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During the last 20years, two-dimensional agarose gel electrophoresis combined with other techniques such as Polymerase Chain Reaction, helicase assay and electron microscopy, helped to characterize plasmid DNA replication and topology. Here we describe some of the most important findings that were made using this method including the characterization of uni-directional replication, replication origin...
Nonviral gene delivery has gained a lot of interest as a promising approach for gene therapy. Despite intensive studies and much progress the outcome of nonviral vectors has remained significantly weaker than that of viral vectors. A weak transfection efficiency of nonviral gene transfection is still limiting their in vivo use. We have tested the possibility to improve the measurement of transfection...
High-altitude wetlands (above 4200m) in the northwest of Argentina are considered pristine and extreme environments. Micrococcus sp. A1, H5, and V7, isolated from such environments, were shown to contain linear megaplasmids, designated pLMA1, pLMH5, and pLMV7, respectively. As known from linear plasmids of other actinomycetes, all three plasmids were resistant to λ exonuclease treatment, which is...
The emergence and spread of pathogenic bacteria that have become resistant to multiple antibiotics through lateral gene transfer have created the need of novel antimicrobials. Toxin–antitoxin (TA) modules, which have been implicated in plasmid maintenance and stress management, are ubiquitous among plasmids from vancomycin or methicillin resistant bacteria. In the Streptococcus pyogenes pSM19035-encoded...
A new insertion variant of IS231 family, designated ISBth4, was identified from Bacillus thuringiensis MEX312. ISBth4 is 2046bp in length and is delimited by two 17bp inverted repeats (IR) with one mismatch, flanked by two perfect 11bp direct repeats (DR). ISBth4 contains two open reading frames (ORFs), ORF1 and ORF2, which encode 72 and 473 amino acids, respectively. Multiple sequence alignments...
We previously firstly discovered that CXCR4 nuclear localization may be responsible for metastasis of renal cell carcinoma (RCC), and that there was a nuclear localization sequence (NLS) that induced CXCR4 to transfer to the nucleus after combining with its ligand SDF-1. Using our previously constructed pEGFP-CXCR4 as the template, corresponding objective regions were amplified. The amplified PCR...
Fusobacterium nucleatum is a Gram-negative anaerobic rod found in dental plaque biofilms, and is an opportunistic pathogen implicated in periodontitis as well as a wide range of systemic abscesses and infections. Genomic analyses of F. nucleatum indicate considerable genetic diversity and a prominent role for horizontal gene transfer in the evolution of the species. Several plasmids isolated from...
The expression of hypoxia-inducible factor-1α (HIF-1α) in heart allografts is an important mechanism in response to ischemia/reperfusion (I/R) injury which represents the single major non-immunologic factor implicated in pathogenesis of chronic graft dysfunction (CGD). Adenoviral mediated overexpression of HIF-1α is a useful way to investigate the molecular mechanisms of I/R injury and the cardiac...
Extraintestinal pathogenic Escherichia coli (ExPEC) are known to cause important diseases of humans and animals, and they have been shown to carry a variety of plasmids associated with increased virulence and decreased antimicrobial susceptibility. Here, the completed DNA sequence of a human uropathogenic E. coli (UPEC; O6:H31 isolate) plasmid, pEC14_114, was determined. The plasmid was 114,222bp...
DNA primase is an enzyme required for replication of both chromosomes and vast majority of plasmids. Guanosine tetra- and penta-phosphate (ppGpp and pppGpp, respectively) are alarmones of the bacterial stringent response to starvation and stress conditions, and act by modulation of the RNA polymerase activity. Recent studies indicated that the primase-catalyzed reaction is also inhibited by (p)ppGpp...
Single plasmids designed to express the six human type I steroid hormone receptors and detect signaling activity are described in this report. These stably replicating plasmids reported ligand-induced transcriptional activation via lacZ assays in Baker’s yeast (Saccharomyces cerevisiae). The ligand concentrations needed to activate signaling in yeast expressing these plasmids spanned five orders of...
Plasmids of the incompatibility group IncP-1 are important agents of horizontal gene transfer and contribute to the spread of antibiotic resistance and xenobiotic degradation within bacterial communities. Even though some prototype plasmids have been studied in much detail, the diversity of this plasmid group was still greatly underestimated until recently, as only two of the five currently known...
Previous studies showed that when pPSX-vioABCDE was used to transform E. coli K12 DH5α the strain retained the plasmid even after 100 generations of unselected growth but produced a low level of the anti-tumour antibiotic violacein. Markedly higher levels of violacein synthesis were obtained from E. coli K12 DH5α pUC18-vioABCDE and Sphingomonas sp. JMP4092 pPSX-vioABCDE. Unfortunately, both strains...
Lactobacillus helveticus R0052 is a bacterium used in commercial probiotic preparations. R0052 contains a small, cryptic plasmid comprised of eight open reading frames, four of which encode proteins of unknown function. Based on the sequence of the replication initiation protein RepA, pIR52-1 is a member of the recently described RepA_N family of Gram-positive theta-replicating plasmids. The repA...
In order to gain information on the significance and functions of small molecular weight plasmids (usually regarded as cryptic) of Bacteroides isolates, we screened 178 European clinical Bacteroides isolates for plasmids and determined the nucleotide sequence of a 5.6kb plasmid. The previously observed frequent plasmid types (Classes I–III; 2.7, 4.2 and 5.6kb, respectively) were found to exhibit different...
Conjugal transfer of plasmid DNA initiates and terminates at a specific non-coding site called the origin of transfer (oriT). Previous analysis of conjugative plasmid pVT745 from Aggregatibacter actinomycetemcomitans suggested that oriT was located adjacent to the operon responsible for initiation of ssDNA transfer. The location of oriT was confirmed by assaying both subclones of the region as well...
Although horizontal gene transfer mediated by plasmids is important to the generation of the genetic variability of Sinorhizobium strains, the barriers which can reduce horizontal gene transfer between bacteria have not yet been studied in Sinorhizobium. We studied the plasmid transfer by electroporation and its restriction in strains of Sinorhizobium meliloti and S. medicae. After conditions for...
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