Monodispersed circular DNA in mammalian cells

The major birch (Betula alba L.) pollen allergen, Bet v 1, has been shown to be homologous to pathogenesis-related proteins in a number of plants. Recently, it was demonstrated that a ginseng protein with high homology to an intracellular pathogenesis-related protein of parsley and to Bet v 1 is a ribonuclease (RNase). Birch pollen extract was separated in an RNase activity gel. Four major RNase bands were excised from the gel, reseparated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and identified by Western blotting with a specific Bet v 1 monoclonal antibody and patient's serum. Thus the monomer and the dimer of Bet v 1 showed RNase activity. Purified recombinant Bet v 1 was shown to degrade plant RNA. The RNase activity of recombinant Bet v 1 was 180 units.mg-1.     

Effect of mimosine on DNA synthesis in mammalian cells

We have designed a general protocol to assess the rate of replicon initiation in mammalian cells in the presence of inhibitors of DNA synthesis. It is based on cross-linking DNA in vivo with trioxsalen, which effectively blocks the movement of the replication forks along DNA, while having little effect on initiation of replication. We applied this protocol to study the effect of the plant amino acid mimosine on the rate of replicon initiation in exponentially growing murine erythroleukemia F4N cells. We found out that during the first 2 h after application of 25-400 microM mimosine, the initiation step was inhibited more efficiently than the overall DNA synthesis. In this respect, the effect of mimosine was similar to that of gamma-ray irradiation and differed from that of hydroxyurea and aphidicolin. The results suggest that in addition to inhibiting the elongation step of DNA synthesis, mimosine inhibits the initiation of DNA replication as well.     

Electropermeabilization of mammalian cells to macromolecules: control by pulse duration

Membrane electropermeabilization to small molecules depends on several physical parameters (pulse intensity, number, and duration). In agreement with a previous study quantifying this phenomenon in terms of flow (Rols and Teissié, Biophys. J. 58:1089-1098, 1990), we report here that electric field intensity is the deciding parameter inducing membrane permeabilization and controls the extent of the cell surface where the transfer can take place. An increase in the number of pulses enhances the rate of permeabilization. The pulse duration parameter is shown to be crucial for the penetration of macromolecules into Chinese hamster ovary cells under conditions where cell viability is preserved. Cumulative effects are observed when repeated pulses are applied. At a constant number of pulses/pulse duration product, transfer of molecules is strongly affected by the time between pulses. The resealing process appears to be first-order with a decay time linearly related to the pulse duration. Transfer of macromolecules to the cytoplasm can take place only if they are present during the pulse. No direct transfer is observed with a postpulse addition. The mechanism of transfer of macromolecules into cells by electric field treatment is much more complex than the simple diffusion of small molecules through the electropermeabilized plasma membrane.     

Targeted recombination with single-stranded DNA vectors in mammalian cells

We studied the ability of single-stranded DNA (ssDNA) to participate in targeted recombination in mammalian cells. A 5' end-deleted adenine phosphoribosyltransferase (aprt) gene was subcloned into M13 vector, and the resulting ssDNA and its double-stranded DNA (dsDNA) were transfected to APRT-Chinese hamster ovary cells with a deleted aprt gene. APRT+ recombinants with the ssDNA was obtained at a frequency of 3 x 10(-7) per survivor, which was almost equal to that with the double-stranded equivalent. Analysis of the genome in recombinant clones produced by ssDNA revealed that 12 of 14 clones resulted from correction of the deletion in the aprt locus. On the other hand, the locus of the remaining 2 was not corrected; instead, the 5' deletion of the vector was corrected by end extension, followed by integration into random sites of the genome. To exclude the possibility that input ssDNA was converted into its duplex form before participating in a recombination reaction, we compared the frequency of extrachromosomal recombination between noncomplementary ssDNAs, and between one ssDNA and one dsDNA, of two phage vectors. The frequency with the ssDNAs was 0.4 x 10(-5), being 10-fold lower than that observed with the ssDNA and the dsDNA, suggesting that as little as 10% of the transfected ssDNA was converted into duplex forms before the recombination event, hence 90% remained unchanged as single-stranded molecules. Nevertheless, the above finding that ssDNA was as efficient as dsDNA in targeted recombination suggests that ssDNA itself is able to participate directly in targeted recombination reactions in mammalian cells.     

Interference of DNA sequence divergence with precise recombinational DNA repair in mammalian cells

Studies done in prokaryotes and eukaryotes have indicated that DNA sequence divergence decreases the frequency of homologous recombination. To determine which step(s) of homologous recombination is sensitive to DNA sequence divergence in mammalian cells we have used an assay that does not rely on the recovery of functional products. The assay is based on the acquisition by homologous recombination of endogenous LINE-1 sequences by exogenous LINE-1 sequences. In parallel experiments, we introduced into mouse cells two gapped exogenous LINE-1 sequences, one from the mouse, L1Md-A2, and the other from the rat, L1Rn-3. Although L1Rn-3 is on average less than 85% homologous to the LINE-1 elements of the mouse, the frequency of homologous recombination with endogenous LINE-1 elements obtained with L1Rn-3 was the same as the one obtained with L1Md-A2 which is on average 95% homologous to the LINE-1 elements of the mouse. The endogenous LINE-1 sequences rescued by L1Rn-3 were 8-18% divergent from L1Rn-3 sequences, whereas those rescued by L1Md-A2 were 2-5% divergent from L1Md-A2 sequences. The gap which had been introduced into the exogenous LINE-1 sequences had been precisely repaired in 50% of the recombinants obtained with L1Md-A2. None of the L1Rn-3 recombinants showed precise gap repair.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cre-loxP-mediated DNA flip-flop in mammalian cells leading to alternate expression of retrovirally transduced genes

Cultured mesangial cells constitutively express alpha-smooth muscle actin (alpha-SMA), a marker of cellular activation. We unexpectedly found that tyrosine kinase pp60v-src, a known activator for a wide range of signalling cascades, suppressed the alpha-SMA expression in mesangial cells. The present study was conducted to elucidate molecular events involved in this phenomenon. Transfection with a reporter plasmid revealed that the serum response element (SRE), the cis-element required for alpha-SMA expression, was constitutively active in mesangial cells. When mesangial cells were transfected with pp60v-src, activity of both SRE and the alpha-SMA promoter was down-regulated. This was associated with depressed levels of phosphorylated extracellular signal-regulated kinases (ERKs), but not c-Jun N-terminal kinase. Selective inhibition of ERKs by PD098059 abrogated constitutive SRE activity, leading to suppressed alpha-SMA expression. These results uncovered a novel potential of pp60v-src for suppression of alpha-SMA via intervention in the ERK-SRE signalling pathway.     

Mechanistic studies of the translational elongation cycle in mammalian mitochondria

Polyclonal antibodies have been prepared against both components of the bovine liver mitochondrial translational elongation factor Tu and Ts complex (EF-Tu x Ts(mt)). The antibodies against EF-Tu(mt) cross-react somewhat with Escherichia coli EF-Tu and wheat germ EF-1alpha. The antibodies against EF-Ts(mt) cross-react little, if at all, with E. coli EF-Ts or with EF-Ts from Euglena gracilis chloroplasts. These polyclonal antibodies have been used to investigate the relative amounts of EF-Tu(mt) and EF-Ts(mt) in bovine liver mitochondria and in cultured cells. The results of this analysis suggest that there is a 1:1 ratio of EF-Tu(mt) to EF-Ts(mt) in mammalian mitochondria. Intermediate complexes formed during the elongation cycle of protein synthesis in bovine liver mitochondria have also been investigated. The EF-Tu x Ts(mt) complex is quite resistant to dissociation by guanine nucleotides. This complex will, however, dissociate in the presence of GTP and Phe-tRNA resulting in the formation of a ternary complex comparable to that observed in prokaryotes. Kinetic data suggest that the use of the ternary complex in chain elongation increases the rate of Phe-tRNA binding to ribosomes, suggesting that it is a true intermediate in the elongation cycle. Sucrose gradient analysis indicates that the binding of EF-Tu(mt) to ribosomes can be detected in the presence of Phe-tRNA and a non-hydrolyzable analog of GTP. These results suggest that, in contrast to previous thinking, the basic features of the elongation cycle in mammalian mitochondria are quite similar to those in prokaryotes.     

A nuclear membrane-associated DNA complex in cultured mammalian cells capable of synthesizing DNA in vitro

A DNA-nuclear membrane complex has been isolated by two different methods from the nuclei of cultured mouse fibroblast (3T3) cells. One method, utilizing the detergent sarkosyl (sodium lauroyl sarkosinate), yields a DNA-nuclear membrane complex (the M band), which contains virtually all of the DNA in the nuclei. However, treatment of the M band by sonication, vortexing, or freeze-thaw reduces the amount of DNA in the complex by approximately 50-80%, depending upon the phase of the cell cycle from which the complex was extracted. The remaining DNA is tightly bound to the nuclear membrane and resists further shearing procedures. Over 90% of the choline-labeled phospholipid present in nuclei is also found in these sheared M bands. The percentage of DNA associated with the nuclear membrane varies during the cell cycle and correlates well with the onset, continuation, and cessation of DNA synthesis. Thus, although DNA-membrane complexes can be detected throughout the cell cycle, the percentage of DNA bound to membrane increases during late G1 and S and decreases during G2. In addition, there are distinct qualitative differences in the type of DNA present in the membrane fraction, with a more highly d(A-T) rich DNA being present in confluent (G0) cells than in cells during the S phase. This d(A-T) rich DNA may be related to the mouse satellite DNA identified by others. The M band can be separated into two DNA-nuclear membrane subfractions by centrifugation through a continuous sucrose gradient. The relative proportions of these two subfractions depend upon the percentage of sarkosyl present in the M band prior to centrifugation, with complete removal of sarkosyl resulting in a very large increase in the sedimentation velocity of the complex and in the formation of only one fraction. Evidence that this is a complex of DNA with membrane is given by the finding that DNA is dissociated from the complex with Pronase, deoxycholate, or high levels of sarkosyl. Removal of virtually all of the DNA with DNase from this rapidly sedimenting complex does not dissociate any of the phospholipid which still sediments rapidly as a single band. A second method, which yields a DNA-membrane fraction from nuclei, utilizes sedimentation of lysed nuclei to equilibrium in CsCl density gradients. This low-density CsCl fraction contains only 10-15% of the total DNA, but contains most of the nascent DNA, which may be chased into a membrane-free fraction. The DNA-membrane fraction from CsCl gradients possesses properties in common with the M-band fraction and can be converted into an M band. DNA membrane complexes from sucrose gradients, as well as the crude M-band preparation and a non-membrane-associated DNA fraction from nuclei can synthesize DNA in vitro without the addition of an external DNA template or DNA polymerase. In contrast to the activity in the non-membrane-associated DNA fraction, the membrane-associated polymerase activity is strongly stimulated by adenosine triphosphate and is unaffected by ethidium bromide...     

Induction of oxidative DNA damage by ferric iron in mammalian cells

Ferric nitrilotriacetate (Fe-NTA) and ferric citrate (Fe-citrate) were compared with respect to their potential to induce oxidative DNA damage in V79 Chinese hamster cells. DNA base modifications, including 8-hydroxyguanine (7,8-dihydro-8-oxoguanine), were quantified by the frequency of lesions recognized by the bacterial Fpg protein (formamidopyrimidine-DNA glycosylase) in combination with the alkaline unwinding assay. Fe-NTA induced oxidative DNA damage in a time- and dose-dependent manner, yielding significant increases in Fpg-sensitive sites above background after incubation for 24 or 48 h with 500 and 250 microM respectively. At both time points the frequency of DNA base modifications exceeded the number of DNA strand breaks. In contrast, neither DNA strand breaks nor Fpg-sensitive sites were detected after treatment with Fe-citrate at concentrations up to 2 microM for 24 or 48 h; this inactivity of Fe-citrate was independent of the molar ratio of iron to ligand (1:1, 1:2, 1:10 or 1:20). The results indicate that the cellular damage induced by ferric iron depends strongly on the actual complex applied, possibly due to differences in the intracellular distribution, which in turn may affect the availability of iron for redox reactions at or in close proximity to the DNA.     

Proteins that participate in nucleotide excision repair of DNA in mammalian cells

The most versatile strategy for repair of damage to DNA, and the main process for repair of UV-induced damage, is nucleotide excision repair. In mammalian cells, the complete mechanism involves more than 20 polypeptides, and defects in many of these are associated with various forms of inherited disorders in humans. The syndrome xeroderma pigmentosum (XP) is associated with mutagen hypersensitivity and increased cancer frequency, and studies of the nucleotide excision repair defect in this disease have been particularly informative. Many of the XP proteins are now being characterized. XPA binds to DNA, with a preference for damaged base pairs. XPC activity is part of a protein complex with single-stranded DNA binding activity. The XPG protein is a nuclease.     

Mismatch repair of heteroduplex DNA intermediates of extrachromosomal recombination in mammalian cells

Previous work indicated that extrachromosomal recombination in mammalian cells could be explained by the single-strand annealing (SSA) model. This model predicts that extrachromosomal recombination leads to nonconservative crossover products and that heteroduplex DNA (hDNA) is formed by annealing of complementary single strands. Mismatched bases in hDNA may subsequently be repaired to wild-type or mutant sequences, or they may remain unrepaired and segregate following DNA replication. We describe a system to examine the formation and mismatch repair of hDNA in recombination intermediates. Our results are consistent with extrachromosomal recombination occurring via SSA and producing crossover recombinant products. As predicted by the SSA model, hDNA was present in double-strand break-induced recombination intermediates. By placing either silent or frameshift mutations in the predicted hDNA region, we have shown that mismatches are efficiently repaired prior to DNA replication.     

Nucleosome assembly during complementary DNA strand synthesis in extracts from mammalian cells

Circular single-stranded phage M13 DNA is used as a template for complementary strand synthesis in cytosolic extracts from proliferating HeLa cells. DNA synthesis is initiated by one or maximally two priming events and typically leads to covalently closed double-stranded reaction products. When carried out in the presence of the nuclear chromatin assembly factor CAF-1, complementary strand synthesis is accompanied by nucleosome assembly. This novel system is very useful for the study of basic biochemical aspects concerning the assembly of nucleosomes. The activity of CAF-1 completely depends on complementary strand synthesis and acts stoichiometrically to promote the assembly of nucleosomes in a noncooperative manner. Apparently, CAF-1 activity is coupled to DNA synthesis via a structural feature of replicating DNA, most likely its partial single strandedness.     

Chromosomal response of insect and mammalian cells to streptonigrin: a comparative study

Respiratory syncytial virus (RSV) infections are characterized by upper or lower respiratory tract symptoms including bronchiolitis and pneumonia. Apnoea may be the first sign of disease in children with RSV infection. The aims of this study were the identification of independent risk factors for RSV associated apnoea and the prediction of the risk for mechanical ventilation in children with RSV associated apnoea. Medical records of children younger than 12 months of age admitted with RSV infection between 1992 and 1995 to the Sophia Children's Hospital, were reviewed. Demographic parameters, clinical features and laboratory parameters (SaO2, pCO2 and pH) were obtained upon admission and during hospitalization. Children with and without apnoea were compared using univariate and multivariate logistic and linear regression analysis. One hundred and eighty-five patients with RSV infection were admitted of whom 38 (21%) presented with apnoea. Patients with apnoea were significantly younger, had a significantly lower temperature, higher pCO2 and lower pH and had on chest radiographs also more signs of atelectasis. The number of patients admitted to the ICU because of mechanical ventilation and oxygen administration was significantly higher in children with RSV associated apnoea. Apnoea at admission was a strong predictor for recurrent apnoea. The relative risk for mechanical ventilation increased with the number of episodes of apnoea: 2.4 (95% CI 0.8-6.6) in children with one episode of apnoea (at admission) versus 6.5 (95% CI 3.3-12.9) in children with recurrent episodes of apnoea. CONCLUSIONS: Age below 2 months is the strongest independent risk factor for RSV associated apnoea. Apnoea at admission increases the risk for recurrent apnoea. The risk for mechanical ventilation significantly increases in children who suffer from recurrent apnoea.     

Cytogenetic effect of ultraviolet rays in mammalian cells at the DNA synthesis stage

The frequency of chromosome aberrations induced by UV light at various wavelengths in the primary culture of mouse embryonic fibroblasts during the S-phase was studied. The aberration frequency is wavelength-dependent and reaches a maximum at 265 nm. The action spectrum for the chromosome aberrations determined at 254, 265, 280 and 302 nm closely conforms to the absorption spectra of thymidine. The value of caffeine potentiation was the same for 265 and 280 nm UV-induced aberrations. This indicates that primary chromosome damages and their transformation in cells are similar at these two wavelengths. The data obtained suggest that the formation of DNA cross-links following thymine dimerization is the first step in the formation of UV-induced chromosome aberrations in mammalian cells at the S phase.     

Increased camptothecin toxicity induced in mammalian cells expressing Saccharomyces cerevisiae DNA topoisomerase I

The yeast Saccharomyces cerevisiae has been useful in establishing the phenotypic effects of specific mutations on the enzymatic activity and camptothecin sensitivity of yeast and human DNA topoisomerase I. To determine whether these phenotypes were faithfully reiterated in higher eukaryotic cells, wild-type and mutant yeast Top1 proteins were epitope-tagged at the amino terminus and transiently overexpressed in mammalian COS cells. Camptothecin preferentially induced apoptosis in cells expressing wild-type eScTop1p yet did not appreciably increase the cytotoxic response of cells expressing a catalytically inactive (eSctop1Y727F) or a catalytically active, camptothecin-resistant eSctop1vac mutant. Using an epitope-specific antibody, immobilized precipitates of eScTop1p were active in DNA relaxation assays, whereas immunoprecipitates of eScTop1Y727Fp were not. Thus, the enzyme retained catalytic activity while tethered to a support. Interestingly, the mutant eSctop1T722A, which mimics camptothecin-induced cytotoxicity in yeast through stabilization of the covalent enzyme-DNA intermediate, induced apoptosis in COS cells in the absence of camptothecin. This correlated with increased DNA cleavage in immunoprecipitates of eScTop1T722Ap, in the absence of the drug. The observation that the phenotypic consequences of expressing wild-type and mutant yeast enzymes were reiterated in mammalian cells suggests that the mechanisms underlying cellular responses to DNA topoisomerase I-mediated DNA damage are conserved between yeast and mammalian cells.     

Rapid evolution of translational control mechanisms in RNA genomes

We have introduced 13 base substitutions into the coat protein gene of RNA bacteriophage MS2. The mutations, which are clustered ahead of the overlapping lysis cistron, do not change the amino acid sequence of the coat protein, but they disrupt a local hairpin, which is needed to control translation of the lysis gene. The mutations decreased the phage titer by four orders of magnitude but, upon passaging, the virus accumulated suppressor mutations that raised the fitness to almost wild-type level. Analysis of the pseudorevertants showed that the disruption of the local hairpin, controlling expression of the lysis gene, had apparently been so complete that its restoration by chance mutations could not be achieved. Instead, alternative foldings initiated by the starting mutations were further stabilized and optimized. Strikingly, in the pseudorevertants analyzed, translational control of the lysis gene had been restored. This feat was accomplished by, on average, four suppressor mutations that generally occurred at codon wobble positions. We also introduced 11 mutations in a hairpin more upstream in the coat protein gene and not implicated in lysis control. Here the titer dropped by three logs, but pseudorevertants with a fitness close to wild-type were soon generated. These pseudorevertants again were the result of the optimization of alternative foldings induced by the mutations. The transition of the secondary structure from wild-type to pseudorevertant could be visualized by structure probing. Our study shows that the folding of the RNA is an important phenotypic property of RNA viruses. However, its distortion can easily be overcome by optimizing alternative base-pairings. These new structures are not qualitatively equivalent to the original one, since they do not successfully compete with the wild-type.     

Effect of split doses of N-methyl-N-nitrosourea on DNA repair synthesis in cultured mammalian cells

DNA repair was measured in human fibroblasts, mouse C3H 10 T 1/2 fibroblsts and rat hepatocytes by the non-semi-conservative incorporation of [3H]-TdR during DNA repair synthesis using liquid scintillation techniques. Confluent monolayers of these cells grown on cover slips were exposed to split doses (125 or 250 microgram/ml) of the mutagenic and carcinogenic alkylating agent MNU and DNA repair synthesis compared with that produced by a single dose (500 microgram/ml). No significant difference in DNA repair capacity was detected in the three cell lines treated with a single dose or split doses of MNU.     

1,N6-ethenodeoxyadenosine, a DNA adduct highly mutagenic in mammalian cells

1,N6-Ethenodeoxyadenosine (epsilon dA) is one of four exocyclic DNA adducts produced by chloroethylene oxide and chloroacetaldehyde, reactive metabolites of vinyl chloride, a human carcinogen. epsilon dA has also been detected in DNA of the liver of humans and untreated animals, suggesting its formation from endogenous sources. The mutagenic potential of epsilon dA was studied using a single-stranded shuttle vector system in several E. coli strains and in simian kidney cells (COS7). This vector system enables quantitative analysis of translesional synthesis past a site-specifically placed DNA adduct in both hosts owing to the lack of the complementary strand. In experiments with five strains of E. coli, a very limited number of targeted mutations (one epsilon dA-->T, one epsilon dA-->dC, and two epsilon dA-->single base deletion) were observed among 756 transformants in hosts preirradiated with UV; no targeted mutations were observed among 563 transformants in nonirradiated hosts. These results indicate that nonmutagenic base pairings of epsilon dA:T are the almost exclusive events in E. coli. In COS7 cells, the frequency of targeted mutations was 70%, consisting of epsilon dA-->dG (63%), epsilon dA-->T (6%), and epsilon dA-->dC (1%), indicating that the insertion of dCMP opposite the adduct is predominant. When compared with the results for 3,N4-ethenodeoxycytidine (epsilon dC), which was studied previously in the same system [Moriya et al. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 11899-11903], the results of this study indicate that the intrinsic mutagenic potency of epsilon dA is comparable to that of epsilon dC in mammalian cells.