpha in DNA replication. Aphidicolin was shown to be a more potent inhibitor of DNA replication than of DNA polymerase alpha or delta activity. Inhibition of DNA replication by various concentrations of BuPdGTP was intermediate between inhibition of purified polymerase alpha or delta activity. Concentrations of BuPdGTP which totally abolished DNA polymerase alpha activity were much less effective in reducing DNA replication, as well as the activity of DNA polymerase delta. Monoclonal antibodies which specifically inhibited polymerase alpha activity reduced, but did not abolish, DNA replication in permeable cells. BuPdGTP, as well as anti-polymerase alpha antibodies, inhibited DNA replication in a nonlinear manner as a function of time. Depending upon the initial or final rates of inhibition of replication by BuPdGTP and anti-alpha antibodies, as little as 50%, or as much as 80%, of the replication activity can be attributed to polymerase alpha. The remaining replication activity (20-50%) is tentatively attributed to polymerase delta, because it was aphidicolin sensitive and resistant to both anti-polymerase alpha antibodies and low concentrations of BuPdGTP. A concentration of BuPdGTP which abolished polymerase alpha activity reduced, but did not abolish, both the synthesis and maturation of nascent DNA fragments.(ABSTRACT TRUNCATED AT 250 WORDS)