Recombination between the dfrA12-orfF-aadA2 cassette array and an aadA1 gene cassette creates a hybrid cassette, aadA8b

Homologous recombination between closely related gene cassettes, such as aadA1 and aadA2, which are 89% identical, can create hybrid cassettes and hybrids of existing cassette arrays. A new cassette array, dfrA12-orfF-aadA8b, which was created by such a recombination event occurring within the aadA2 cassette in the dfrA12-orfF-aadA2 array, has been identified.     

Novel spectinomycin/streptomycin resistance gene, aadA14, from Pasteurella multocida

A novel spectinomycin/streptomycin resistance gene, designated aadA14, was detected on the mobilizable 5,198-bp plasmid pCCK647 from Pasteurella multocida. The aadA14 gene encodes an aminoglycoside adenylyltransferase of 261 amino acids. Sequence comparisons revealed that the AadA14 protein showed less than 60% identity to the AadA proteins known so far.     

Ultrasound-mediated gene delivery: kinetics of plasmid internalization and gene expression.

Sonoporation is an approach that can be used to transfer DNA or drugs into cells. However, very little is known about the mechanism of ultrasound-mediated membrane permeabilization. In this investigation, DNA transport post-sonoporation and the subsequent plasmid internalization and protein expression kinetics have been studied. Using a plasmid encoding for the green fluorescent protein (GFP), labelled or not with an intercalating agent (YOYO-1), it was found that, as compared to lipofection that requires endocytosis, sonoporation allowed a rapid and direct transfer of naked DNA into the cell cytoplasm probably via ultrasound-induced pores in the membrane. The kinetics of protein expression were significantly faster for sonoporation than for lipofection, the mechanism of which requires endocytosis. However, unprotected DNA in the cytoplasm could be degraded by resident cytosolic DNases, thereby decreasing ultrasound-mediated gene delivery efficiency.     

Emergence of aminoglycoside resistance genes aadA and aadE in the genus Campylobacter.

Resistance to streptomycin or spectinomycin or both in five Campylobacter coli strains, two Campylobacter jejuni strains, and a Campylobacter-like strain was studied by enzymatic assays and dot blot hybridization. Resistance was due to 6- or 3",9-aminoglycoside adenylyltransferases and to new types of phospho- and adenylyltransferases.     

Controlled plasmid gene transfer to murine renal carcinoma by hexadecylphosphocholine.

We report here that the anticancer drug hexadecylphosphocholine (HPC) can control plasmid DNA-mediated gene transfer to renal carcinoma following intratumoral administration. Significant improvement of gene expression levels could be achieved depending on HPC dose administered. Optimal concentration of HPC co-injected with plasmid DNA was found to be 0.2% (w/v) showing up to a 10-fold increase in reporter gene expression levels when compared to DNA administered alone. In vivo gene transfer activity of HPC was not affected by the nature of the diluent used, i.e. glucose-based or saline-based isotonic solutions. Although in vitro transfection activity of HPC formulations could not be evidenced, a liposome leakage assay revealed that HPC could significantly destabilize stable lipid membranes suggesting that a possible membrane permeation enhancer activity of HPC combined to the physical stress induced by the intratumor injection may facilitate plasmid DNA entry inside the cells resulting in increased gene expression. HPC/plasmid formulations represent new and attractive non-viral gene delivery systems with potential in cancer gene therapy and vaccination.     

Extrachromosomal plasmid vectors for gene therapy

Extrachromosomal DNA is becoming widely utilized as a gene therapy vector. Plasmid DNA offers multiple advantages over viral gene therapy vectors, including large packaging capacity, stability without integration and reduced toxicity. Furthermore, plasmid DNA can be delivered to many different tissues, using a variety of delivery techniques currently being developed. This review will discuss the advantages of extrachromosomal DNA as a gene therapy vector, highlighting recent advances and successes in its use in vivo.     

Nosocomial spread of an amikacin resistance gene on both a mobilized, nonconjugative plasmid and a conjugative plasmid.

Resistance to amikacin among members of the family Enterobacteriaceae at a hospital in Venezuela rose from 2% in 1979 to 5% in 1984 and 10% in 1985 as amikacin usage rose 20-fold to exceed gentamicin usage. Resistance to gentamicin remained at 25 to 27%. We examined the plasmids from 21 isolates obtained in 1984 and 1985. Nine of eleven in 1984 and three of ten in 1985 carried aacA and sul on a 3.8-kb BamHI fragment of pBWH300, a 10.4-kb nonconjugative plasmid that had been mobilized into strains of six species by at least two different coresident conjugative plasmids. Six 1985 isolates of two species carried these genes on a similar BamHI fragment of the 104-kb conjugative plasmid pBWH303. One isolate in 1984 and one in 1985 carried the 69-kb conjugative plasmid pBWH301, which had aacA as the promoter-proximal gene of an operon that also encompassed the cat and aadB resistance genes. Another conjugative plasmid, pBWH302, was found in a single isolate. It carried a different aacA allele on the functional transposon Tn654, which appeared to be closely related to Tn1331, a transposon previously isolated in Argentina and Chile. Increased selection may thus have led to dissemination of an endemic aacA allele on two endemic plasmids, one spread by mobilization, with occasional intrusion of additional aacA alleles from outside.     

pCOR: a new design of plasmid vectors for nonviral gene therapy.

A totally redesigned host/vector system with improved properties in terms of safety has been developed. The pCOR plasmids are narrow-host range plasmid vectors for nonviral gene therapy. These plasmids contain a conditional origin of replication and must be propagated in a specifically engineered E. coli host strain, greatly reducing the potential for propagation in the environment or in treated patients. The pCOR backbone has several features that increase safety in terms of dissemination and selection: (1) the origin of replication requires a plasmid-specific initiator protein, pi protein, encoded by the pir gene limiting its host range to bacterial strains that produce this trans-acting protein; (2) the plasmid's selectable marker is not an antibiotic resistance gene but a gene encoding a bacterial suppressor tRNA. Optimized E. coli hosts supporting pCOR replication and selection were constructed. High yields of supercoiled pCOR monomers were obtained (100 mg/l) through fed-batch fermentation. pCOR vectors carrying the luciferase reporter gene gave high levels of luciferase activity when injected into murine skeletal muscle.     

Angiographically guided utero-placental gene transfer in rabbits with adenoviruses, plasmid/liposomes and plasmid/polyethyleneimine complexes.

We examined the feasibility of gene transfer to rabbit placenta using adenoviruses, plasmid/liposomes and plasmid/polyethyleneimine (PEI) complexes. Pregnant New Zealand White rabbits (n = 17) were anesthetized and local gene transfer was done via a catheter inserted in uterine arteries under direct angiographic control. Either nuclear targeted LacZ adenoviruses (1.0 x 10(10) p.f.u.), nuclear targeted LacZ plasmid (500 microg)/liposome (DOTMA:DOPE 1:1) complexes or nuclear targeted LacZ plasmid (250 microg)/PEI (25 kDa) complexes (charge ratio +/-4) were used. Animals were killed 3 days later and detection of the transgene expression was done by X-gal staining and RT-PCR. Adenovirus-mediated gene transfer resulted in a high transfection efficiency (34 +/- 10%) in placental trophoplastic cells. Very little, if any, transfection was seen in fetal membranes. Plasmid/liposomes and plasmid/PEI complexes led to a very low (<0.01%) transfection efficiency in trophoblastic cells, but some transfection was seen in fetal membranes. A total of 25 fetuses were analyzed for the presence of transgene at the time of death. In most fetuses expression of the LacZ gene was below the sensitivity of the X-gal staining, but expression was detected by PCR in 50%, 50% and 42% of the analyzed fetuses after adenoviral, plasmid/PEI and plasmid/liposome gene transfer, respectively. No major inflammatory changes were present in the transfected placentas as analyzed by general histology and macrophage- and T cell-specific immunostainings. We conclude that catheter-mediated intravascular gene transfer with adenoviruses can be used for the transfection of placental trophoplastic cells, but plasmid complexes are inefficient for this purpose. However, selective angiographically guided gene transfer also led to leakage of the vector to fetuses. Therefore, if gene therapy is developed for the treatment of placental disorders, the gene-vector combination should not be harmful to the fetus and the expression of the transgene should only occur in placenta.     

Class 1 integron containing a new gene cassette,aadA10, associated with Tn1404 from R151

The carbenicillin, gentamicin, kanamycin, streptomycin, spectinomycin, sulfonamide, and tobramycin resistance determinants found on Pseudomonas aeruginosa plasmid R151 have previously been shown to translocate to another plasmid, R388, and it was inferred that a transposon, Tn1404, carried the resistance determinants. Sequencing of the cassette array from the plasmid known as R388::Tn1404 revealed two known gene cassettes, oxa10 and aadB, and a previously unidentified cassette determining resistance to streptomycin and spectinomycin, here designated aadA10, in the order oxa10-aadB-aadA10. These cassettes replaced the dfrB2-orfA cassette array of R388, indicating that movement of the resistance determinants from R151 to R388 resulted from recombinational exchange between two class 1 integrons rather than transposition. The AadA10 protein is most closely related to AadA6 (85% identical) and AadA7 (80% identical). The aadA10 cassette found here has only a simple site containing a 7-bp spacer derived from attI1 in place of a 59-base element and is likely to represent a derivative of the complete cassette. IntI1-mediated deletion of the aadA10 cassette was not detected, indicating that this single simple site is either inactive or only weakly active.     

High-efficiency plasmid gene transfer into dystrophic muscle

The efficiency of plasmid gene transfer in skeletal muscle is significantly enhanced by pretreatment with hyaluronidase and the application of an electrical field to the muscle following the injection of plasmid DNA, a process referred to as electrotransfer. However, the presence of increased levels of connective tissue in muscular dystrophies, such as Duchenne muscular dystrophy (DMD), may affect the efficiency of this process. Here we demonstrate that the efficiency of electrotransfer is not affected by increased levels of connective tissue in the mdx mouse model of DMD and that any damage induced by the electrotransfer process is not exacerbated in the dystrophic phenotype. However, increasing the concentration of hyaluronidase does not improve transfection efficiencies further. Unlike direct injection of plasmid DNA, the efficiency of electrotransfer is not dependent upon the sex and age of mice used. The combined treatment of hyaluronidase and electrotransfer results in highly efficient gene transfer in dystrophic muscle with limited muscle damage.     

DNA sequence analysis of regions surrounding blaCMY-2 from multiple Salmonella plasmid backbones

The emergence in the United States of resistance to expanded-spectrum cephalosporin (e.g., ceftriaxone) within the salmonellae has been associated primarily with three large (>100-kb) plasmids (designated types A, B, and C) and one 10.1-kb plasmid (type D) that carry the blaCMY-2 gene. In the present study, the distribution of these four known blaCMY-2-carrying plasmids among 35 ceftriaxone-resistant Salmonella isolates obtained from 1998 to 2001 was examined. Twenty-three of these isolates were Salmonella enterica serotype Newport, 10 were Salmonella enterica serotype Typhimurium, 1 was Salmonella enterica serotype Agona, and 1 was Salmonella enterica serotype Reading. All 23 serotype Newport isolates carried a type C plasmid, and 5, 4, and 1 serovar Typhimurium isolate carried type B, A, and C plasmids, respectively. Both the serotype Agona and serotype Reading isolates carried type A plasmids. None of the isolates carried a type D plasmid. Hybridization data suggested that plasmid types A and C were highly related replicons. DNA sequencing revealed that the region surrounding blaCMY-2 was highly conserved in all three plasmid types analyzed (types B, C, and D) and was related to a region surrounding blaCMY-5 from the Klebsiella oxytoca plasmid pTKH11. These findings are consistent with a model in which blaCMY-2 has been disseminated primarily through plasmid transfer, and not by mobilization of the gene itself, to multiple Salmonella chromosomal backbones.     

Nucleotide sequence of the SHV-5 beta-lactamase gene of a Klebsiella pneumoniae plasmid.

The nucleotide sequence of the SHV-5 beta-lactamase gene, subcloned from a plasmid of Klebsiella pneumoniae, was determined. The amino acid changes thought to be responsible for the extended substrate profile of SHV-5 are Gly----Ser234 and Glu----Lys235. SHV-5 is identical to SHV-4, except for Leu----Arg201, which accounts for the difference in apparent pI of the two enzymes.     

Hydrogel pullulan nanoparticles encapsulating pBUDLacZ plasmid as an efficient gene delivery carrier.

This study provides a method for enhancing the delivery of nucleic acid molecules to cells by encapsulating it inside the hydrogel pullulan nanoparticles. In this study, pullulan nanoparticles encapsulating pBUDLacZ plasmid has been prepared inside the aqueous droplets of w/o microemulsions. Transmission electron microscopy (TEM) image showed that the particles are spherical in shape with size of 45+/-0.80 nm diameter. Cell cytotoxicity studies as determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay demonstrated that cells incubated with nanoparticles remained more than 100% viable at nanoparticle concentration as high as 1000 microg/ml. From scanning electron microscope images, it was observed that the nanoparticles were internalised and the cells exhibited vacuoles in the cell body due to nanoparticle internalisation. Endocytosis of nanoparticles resulted in disruption of F-actin and beta-tubulin cytoskeleton of human fibroblasts. The efficacy of transfection in vitro on HEK293 and COS-7 cells demonstrated cell type dependence, with COS cells having a higher gene expression. The beta-gal expression in COS-7 cells by pullulan nanoparticle was comparable to commercially available Lipofectamine 2000. The results of this study are encouraging for the development of pullulan nanoparticles as an intracellular delivery system for drugs and genes.     

Prevention of experimental allergic encephalomyelitis by intramuscular gene transfer with cytokine-encoding plasmid vectors.

Antiinflammatory cytokines such as transforming growth factor beta1 (TGF-beta1) and interleukin 4 (IL-4) can protect from autoimmune diseases. To study the immunoregulatory effects of these cytokines in vivo, we used a method of gene therapy that permits continuous cytokine delivery over a period of weeks. We injected naked plasmid DNA expression vectors encoding either TGF-beta1 (pVR-TGF-beta1) or an IL-4-IgG1 chimeric protein (pVR-IL-4-IgG1) intramuscularly. This resulted in production of TGF-beta1 or IL-4-IgG1, respectively, and protection from myelin basic protein (MBP)-induced experimental allergic encephalomyelitis (EAE). TGF-beta1 gene delivery had pronounced downregulatory effects on T cell proliferation and production of interferon gamma (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha), on in vitro restimulation with MBP. IL-4-IgG1 vector administration also suppressed these responses, although much less than TGF-beta1, and enhanced secretion of endogenous IL-4. Therapy resulted in a significant decrease in the severity of histopathologic inflammatory lesions. In the CNS, treatment with either vector suppressed IL-12 and IFN-gamma mRNA expression, while IL-4 and TGF-beta1 mRNA levels were increased compared with control mice. Thus, cytokine plasmid treatment appeared to inhibit MBP-specific pathogenic Thl responses, while enhancing endogenous secretion of protective cytokines. We demonstrate that gene therapy with these vectors is an effective therapeutic strategy for EAE.     

新质粒介导喹诺酮耐药基因qnrB24的基因分析

目的对新的质粒介导喹诺酮耐药基因(qnrB24)进行相关研究和分析。方法对新发现的qnrB24基因阳性菌株进行转移接合实验,了解qnrB24对喹诺酮类药物的耐药性,碱裂解法提取接合子质粒,Southern blot明确质粒大小。采用热不对称交错聚合酶链反应(PCR)技术研究基因5′端以及3′端未知侧翼碱基序列。结果这个突变基因命名为qnrB24(Genbank登录号HM192542)。药敏试验结果显示携带qnrB24基因接合子对环丙沙星的最小抑菌浓度(MIC)值为0.125μg/mL,左氧氟沙星MIC值为0.190μg/mL。接合子对常见氟喹诺酮类抗菌药物的敏感性较大肠杆菌J53受体菌下降约8~11倍,接合子耐药水平低于临床分离菌株。Southern杂交显示该基因位于约60.0Kb质粒上。热不对称交错PCR结果显示,qnrB24基因5′端为假定的转座酶。结论 qnrB24通过质粒介导引起细菌对喹诺酮类药物的耐药性上升,容易发展成高水平耐药,因此需要监测其在细菌中的流行性。     

脂氟显微泡及质粒浓度对基因转染效率影响的研究

目的 研究脂氟显微泡及质粒浓度对pcDNA6.2-GW/EmGFP重组质粒转染效率及人肝癌HepG₂细胞死亡率的影响,初步探索转染最佳条件及shRNA重组质粒对靶基因survivin的抑制效果。方法 将重组质粒加入各组,设置6个质粒浓度梯度组。向各组加入脂氟显微泡混悬液,设置6个微泡浓度梯度组。对照组不加入微泡和质粒。根据前期优化结果,采用治疗性超声(1.2W/cm₂、占空比20%)辐照90s。48h后使用荧光显微镜及流式细胞仪检测细胞GFP表达效率。细胞死亡率通过台盼蓝染色计数获得。设立阳性质粒组P(+)和阴性质粒组P(－),western blot检测靶基因蛋白表达水平。 结果 当脂氟显微泡浓度小于等于 150μl/ml 时,转染率随微泡浓度增加而增加;当大于 150μl/ml 时,转染率却随微泡浓度增加而降低(p<0.05)。HepG₂细胞死亡率随微泡浓度增高,呈上升趋势(p<0.05)。在一定范围(20μl/ml)内,增加质粒浓度可以提高转染效率(p<0.05);但继续增加质粒浓度对基因转染率无明显影响(p>0.05)。质粒浓度的改变对细胞死亡率无明显影响。Western blot结果表明, P(+)组survivin基因表达受到明显抑制。结论 超声联合微泡可以有效介导基因转染,当微泡浓度为 150μl/ml,质粒浓度为 20μl/ml时,基因转染率最高,细胞死亡率较低。阳性重组质粒P(+)可下调靶基因表达水平,为后续基因转染的相关研究奠定基础。