The tumor suppressor p53 can reduce stable transfection in the presence of irradiation

The tumor suppressor p53 is believed to play an essential role in maintaining genome stability. Although it is currently unknown how p53 is involved in this important biological safeguard, several previous publications indicate that p53 can help to maintain genome integrity through the recombination-mediated DNA repair process. The integration of linearized plasmid DNA into the host chromosome utilizes the same repair process, and the frequency can be measured by clonogenic assays in which cells that were stably transfected by plasmid integration can be scored by their colony-forming abilities. To gain insight into whether p53 has a direct role in plasmid integration into the host chromosome, we determined the frequency of stable transfection with CHO cells expressing either wild-type or mutant p53 in the presence and absence of irradiation. We found that low-dose irradiation (50 to 100 cGy) increased stable transfection frequencies in CHO cells regardless of their p53 status. However, the increase of transfection frequency was significantly lower in CHO cells expressing wild-type p53. Our data thus suggest that wild-type p53 can suppress plasmid DNA integration into the host genome. This p53 function may play a direct and significant role in maintaining genome stability.     

Involvement of a plasmid in exopolysaccharide production by Klebsiella oxytoca

A Klebsiella oxytoca isolate which can produce significant levels of an exopolysaccharide using whey as a growth substrate has been reported. The plasmid profile of this isolate was shown to be different from that of the non-exopolysaccharide-producing K. oxytoca ATCC 43863. Irreversible curing of the single plasmid in the K. oxytoca isolate was achieved using 30 g acriflavin/ml. The ability to produce the exopolysaccharide was lost with the curing of the plasmid (parent strain produced a medium viscosity of 1260 cP at 1 s^–1 compared to 1.6 cP at 200 s^–1 produced by the cured strain). However, the ability to metabolize lactose was not significantly affected by curing, and both the parent and the cured strain produced similar levels of viable cells (~10⁹ cfu/ml) after 62 h growth on lactose-rich medium. The exopolysaccharide-producing ability of the isolate was stable for at least 139 generations.     

Plasmid-mediated florfenicol and ceftriaxone resistance encoded by the floR and bla(CMY-2) genes in Salmonella enterica serovars Typhimurium and Newport isolated in the United States

Multidrug resistance plasmids carrying the bla(CMY-2) gene have been identified in Salmonella enterica serovars Typhimurium and Newport from the United States. This gene confers decreased susceptibility to ceftriaxone, and is most often found in strains with concomitant resistance to ampicillin, chloramphenicol, streptomycin, sulfamethoxazole and tetracycline. The bla(CMY-2)-carrying plasmids studied here were shown to also carry the florfenicol resistance gene, floR, on a genetic structure previously identified in Escherichia coli plasmids in Europe. These data indicate that the use of different antimicrobial agents, including phenicols, may serve to maintain multidrug resistance plasmids on which extended-spectrum cephalosporin resistance determinants co-exist with other resistance genes in Salmonella.     

Isolation and sequencing of the replication region of plasmid pBFp1 isolated from a marine biofilm

A 24 kb plasmid, pBFp1, encoding mercury resistance was previously isolated from a marine biofilm. Isolation and sequencing of a 4280 bp DNA fragment containing the plasmid replicon (rep-pBFp1) revealed a putative open reading frame encoding a RepA protein and an oriV-like region containing an A+T rich sub-region, iterons, and DnaA boxes. Sequence comparisons showed significant similarities to the incW plasmid pSa both for the RepA amino acid sequence and in the iteron DNA sequence. Plasmid pBFp1 was also shown to be incompatible with pSa in standard incompatibility testing. A probe from the repA gene of pBFp1 was further made and tested on a collection of plasmids exogenously isolated from marine habitats in a previous study.     

Polyelectrolyte complexes as a tool for purification of plasmid DNA. Background and development

The demand for highly purified plasmids in gene therapy and plasmid-based vaccines requires large-scale production of pharmaceutical-grade plasmid. Plasmid DNA was selectively precipitated from a clarified alkaline lysate using the polycation poly(N,N'-dimethyldiallylammonium) chloride which formed insoluble polyelectrolyte complex (PEC) with the plasmid DNA. Soluble PECs of DNA with polycations have earlier been used for cell transformation, but now the focus has been on insoluble PECs. Both DNA and RNA form stable PECs with synthetic polycations. However, it was possible to find a range of salt concentration where plasmid DNA was quantitatively precipitated whereas RNA remained in solution. The precipitated plasmid DNA was resolubilised at high salt concentration and the polycation was removed by gel-filtration.     

Anaerobic and aerobic continuous cultures of<Emphasis Type="Italic"> Saccharomyces cerevisiae</Emphasis>: comparison of plasmid stability and<Emphasis Type="Italic"> EXG1</Emphasis> gene expression

Two bioreactor continuous cultures, at anaerobic and aerobic conditions, were carried out using a recombinant Saccharomyces cerevisiae strain that over-expresses the homologous gene EXG1. This recombinant system was used to study the effect of dissolved oxygen concentration on plasmid stability and gene over-expression. Bioreactor cultures were operated at two dilution rates (0.14 and 0.03 h^–1) to investigate the effect of other process parameters on EXG1 expression. Both cultures suffered severe plasmid instability during the first 16 generations. Segregational plasmid loss rate for the aerobic culture was two-fold that of the anaerobic operation. In spite of this fact, exo--glucanase activity at aerobic conditions was 12-fold that of the anaerobic culture. This maximal activity (30 U ml^–1) was attained at the lowest dilution rate when biomass reached its greatest value and glucose concentration was zero.     

Molecular classification of cancer types from microarray data using the combination of genetic algorithms and support vector machines

Simultaneous multiclass classification of tumor types is essential for future clinical implementations of microarray-based cancer diagnosis. In this study, we have combined genetic algorithms (GAs) and all paired support vector machines (SVMs) for multiclass cancer identification. The predictive features have been selected through iterative SVMs/GAs, and recursive feature elimination post-processing steps, leading to a very compact cancer-related predictive gene set. Leave-one-out cross-validations yielded accuracies of 87.93% for the eight-class and 85.19% for the fourteen-class cancer classifications, outperforming the results derived from previously published methods.     

Nitrite reduction in Paracoccus sp. is affected by a novel plasmid pYR1

Two relatively low-copy plasmids of 9 and 16 kb were found to comprise the extrachromosomal DNA of a Paracoccus strain. Reduction of nitrate by plasmid-cured cells resulted in a significant intermediate nitrite accumulation as compared to wild-type cells. By examining nitrate reduction by transformants containing one of the two plasmids, it was found that nitrite accumulation was influenced by the 9.0-kb plasmid, designated as pYR1. Subcloning analysis showed that a 1.8-kb fragment of this plasmid affected nitrite accumulation. Sequence analysis of this fragment revealed the presence of five open reading frames. One of the six deduced proteins showed a strong homology to ABC transporters.