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At least 649 records · Page 36Linked to original sources
PubMed1986

Hyperresistance to DNA damaging agents in yeast.

In order to study resistance to DNA damaging agents, yeast DNA segments conferring hyperresistance in this organism to such genotoxic agents were selected for among yeast cells transformed by a yeast genome library based on the multi-copy vector plasmid YEp13. Genetic variants hyperresistant to 4-nitroquinoline-N-oxide, formaldehyde, and alkylating agents were isolated and the respective hyperresistance determinants shown to co-segregate with the vector plasmid. Phenotypical characterization indicated different degrees of resistance, few cases of cross-resistance and differing structural stability of the cloned DNA. By transfer to E. coli and subsequent retransformation of yeast a number of plasmids was shown to stably carry the genetic information for hyperresistance.

4-Nitroquinoline-1-oxide↗
PubMedDate not supplied

Plasmid patterns and in-vitro susceptibility of penicillinase-producing Neisseria gonorrhoeae with emphasis on thiamphenicol: results of surveillance in The Netherlands.

Susceptibility to thiamphenicol was tested, and plasmids were characterized for 1925 strains of penicillinase-producing Neisseria gonorrhoeae (PPNG) isolated in The Netherlands in 1981 and 1982. Of the 1925 isolates, 771 harbored the so-called "Asia" (4.5-megadalton) resistance plasmid and 1154 had the so called "Africa" (3.2-megadalton) plasmid. Isolates in the first group were usually inhibited by 0.25 microgram of thiamphenicol ml, while the MIC for the latter isolates was 1-2 micrograms/ml. A similar bimodal distribution of MICs has been observed for non-PPNG isolates. In the period covering this study, a substantial shift from predominance of the "Africa" type to that of the "Asia" type of N. gonorrhoeae was observed.

Africa↗
PubMed1989

Construction of plasmids containing a unique acetylaminofluorene adduct located within a mutation hot spot. A new probe for frameshift mutagenesis.

N-2-acetylaminofluorene (AAF), a potent rat liver carcinogen, binds primarily to the C-8 position of guanine residues. In a bacterial forward mutation assay, more than 90% of the mutations induced by -AAF adducts are frameshift mutations located at specific sites: the so-called mutation hot spots. We are particularly interested in a class of -2 frameshift mutations occurring within a specific sequence, the NarI sequence. The NarI site, GGCGCC, contains three guanine residues that are approximately equally reactive toward -AAF substitution. To study further the mechanism by which mutations are induced by -AAF adducts at this site, we designed a new plasmid probe. In this paper we describe the construction and the effectiveness of this probe, pSM14, which provides a simple phenotypic test for detecting frameshift mutations within the NarI site. The construction and the characterization of plasmids with a single -AAF adduct in each of the three positions of the NarI site are also described. The strategy of construction that was used involves the ligation of oligonucleotides containing a single adduct in a NarI site into a gapped-duplex pSM14 plasmid. Plasmids that have successfully integrated the oligonucleotides by ligation at both the 5' and the 3' ends were purified by centrifugation on CsCl gradients. These constructs have been used in single adduct mutation studies.

2-Acetylaminofluorene↗
PubMed1993

Linear plasmids and chromosomes in bacteria.

Linear plasmids and chromosomes were unknown in prokaryotes until recently but have now been found in spirochaetes, Gram-positive bacteria, and Gram-negative bacteria. Two structural types of bacterial linear DNA have been characterized. Linear plasmids of the spirochaete Borrelia have a covalently closed hairpin loop at each end and linear plasmids of the Gram-positive filamentous Streptomyces have a covalently attached protein at each end. Replicons with similar structures are more frequent in eukaryotic cells than in prokaryotes. Linear genomic structures are probably more common in bacteria than previously recognized, however, and some replicons may interconvert between circular and linear isomers. The molecular biology of these widely dispersed elements provides clues to explain the origin of linear DNA in bacteria, including evidence for genetic exchange between prokaryotes and eukaryotes.

Bacteria↗
PubMedDate not supplied

Molecular epidemiology, based on plasmid profiles, of Haemophilus ducreyi infections in the United States. Results of surveillance, 1981-1990.

To determine the distribution and extent of temporal changes in Haemophilus ducreyi strains in the United States, 342 isolates of H. ducreyi collected in 18 urban areas in the United States between 1981 and 1990 were characterized by plasmid content. Isolates possessed either no plasmid (29.8%), the 5.7-Mdal plasmid (50.9%), both the 4.4- and 5.7-Mdal plasmids (6.1%), the 3.2-Mdal plasmid (12.9%), or a combination of 1.8-, 2.6-, 2.8-, and 3.2-Mdal plasmids (1 case out of 342). Isolates that possessed no plasmid or that possessed the 5.7-Mdal plasmid had the widest geographic distribution; only isolates from cities on the West Coast possessed the 3.2-Mdal plasmid. Evidence was found of temporal changes in H. ducreyi strain populations among isolates from both Tampa and New Orleans.

Epidemiologic Methods↗
PubMed1983

Characterization of a chimeric beta-lactamase plasmid of Neisseria gonorrhoeae which can function in Escherichia coli.

A chimeric beta-lactamase encoding plasmid, containing the 4.4 Mdal beta-lactamase plasmid and the 2.6 Mdal cryptic plasmid of Neisseria gonorrhoeae has been characterized by physical and biological methods. Digestion with restriction enzymes indicates the presence of the following restriction sites: 1 site: AccI, AvaI, HgiAI, HincII, MstI, PstI, PvuII, XbaI and XorI; 2 sites: HindIII and BamHI; 3 sites: BclI, Sau96I and AvaII; 6 sites: HinfI; greater than 8 sites: AluI, BbvI, DdeI, HgaI, HhaI, HpaII, MspI, Sau3A, TacI and TaqI. No restriction sites were found for the following: BglI, BglII, BstEII, EcoRI, EcoRII, HpaI, HphII, KpnI, SacI, SalI, SmaI, SstI, SstII, and XhoI. Five plasmid specific proteins have been identified by DNA directed in vitro protein synthesis (43K, 41K, 30K, 16K and 14K). The location on the physical map of the coding regions for each of these proteins has been determined by the following methods: using plasmid DNA restricted by various enzymes in an in vitro protein synthesis system; identifying promoter-containing regions by digesting plasmid DNA with DdeI, adding RNA polymerase and then determining which fragments are retained by nitrocellulose. This plasmid contains both parental phenotypes in that it encodes penicillin resistance and possesses the sequence necessary for uptake in the gonococcus. Transformation data indicate that this plasmid can function in both E. coli and N. gonorrhoeae and that growth in E. coli has no effect on the plasmid's ability to transform the gonococcus.

Bacterial Proteins↗
PubMed2003

Identification and characterization of a new staphylococcal enterotoxin-related putative toxin encoded by two kinds of plasmids.

We identified and characterized a novel staphylococcal enterotoxin-like putative toxin, which is named SER. Nucleotide sequencing analysis of the ser gene revealed that ser was most closely related to the seg gene. The ser gene product, SER, was successfully expressed as a recombinant protein in an Escherichia coli expression system, and recombinant SER (rSER) showed significant T-cell stimulation activity. The SER production in ser-harboring Staphylococcus aureus strains was confirmed by Western blot analysis using anti-rSER antibody. Moreover, ser was seen to be encoded by at least two types of plasmids. In particular, one kind of plasmid encoding the ser gene has been known as a sed- and sej-carrying pIB485-related plasmid.

Amino Acid Sequence↗
PubMed1991

[Characteristics of the R-plasmid pM3 (IncP-9) of a broad circle of hosts].

A new broad host range plasmid pM3 (IncP-9) was found in a facultative methylotrophic bacteria Pseudomonas putida and described. The pM3 plasmid is characterized by thermo-instability in Enterobacteriaceae family of bacteria at 36 degrees C or higher temperatures. It is also unable to be inherited as an autonomous element in the obligate methylotrophic bacteria Methylobacillus M75. The peculiarities of plasmid inheritance make possible to use it as a tool for genetic research, for instance, to construct the donor strains in Methylobacillus M75 able to mobilize the chromosomal genes for conjugational transfer in isogenic systems of crosses.

Chromosomes, Bacterial↗
PubMed1996

Nucleotide sequence and characterization of the trbABC region of the IncI1 Plasmid R64: existence of the pnd gene for plasmid maintenance within the transfer region.

A 6.72-kb DNA sequence between the exc gene and the oriT operon within the transfer region of IncI1 plasmid R64 was sequenced and characterized. Three novel transfer genes, trbA, trbB, and trbC, were found in this region, along with the pnd gene responsible for plasmid maintenance. The trbABC genes appear to be organized into an operon located adjacent to the oriT operon in the opposite orientation. The trbA and trbC genes were shown to be indispensable for R64 plasmid transfer, while residual transfer activity was detected in the case of R64 derivatives carrying the trbB++ deletion mutation. The T7 RNA polymerase-promoter system revealed that the trbB gene produced a 43-kDa protein and the trbC gene produced an 85-kDa protein. The nucleotide sequence of the pnd gene is nearly identical to that of plasmid R483, indicating a function in plasmid maintenance. The plasmid stability test indicated that the mini-R64 derivatives with the pnd gene are more stably maintained in Escherichia coli cells under nonselective conditions than the mini-R64 derivatives without the pnd gene. It was also shown that the R64 transfer system itself is involved in plasmid stability to a certain degree. Deletion of the pnd gene from the tra+ mini-R64 derivative did not affect transfer frequency. DNA segments between the exc and trbA genes for IncI1 plasmids R64, Colb-P9, and R144 were compared in terms of their physical and genetic organization.

Amino Acid Sequence↗
PubMed1990

The Streptomyces aureofaciens plasmid pIMB R8 and its use for shuttle vector construction.

The cryptic low copy plasmid designated as pIMB R8 was isolated from an industrial strain of the chlortetracycline producer Streptomyces aureofaciens R8/26. Using restriction endonucleases the pIMB R8 plasmid was characterized to have 15 kb. Subcloning of the Bam HI fragments of pIMB R8 into replication probe vector resulted in the identification of the replication part. The 0.7 kb Bc/I--Bg/I fragment is sufficient for normal replication, but produces about fourty times high plasmid copy numbers than the original pIMB R8. Using the replication part of pIMB R8 was constructed several shuttle cloning vectors for the E. coli-streptomycete system.

Cloning, Molecular↗
PubMed2005

Characterization of putative rolling-circle plasmids from the Gram-negative bacterium Xylella fastidiosa and their use as shuttle vectors.

This study was initiated to characterize a small Xylella fastidiosa (X. fastidiosa) plasmid and attempt to create a X. fastidosa/Escherichia coli shuttle vector that was stable in planta. Restriction enzyme analysis of a 1.3kb plasmid DNA from a grape-infecting strain of X. fastidiosa (UCLA) revealed the presence of three similar, but genetically distinct, plasmids, pUCLAs. Evidence that suggests the pUCLA plasmids replicate via a rolling-circle (RC) mechanism include: (i) the presence of ssDNA in X. fastidiosa cells; (ii) the presence of conserved motifs in the predicted ORF1 that are typical of initiator (Rep) proteins associated with RC replication; (iii) high amino acid identity between the putative Rep proteins of pUCLAs and Pf3, a filamentous bacteriophage of Pseudomonas aeruginosa that replicates by a RC mechanism; and (iv) the presence of a putative origin of replication upstream of ORF1 that has the potential to form secondary hairpin structures. One DNA motif present in pUCLA shared sequence similarity to known nicking sites in the origins of replication of other RC plasmids and phages. The shuttle vector, pXF001, successfully transformed grape X. fastidiosa strains and was found to be present as autonomous, structurally unchanged DNA molecules in X. fastidiosa. However, pXF001 was not stably maintained in X. fastidiosa without antibiotic selection.

Amino Acid Sequence↗
PubMed1987

Initiation signals for the conversion of single stranded to double stranded DNA forms in the streptococcal plasmid pLS1.

We have characterized a region in the streptococcal plasmid pLS1 located between nucleotides 4103 and 4218 which is a signal involved in the conversion of single stranded intermediates of replication to double stranded plasmid forms. This region has a large axis of dyad symmetry resulting in the formation of a secondary structure as revealed by the location of endonuclease S1-cleavage sites in supercoiled covalently closed circular pLS1 DNA. Deletions affecting this region caused a fivefold reduction in plasmid copy number, plasmid instability and the accumulation of single-stranded DNA intermediates. The conversion signal of pLS1 has homologues in other staphylococcal plasmids, sharing a consensus sequence located in the loop of the signal. Computer assisted analysis showed that the signal detected in pLS1 has a high degree of homology with the complementary strand origin of the Escherichia coli single stranded bacteriophages phi X174 and M13.

Bacteriophage phi X 174↗
PubMed1993

Nucleotide sequence and characterization of the traABCD region of IncI1 plasmid R64.

A 3.6-kb BglII-SmaI segment of the transfer region of IncI1 plasmid R64drd-11 was sequenced and characterized. Analysis of the DNA sequence indicated the presence of four genes, traA, traB, traC, and traD, in this region. The expression of the traB, traC, and traD genes was examined by maxicell experiments and that of the traA gene was examined by constructing the traA-lacZ fusion gene. The introduction of frameshift mutations into the four genes indicated that the traB and traC genes are essential for conjugal transfer in liquid medium and on a solid surface. Both were also required for the formation of the thin pilus, which is the receptor for phages I alpha and PR64FS. Upstream of the traA gene, a promoter sequence for sigma 70 of E. coli RNA polymerase was identified by S1 nuclease mapping and primer extension experiments.

Amino Acid Sequence↗
PubMed2004

Identification and characterization of a linear-plasmid-encoded factor H-binding protein (FhbA) of the relapsing fever spirochete Borrelia hermsii.

In North America, tick-borne relapsing fever (TBRF) is caused by the spirochete species Borrelia hermsii, Borrelia parkeri, and Borrelia turicatae. We previously demonstrated that some isolates of B. hermsii and B. parkeri are capable of binding factor H and that cell-bound factor H can participate in the factor I-mediated cleavage of C3b. Isolates that bound factor H expressed a factor H-binding protein (FHBP) that we estimated to be approximately 19 to 20 kDa in size and thus, pending further characterization, temporarily designated FHBP19. Until this report, none of the FHBPs of the TBRF spirochetes had been characterized. Here we have recovered the gene encoding the FHBP of B. hermsii YOR from a lambda ZAP II library and determined its sequence. The gene encodes a full-length protein of 22.7 kDa, which after processing is predicted to be 20.5 kDa. This protein, which we redesignate factor H-binding protein A (FhbA), is unique to B. hermsii. Two-dimensional pulsed-field gel electrophoresis and hybridization analyses revealed that the B. hermsii gene encoding FhbA is a single genetic locus that maps to a linear plasmid of approximately 220 kb. The general properties of FhbA were also assessed. The protein was found to be surface exposed and lipidated. Analysis of the antibody response to FhbA in infected mice revealed that it is antigenic during infection, indicating expression during infection. The identification and characterization of FhbA provides further insight into the molecular mechanisms of pathogenesis of the relapsing fever spirochetes.

Amino Acid Sequence↗
PubMed1990

Evidence for two types of cytotoxic necrotizing factor in human and animal clinical isolates of Escherichia coli.

We have characterized the in vitro and in vivo toxic properties of cell sonic extracts from 22 animal and human clinical isolates of Escherichia coli that caused both necrosis in the rabbit skin and multinucleation in tissue cultures, two toxic properties previously reported as being specific for E. coli cytotoxic necrotizing factor (CNF). Two distinct toxic phenotypes were observed. Type 1, which was displayed by originally described CNF strains, was characterized by extensive multinucleation and rounding of cells in HeLa cell culture assays, moderate necrosis in the rabbit skin test, and absence of necrosis in the mouse footpad test. Type 2, which has recently been shown to be associated with E. coli Vir plasmid, was characterized by moderate multinucleation, by polymorphism and elongation of HeLa cells, and by an intense necrotic response in both the rabbit skin test and the mouse footpad test. The distinction between the two cytotoxins accounting for these effects (CNF 1 and CNF 2), together with their partial relatedness, was confirmed by seroneutralization studies of both cytopathic effects and necrosis in the rabbit skin test. In addition, type 2 extracts were more lethal in the mouse intraperitoneal test and induced a moderate, although not totally repetitive, fluid accumulation in the ileal loop test. The original toxic properties of these recently recognized categories of E. coli strains, together with their association with enteritis and septicemia, suggest that these strains may play a significant role in pathology.

Animals↗
PubMed2006

Characterization of a higBA toxin-antitoxin locus in Vibrio cholerae.

Toxin-antitoxin (TA) loci, which were initially characterized as plasmid stabilization agents, have in recent years been detected on the chromosomes of numerous free-living bacteria. Vibrio cholerae, the causative agent of cholera, contains 13 putative TA loci, all of which are clustered within the superintegron on chromosome II. Here we report the characterization of the V. cholerae higBA locus, also known as VCA0391/2. Deletion of higA alone was not possible, consistent with predictions that it encodes an antitoxin, and biochemical analyses confirmed that HigA interacts with HigB. Transient exogenous expression of the toxin HigB dramatically slowed growth of V. cholerae and Escherichia coli and reduced the numbers of CFU by several orders of magnitude. HigB toxicity could be counteracted by simultaneous or delayed production of HigA, although HigA's effect diminished as the delay lengthened. Transcripts from endogenous higBA increased following treatment of V. cholerae with translational inhibitors, presumably due to reduced levels of HigA, which represses the higBA locus. However, no higBA-dependent cell death was observed in response to such stimuli. Thus, at least under the conditions tested, activation of endogenous HigB does not appear to be bactericidal.

Amino Acid Sequence↗
PubMed1997

Regulation of the rat cardiac troponin I gene by the transcription factor GATA-4.

Troponin I is a thin-filament contractile protein expressed in striated muscle. There are three known troponin I genes which are expressed in a muscle-fibre-type-specific manner in mature animals. Although the slow skeletal troponin I isoform is expressed in fetal and neonatal heart, the cardiac isoform is restricted in its expression to the myocardium at all developmental stages. To study the regulation of this cardiac-specific and developmentally regulated gene in vitro, the rat cardiac troponin I gene was cloned. Transient transfection assays were performed with troponin I-luciferase fusion plasmids to characterize the regulatory regions of the gene. Proximal regions of the upstream sequence were sufficient to support high levels of expression of the reporter gene in cardiocytes and relatively low levels in other cell types. The highest luciferase activity in the cardiocytes was noted with a plasmid that included the region spanning -896 to +45 of the troponin I genomic sequence. Co-transfection of GATA-4, a recently identified cardiac transcription factor, with troponin I-luciferase constructs permitted high levels of luciferase expression in non-cardiac cells. Electrophoretic mobility-shift assays demonstrated specific binding of GATA-4 to oligonucleotides representative of multiple sites of the troponin I sequence. Mutation of a proximal GATA-4 DNA-binding site decreased transcriptional activation in transfected cardiocytes. These results indicate that the proximal cardiac troponin I sequence is sufficient to support high levels of cardiac-specific gene expression and that the GATA-4 transcription factor regulates troponin I-luciferase expression in vitro.

3T3 Cells↗
PubMed2004

Characterization and biological evaluation of a microparticle adjuvant formulation for plasmid DNA vaccines.

We describe the physiochemical characterization and immunological evaluation of plasmid DNA vaccine formulations containing a nonionic triblock copolymer adjuvant (CRL1005) in the presence and absence of a cationic surfactant, benzalkonium chloride (BAK). CRL1005 forms particles of 1-10 microns upon warming above its phase-transition temperature (approximately 6-8 degrees C) and the physical properties of the particles are altered by BAK. DNA/CRL1005 vaccines formulated with and without BAK were evaluated in rhesus macaques to determine the effect of CRL1005 and BAK on the ability of plasmid DNA to induce a cellular immune response. Immunogenicity results indicate that the addition of CRL1005 to human immunodeficiency virus-1 gag plasmid DNA formulated in phosphate-buffered saline leads to an enhancement in the gag-specific cellular immune response. Moreover, the addition of BAK to human immunodeficiency virus-1 gag plasmid DNA/CRL1005 formulations produces an additional enhancement in gag-specific cellular immunity. In vitro characterization studies of DNA/CRL1005 formulations indicate no detectable binding of DNA to CRL1005 particles in the absence of BAK, suggesting that the enhancement of cellular immunity induced by DNA/CRL1005 formulations is not due to enhanced DNA delivery. In the presence of BAK, however, results indicate that BAK binds to CRL1005 particles, producing cationic microparticles that bind DNA through electrostatic interactions. If BAK is present at the phase-transition temperature, it reduces the particle size from approximately 2 microns to approximately 300 nm, presumably by binding to hydrophobic surfaces during particle formation. Zeta potential measurements indicate that the surface charge of CRL1005-BAK particles changes from positive to negative upon DNA binding, and DNA bound to the surface of CRL1005-BAK particles was visualized by fluorescence microscopy. These results indicate that the addition of BAK to DNA/CRL1005 formulations leads to the formation of approximately 300 nm CRL1005-BAK-DNA particles that enhance the cellular immune response in rhesus monkeys.

Adjuvants, Pharmaceutic↗