Mutagenic effects of gamma-rays on plasmid DNA in Escherichia coli

A purified and dried DNA of plasmid pKO482 (galK+) is 10 times more resistant to the inactivating action of 60Co-gamma-rays than that of lambda phage. gamma-Irradiation of the plasmid DNA induces forward mutations of galK, the frequency of which increases linearly with the dose. The efficiency of the mutagenic action of gamma-rays on the plasmid galK locus is 10(-12) per 1 rad and per 1 base pair. The mutagenic effect of gamma-radiation but slightly depends upon bacterial recA+ gene and upon the SOS-repair system induced by UV-irradiation of the recipient cells. It is assumed that the premutational lesions induced in the purified DNA by the direct effect of gamma-radiation are fixed into mutations by misreplication.     

Lethal and mutagenic effects of 8-methoxypsoralen-induced lesions on plasmid DNA

The genotoxic effect of 8-methoxypsoralen damages (monoadducts and crosslinks) on plasmid DNA was studied. pBR322 DNA was treated with several concentrations of 8-methoxypsoralen plus fixed UVA light irradiation. After transformation into E. coli cells with different repair capacities (uvrA, recA and wild-type), plasmid survival and mutagenesis in ampicillin- and tetracycline-resistant genes were analysed. Results showed that crosslinks were extremely lethal in all 3 strains; indeed, it seemed that they were not repaired even in proficient bacteria. Monoadducts were also found to be lethal although they were removed to some extent by the excision-repair pathway (uvrA-dependent). Damaged plasmid DNA appeared to induce mutagenic repair, but only in the wild-type strain. In order to study the influence of the SOS response on plasmid recovery, preirradiation of the host cells was also performed. Preirradiation of the uvrA or wild-type strains significantly increased plasmid recovery. Consistent with the expectations of SOS repair, no effect was observed in preirradiated recA cells. Plasmid recovery in the excision-deficient strain was mainly achieved by the mutagenic repair of some fraction of the lesions, probably monoadducts. The greatest increase in plasmid recovery was found in the wild-type strain. This likely involved the repair of monoadducts and some fraction of the crosslinks. We conclude that repair in preirradiated repair-proficient cells is carried out mainly by an error-free pathway, suggesting enhancement of the excision repair promoted by the induction of SOS functions.     

Biological effects of stevioside on the survival of Escherichia colistrains and plasmid DNA

Stevioside is widely used daily in many countries as a non-caloric sugar substitute. Its sweetening power is higher than that of sucrose by approximately 250–300 times, being extensively employed as a household sweetener, or added to beverages and food products. The purpose of this study was to ascertain stevioside genotoxic and cytotoxic potentiality in different biological systems, as its use continues to increase. Agarose gel electrophoresis and bacterial transformation were employed to observe the occurrence of DNA lesions. In addition to these assays, Escherichia coli strains were incubated with stevioside so that their survival fractions could be obtained. Results show absence of genotoxic activity through electrophoresis and bacterial transformation assays and drop of survival fraction of E. coli strains deficient in rec A and nth genes, suggesting that stevioside (i) is cytotoxic; (ii) could need metabolization to present deleterious effects on cells; (iii) is capable of generating lesions in DNA and pathways as base excision repair, recombination and SOS system would be important to recover these lesions.     

Sterilizing filtration of plasmid DNA: effects of plasmid concentration, molecular weight, and conformation

Plasmid DNA purification development has been driven by the increased need for large quantities of highly purified, sterile plasmid DNA for clinical studies. Detailed characterization and development of the terminal sterile filtration process step is often limited due to time constraints and the scarcity of sufficient quantities of purified plasmid. However, the large size of the plasmid molecule and variations in conformation can lead to significant yield losses if this process step is not optimized. In this work, the gradual pore-plugging model of flow decay was found to be valid for plasmid DNA by using an ultra scaledown apparatus (1-4 cm(2) filter area). Filtration capacity was found to be insensitive to pressure. Multiple filter types were screened and both source and composition of materials were found to affect filter capacity dramatically. The filter capacity for plasmid was improved by increasing plasmid concentrations as well as by modifying buffer conditions to reduce the apparent size of the plasmid. Filtration capacities varied over a greater than 2 log range when plasmids with sizes ranging from 5.5 to 11 kb and supercoiled plasmid content of 55-95% were explored. Larger plasmids and feeds with lower supercoiled contents led to reduced capacities. These results can be used to define conditions for scale-up of plasmid sterile filtration, as evidenced by processing a 30 g lot using a filtration area of 1,000 cm(2), with a 96% yield, based on filtration capacity data from 4 cm(2) test filters.     

Further studies on the lethal and mutagenic effects of 8-methoxypsoralen-induced lesions on plasmid DNA

Our previous results on the genotoxic effect of 8-methoxypsoralen-induced lesions on pBR322 suggested an important involvement of an inducible error-free repair pathway in the repair of plasmid lesions. We present herein further results obtained in order to explore that possibility, together with a more general report on the subject. pBR322 treated with increasing concentrations of 8-MOP plus fixed UVA light irradiation was used to transform several E. coli strains differing in their repair capacities, and plasmid survival and mutagenesis were determined. Survival results suggested that crosslinks were completely lethal in pBR322 whereas monoadducts were partially removed from plasmid DNA mainly through an error-free excision pathway. A mutagenic repair pathway did not show a significant contribution to the total repair process. Cell preirradiation stimulated plasmid recovery in recA+ strains, including the umuC strain, thus confirming our previous results indicating that an inducible error-free repair had occurred. Globally, our results showed a strong requirement on the excision pathway for the repair of psoralen-damaged plasmid DNA. In contrast, the recA dependent pathway was needed only for SOS induction. After a theoretical correction of the data for estimating the effect only due to 8-MOP adducts, a different pattern of repair mechanisms appeared to be involved.     

DNA inversion on conjugative plasmid pVT745

Plasmid pVT745 from Actinobacillus actinomycetemcomitans strain VT745 can be transferred to other A. actinomycetemcomitans strains at a frequency of 10(-6). Screening of transconjugants revealed that the DNA of pDMG21A, a pVT745 derivative containing a kanamycin resistance gene, displayed a structural rearrangement after transfer. A 9-kb segment on the plasmid had switched orientation. The inversion was independent of RecA and required the activity of the pVT745-encoded site-specific recombinase. This recombinase, termed Inv, was highly homologous to invertases of the Din family. Two recombination sites of 22 bp, which are arranged in opposite orientation and which function as DNA crossover sequences, were identified on pVT745. One of the sites was located adjacent to the 5' end of the invertase gene, inv. Inversion of the 9-kb segment on pVT745 derivatives has been observed in all A. actinomycetemcomitans strains tested except for the original host, VT745. This would suggest that a host factor that is either inactive or absent in VT745 is required for efficient recombination. Inactivation of the invertase in the donor strain resulted in a 1,000-fold increase in the number of transconjugants upon plasmid transfer. It is proposed that an activated invertase causes the immediate loss of the plasmid in most recipient cells after mating. No biological role has been associated with the invertase as of yet.     

Effect of DNA topology on plasmid DNA repair in vivo

Escherichia coli nucleotide excision repair (NER) is responsible for removing bulky DNA adducts by dual incisions of the UvrABC endonuclease. Although the activity of the UvrAB complex which can induce DNA conformational change is employed in NER, the involvement of DNA topology and DNA topoisomerases remains unclear. We examined the effect of topoisomerase inhibitions on a NER in vivo system. The repair analysis of intracellular plasmid revealed that the DNA damage on positive supercoils generated by gyrase inhibition remained unrepaired, whereas the DNA damage was repaired in topoisomerase I mutants. These results suggest that DNA topology affects the NER process and the removal of positive supercoils by gyrase is vital for the efficiency of the E. coli NER system.     

Monte Carlo simulation of DNA strand-break induction in supercoiled plasmid pBR322 DNA from indirect effects

We present a new Monte Carlo simulation code system (DBREAK) of the detailed events that occur when ionizing radiation interacts with water and DNA molecules. The model treats the initial energy deposition by radiation, the formation of chemically active species, subsequent diffusion-controlled chemical reactions, and induction of DNA strand breaks. DBREAK assumes one-hit single-strand break (SSB) and two-hit double-strand break (DSB) mechanisms. A high-resolution model of plasmid DNA structure has been introduced. The calculated results are compared with the results of previously performed experiments of the same type. Under aerobic conditions, 89.4% of the DNA damage was attributed to OH-radical and 10.5% and 0.1% to e^– _aq and H, respectively. We also compared the differences between liquid-water track structure and gas-phase-water track structure. The calculated yield of SSBs by liquid-water track structure exceeded that of gas-phase-water track structure by a factor of 1.2. Received: 13 February 1997 / Accepted in revised form: 26 August 1997     

PCR-mediated deletion of plasmid DNA

The PCR-mediated plasmid DNA deletion method is a simple approach to delete DNA sequences from plasmids using only one round of PCR, with two primers, and without ligation or purification prior to in vivo recombination. By using only PCR, the method is sequence independent and, as shown in this study, is applicable to various sizes of plasmids and deletions using minimal primer design.     

In vivo plasmid DNA electrotransfer

In vivo electrotransfer is a physical technique for gene delivery in various mammalian tissues, which involves the injection of plasmid DNA into a target tissue and administration of an electric field. Its ease of performance, as well as recent understanding of its mechanism and applications to different mammalian tissues such as skeletal muscle, liver, brain and tumors, makes it a powerful technique. It could be used in gene therapy and as a laboratory tool to study gene functions.     

Affinity adsorption of plasmid DNA

The development of a protein-mediated dual functional affinity adsorption of plasmid DNA is described in this work. The affinity ligand for the plasmid DNA comprises a fusion protein with glutathione-S-transferase (GST) as the fusion partner with a zinc finger protein. The protein ligand is first bound to the adsorbent by affinity interaction between the GST moeity and gluthathione that is covalently immobilized to the base matrix. The plasmid binding is then enabled via the zinc finger protein and a specific nucleotide sequence inserted into the DNA. At lower loadings, the binding of the DNA onto the Fractogel, Sepharose, and Streamline matrices was 0.0078 +/- 0.0013, 0.0095 +/- 0.0016, and 0.0080 +/- 0.0006 mg, respectively, to 50 microL of adsorbent. At a higher DNA challenge, the corresponding amounts were 0.0179 +/- 0.0043, 0.0219 +/- 0.0035, and 0.0190 +/- 0.0041 mg, respectively. The relatively constant amounts bound to the three adsorbents indicated that the large DNA molecule was unable to utilize the available zinc finger sites that were located in the internal pores and binding was largely a surface adsorption phenomenon. Utilization of the zinc finger binding sites was shown to be highest for the Fractogel adsorbent. The adsorbed material was eluted with reduced glutathione, and the eluted efficiency for the DNA was between 23% and 27%. The protein elution profile appeared to match the adsorption profiles with significantly higher recoveries of bound GST-zinc finger protein.     

Rapid screening for plasmid DNA

Summary A procedure is described for demonstrating plasmid DNA and its molecular weight, based on rate zonal centrifugation of unlabelled DNA in neutral sucrose gradients containing a low concentration of ethidium bromide. Each DNA species is then visualized as a discrete fluorescent band when the centrifuge tube is illuminated with ultra-violet light. Plasmids exist as closed circular and as relaxed circular molecules, which sediment separately, but during preparation of lysates, closed circular molecules are nicked so that each plasmid forms only a single band of relaxed circles within the gradient.     

Effect of shear on plasmid DNA in solution

This study was designed to evaluate the effect of shear on the supercoiled circular (SC) form of plasmid DNA. The conditions chosen are representative of those occurring during the processing of plasmid-based genes for gene therapy and DNA vaccination. Controlled shear was generated using a capillary rheometer and a rotating disk shear device. Plasmid DNA was tested in a clarified alkaline lysate solution. This chemical environment is characteristic of the early stages of plasmid purification. Quantitative data is reported on shear degradation of three homologous recombinant plasmids of 13, 20 and 29 kb in size. Shear sensitivity increased dramatically with plasmid molecular weight. Ultrapure plasmid DNA redissolved in 10 mM Tris/HCl, 1 mM EDTA pH 8 (TE buffer) was subjected to shear using the capillary rheometer. The shear sensitivity of the three plasmids was similar to that observed for the same plasmids in the clarified alkaline lysate. Further experiments were carried out using the 20 kb plasmid and the rotating disk shear device. In contrast with the capillary rheometer data, ultrapure DNA redissolved in TE buffer was up to eight times more sensitive to shear compared to plasmid DNA in the clarified alkaline lysate. However, this enhanced sensitivity decreased when the ionic strength of the solution was raised by the addition of NaCl to 150 mM. In addition, shear damage was found to be independent of plasmid DNA concentration in the range from 0.2 7g/ml to 20 7g/ml. The combination of shear and air-liquid interfaces caused extensive degradation of the plasmid DNA. The damage was more evident at low ionic strength and low DNA concentration. These findings show that the tertiary structure of plasmid DNA can be severely affected by shear forces. The extent of damage was found to be critically dependent on plasmid size and the ionic strength of the environment. The interaction of shear with air-liquid interfaces shows the highest potential for damaging SC plasmid DNA during bioprocesses.     

Conservation of IS66 homologue of octopine Ti plasmid DNA in Rhizobium fredii plasmid DNA

Summary DNA sequences homologous to the T-DNA region of the octopine Ti plasmid from Agrobacterium tumefaciens are found in various fast-growing Rhizobium fredii strains. The largest fragment (BamHI fragment 2) at the right-boundary region of the core T-DNA hybridizes to more than one plasmid present in R. fredii. However, one smaller fragment (EcoRI fragment 19a) adjacent to the core T-DNA shows homology only with the plasmid carrying the symbiotic nitrogen-fixation genes (pSym). Hybridization data obtained with digested R. fredii USDA193 pSym DNA suggests that the homology is mainly with two HindIII fragments, 1.7 kb and 8.8 kb in size, of the plasmid. The 1.7 kb HindIII fragment also hybridizes to two regions of the virulence plasmid of A. tumefaciens, pAL1819, a deletion plasmid derived from the octopine Ti plasmid, pTiAch5. Hybridization studies with an insertion element IS66 from A. tumefaciens indicate that the 1.7 kb HindIII fragment of R. fredii plasmid, homologous to the T-DNA and the virulence region of Ti plasmid, is itself an IS66 homologue.