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PubMedDate not supplied

Construction of recombinant plasmids containing rat thyroglobulin mRNA sequences.

Two plasmids containing rat thyroglobulin cDNA sequences have been constructed and characterized. A plasmid with a 500-bp insert (pRT6) was isolated and identified as thyroglobulin-specific on the basis of the tissue specificity of the inserted sequence and of its ability to retain thyroglobulin mRNA on a nitrocellulose filter. The cDNA insert in pRT6 was subsequently used to screen a rat thyroid cDNA library constructed with large cDNA. A plasmid was found containing a 1700-bp insert. The polarity and the fidelity of the insert is demonstrated by S1 mapping.

Animals↗
PubMed1984

Cloning of cDNA sequences for murine ATP-citrate lyase. Construction of recombinant plasmids using an immunopurified mRNA template and evidence for the nutritional regulation of ATP-citrate lyase mRNA content in mouse liver.

Mouse liver mRNA that was enriched in sequences coding for ATP-citrate lyase by polysome immunoadsorption was used as a template for cDNA synthesis. Double-stranded cDNA sequences were inserted into the plasmid pBR322 and cloned in Escherichia coli RR1. Twenty-seven plasmids containing putative cDNA sequences for ATP-citrate lyase were identified by differential hybridization with single-stranded 32P-cDNAs synthesized from immunopurified mRNA, sucrose gradient-purified ATP-citrate lyase mRNA, and mRNA isolated from the livers of mice that were nutritionally induced or de-induced for ATP-citrate lyase biosynthesis. A subgroup of five recombinant plasmids was characterized further in hybridization-selection experiments. Each of these plasmids positively selected ATP-citrate lyase mRNA as determined by in vitro translation and specific immunoprecipitation. The length of ATP-citrate lyase mRNA was estimated to be 4900 bases in a Northern blot analysis. A 32P-cDNA probe derived from a 1500-base pair insert was used to investigate the basis for the 20-30-fold induction of ATP-citrate lyase that occurs when starved animals are fed a high carbohydrate/low fat diet. Dot-blot hybridization analysis disclosed that the relative content of liver ATP-citrate lyase mRNA increased 25-fold after 15 h of refeeding, indicating that the synthesis of the lipogenic enzyme is controlled at a pretranslational level in the nutritional paradigm.

ATP Citrate (pro-S)-Lyase↗
PubMed1978

Construction and characterization of an Escherichia coli plasmid bearing a functional gene G of bacteriophage phiX174.

In order to study the mutagenic effects of site-specific, covalent modifications of biologically active DNA, we need host cells that are permissive for any type of mutation that might be produced in vivo from the modified DNA. Specifically, we require a general, in vivo complementation system for the bacteriophage phiX174 gene G, an essential gene that we have chosen for our initial studies of chemical mutagenesis. Toward this end, we have constructed a plasmid (pphiXG) that carries a functional copy of phiX174 gene G. Three different bacterial strains that are nonpermissive for am9, a gene G amber mutant, have been transformed with pphiXG. The transformants are now permissive for this gene G mutant, but not for the gene A or E mutants that have been tested. This paper describes the construction and the biochemical characterization of this plasmid, pphiXG, and describes some of the biological properties exhibited by the pphiXG-bearing strains.

Coliphages↗
PubMedDate not supplied

Plasmid profile analysis and antibiotic resistance of Salmonella strains from clinical isolates in Cluj-Napoca.

Resistance patterns, plasmid profiles and the genetic resistance determinants were investigated in 38 isolates of Salmonella enterica serotype Typhimurium and 19 isolates of Salmonella enterica serovar Enteritidis derived from children hospitalized in two clinics in Cluj-Napoca, during the period of 1995-1997. Incidence of plasmid and antibiotic resistance was very high in Salmonella typhimurium isolates. All strains were resistant to almost all antibiotics tested but susceptible to the third generation cephalosporines and fluoroquinolones. We identified three resistance patterns and six plasmid profiles. Each plasmid profile was characterized by the presence of two large plasmids of 150-180 Kbp. Approximately 60% of strains harbored three or four small plasmids of 1.3 to 9.5 Kbp. The plasmids of 8.5 Kbp encoded resistance to beta-lactam antibiotics and were non-conjugative. The other small plasmids were cryptic and also non-conjugative. Salmonella enteritidis isolates were susceptible to many antibiotics, except Tetracycline and Trimethoprim-Sulfamethoxazole. We identified three different resistance patterns but nine plasmid profiles. All plasmid profiles were characterized by the presence of a large plasmid (> 100 Kbp). The number and the diversity of small plasmids were higher than in S. typhimurium strains. There was no parallelism between resistance and plasmid profile: for the same resistance pattern a number of two or three plasmid profiles were found. Our conclusions are that Salmonella typhimurium strains were multiresistant to antibiotics and that many genetically different strains of Salmonella typhimurium and Salmonella enteritidis were responsible for gastroenteritis in children from Cluj County. The increasing antibiotic resistance highlights the need for more refined methods in genetic and epidemiological characterization of bacteria involved in gastrointestinal infections.

Anti-Bacterial Agents↗
PubMed1988

Shigella flexneri invasion plasmid antigens B and C: epitope location and characterization with monoclonal antibodies.

Invasion plasmid antigens B (IpaB) and C (IpaC) are associated with the ability of shigellae to invade cultured mammalian cells. Monoclonal antibodies against IpaB and IpaC polypeptides were produced and used in a whole-cell enzyme-linked immunosorbent assay to show that both IpaB and IpaC polypeptides were exposed on the surface of virulent shigellae. Moreover, these surface epitopes were shown to be highly conserved among different serotypes of Shigella spp. and enteroinvasive Escherichia coli. Cross-reactive epitopes were not found on noninvasive Shigella strains or on other enteric bacteria including Salmonella, Yersinia, Campylobacter, Vibrio, and Aeromonas spp. and various pathogenic strains of E. coli. The monoclonal antibodies were used in competitive binding assays to define three unique epitopes of the IpaB polypeptide and four unique epitopes of the IpaC polypeptide. Epitope locations and their corresponding DNA-encoding regions were defined by examining the IpaB and IpaC products expressed by lambda gt11 recombinants and by constructing a genetic map of the insert DNAs of these recombinants. Three IpaB epitopes (2F1, 1H4, 4C8) were found to be encoded on three contiguous DNA regions comprising a 700-base-pair (bp) segment that corresponded to the amino-terminal end of the IpaB polypeptide. Similarly, a 640-bp DNA segment that corresponded to the amino-terminal end of the IpaC polypeptide was found to encode three clustered IpaC epitopes (5H1, 9B6, 5B1). Approximately 50 bp downstream from this region a fourth IpaC epitope-encoding region (2G2) was found. The effect of the monoclonal antibodies on plaque formation by virulent Shigella flexneri on a monolayer of cultured mammalian cells (a sensitive measure of invasiveness) was determined. Only the IpaB-specific monoclonal antibody 2F1 was able to reduce the plaque-forming capacity by greater than 50%, suggesting that this epitope of the IpaB polypeptide is involved in the invasion process.

Antibodies, Bacterial↗
PubMed2005

Characterization of two small cryptic plasmids from Pseudomonas sp. strain S-47.

Two small cryptic plasmids, p47L and p47S, identified in Pseudomonas sp. S-47 were characterized by determination of DNA sequences and physical and functional maps. They are 3084 and 1782 bp in length, respectively, with GC contents of 63.55 and 65.21%. The detection of single-strand DNAs of both plasmids indicates that they replicate by a rolling-circle mechanism. The deduced polypeptide encoded by the rep gene of p47L is homologous with Rep proteins of plasmids belonging to the pIJ101/pJV1 family, which are known to replicate by the rolling-circle mechanism. Despite containing a homologous signature with Rep proteins of rolling-circle replicating (RCR) plasmids in the pT181 family, the Rep of p47S lacks significant homology with Rep proteins of this family and is missing a region similar to the family's replication origin (dso). Based on the rep sequence comparisons, p47L falls into a previously defined plasmid family whereas p47S defines a new family of RCR plasmid.

Amino Acid Sequence↗
PubMed2001

[Characterization of cefoperazone resistance gene on plasmid pFC in E. coli HX88108].

OBJECTIVE: To investigate the characterization of cefoperazone resistance gene (CPZr) on plasmid pFC in E. coli HX88108 and inquire into the mechanism of resistance to CPZ at the molecular level. METHODS: E. coli HX88108 strain which demonstrated high-level resistance to cefoperazone (MIC, > 512 micrograms/ml) was isolated from a severely infected patient in 1988. Five plasmids coexisting in the strain were designated pFC, pFT1; pFT2, pFT3 and pFX, respectively. Four plasmids except pFX conferred CPZ resistance. Cefoperazone resistance gene (CPZr) has been cloned from plasmid pFC. beta-lactamase assays with Nitrocefin were performed. RESULTS: The expression product of CPZr was beta-lactamase. The high level beta-lactamase enzymatic activities against cephaloridine of CPZr transformants which were detected spectrophotometrically at 260 nm wave length demonstrated high level similarities to that of pFC. MICs of 18 antibiotics were determined according to a guideline of NCCLS by broth dilution method. CPZr transformants showed moderate level resistance to ampicillin, cefazolin, cefazolin, cefamandole and CPZ (MIC, 64 micrograms/ml). Meanwhile, susceptibility testing results demonstrated that the level of resistance to CPZ of pFC transformant in this study (MIC, 64 micrograms/ml) was much lower than that in 1988 (MIC, > 512 micrograms/ml) and resistance to nofloxacin and aminoglycosides was not observed. Induction experiment and temperature-sensitive mutation of CPZ resistance were performed. CPZr colonal strains revealed the higher-level of resistance to CPZ (MIC, 512 micrograms/ml) due to antibiotic CPZ induction rather than temperature sensitive mutation. CONCLUSION: This observation suggests that resistance to antibiotics encoded by plasmid might have been lower or lost under no antibiotic stress in a certain period, but higher under heavy stress.

Cefoperazone↗
PubMed1994

Identification and characterization of an Enterococcus faecalis plasmid pAD1-encoded stability determinant which produces two small RNA molecules necessary for its function.

A determinant, designated par, essential for stable maintenance for an autonomously replicating fragment of the Enterococcus faecalis plasmid pAD1, was identified by transposon mutagenesis, and its DNA sequence was determined. The position of flanking transposon inserts with no effect on stability indicates that par is encoded on no more than approximately 720 bp of DNA. This region contains no large open reading frames (> 62 amino acids) but does contain a number of direct and inverted repeats and GC- and AT-rich boxes. Disruption of these elements by transposon insertion, or deletion of the entire cluster of elements, resulted in a loss of plasmid stability but did not affect replication or copy number in E. faecalis. Characterization of selected mutants suggested that some manipulations of par may interfere with essential plasmid replication functions and/or be lethal to the host cell. Northern blot analysis revealed that two small RNA molecules of approximately 250 and 145 nucleotides homologous to par are produced by cells containing the complete pAD1 replicon. Mutations affecting par that resulted in a decrease in plasmid stability also resulted in changes in the pattern of production of the par RNAs, suggesting that these RNAs are important for par function.

Base Sequence↗
PubMed1993

Characterization of a small cryptic plasmid from Salmonella enteritidis that affects the growth of Escherichia coli.

We examined the plasmid content of 25 clinical isolates of Salmonella enteritidis, and detected the presence of small plasmids (3-5.3 kb) in 9 of them, alone, or in addition to the large, so-called virulence plasmid. A 5.3-kb plasmid isolated as unique extrachromosomal DNA from a strain responsible for a high-mortality outbreak was characterized by restriction mapping and cloning. The plasmid replicon was localized in a 1.7-kb fragment, that hybridized with three of the small plasmids detected in S. enteritidis, and with another small plasmid from Salmonella typhimurium. A strain of Escherichia coli carrying this plasmid, or a cloned 3.7-kb PvuII restriction fragment, showed a slower growth rate, especially in minimal medium, as well as a noticeable increase in DNA methyltransferase activity.

Blotting, Southern↗
PubMed2005

Protein diversity confers specificity in plasmid segregation.

The ParG segregation protein (8.6 kDa) of multidrug resistance plasmid TP228 is a homodimeric DNA-binding factor. The ParG dimer consists of intertwined C-terminal domains that adopt a ribbon-helix-helix architecture and a pair of flexible, unstructured N-terminal tails. A variety of plasmids possess partition loci with similar organizations to that of TP228, but instead of ParG homologs, these plasmids specify a diversity of unrelated, but similarly sized, partition proteins. These include the proteobacterial pTAR, pVT745, and pB171 plasmids. The ParG analogs of these plasmids were characterized in parallel with the ParG homolog encoded by the pseudomonal plasmid pVS1. Like ParG, the four proteins are dimeric. No heterodimerization was detectable in vivo among the proteins nor with the prototypical ParG protein, suggesting that monomer-monomer interactions are specific among the five proteins. Nevertheless, as with ParG, the ParG analogs all possess significant amounts of unordered amino acid residues, potentially highlighting a common structural link among the proteins. Furthermore, the ParG analogs bind specifically to the DNA regions located upstream of their homologous parF-like genes. These nucleoprotein interactions are largely restricted to cognate protein-DNA pairs. The results reveal that the partition complexes of these and related plasmids have recruited disparate DNA-binding factors that provide a layer of specificity to the macromolecular interactions that mediate plasmid segregation.

Bacterial Proteins↗
PubMed2006

Cryptic plasmids isolated from Campylobacter strains represent multiple, novel incompatibility groups.

Three small, cryptic plasmids from the multi-drug-resistant (MDR) Campylobacter coli strain RM2228 and one small, cryptic plasmid from the MDR Campylobacter jejuni strain RM1170 were sequenced and characterized. pCC2228-1 has some similarity to Firmicutes RepL family plasmids that replicate via a rolling-circle mechanism. pCC2228-2 is a theta-replicating, iteron-containing plasmid (ICP) that is a member of the same incompatibility (Inc) group as previously described Campylobacter shuttle vectors. The other two ICPs, pCC2228-3 and pCJ1170, represent a second novel Inc group. Comparison of the four plasmids described in this study with other characterized plasmids from C. jejuni, C. coli, C. lari, and C. hyointestinalis suggests that cryptic plasmids in Campylobacter may be classified into as many as nine Inc groups. The plasmids characterized in this study have several unique features suitable for the construction of novel Campylobacter shuttle vectors, e.g., small size, absence of many common multiple-cloning site restriction sites, and Inc groups not represented by current Campylobacter shuttle plasmids. Thus, these plasmids may be used to construct a new generation of Campylobacter shuttle vectors that would permit transformation of environmental Campylobacter isolates with an existing repertoire of native plasmids.

Bacterial Proteins↗
PubMed2004

Characterization of the novel Fusobacterium nucleatum plasmid pKH9 and evidence of an addiction system.

Fusobacterium nucleatum is an important oral anaerobic pathogen involved in periodontal and systemic infections. Studies of the molecular mechanisms involved in fusobacterial virulence and adhesion have been limited by lack of systems for efficient genetic manipulation. Plasmids were isolated from eight strains of F. nucleatum. The smallest plasmid, pKH9 (4,975 bp), was characterized and used to create new vectors for fusobacterial genetic manipulation. DNA sequence analysis of pKH9 revealed an open reading frame (ORF) encoding a putative autonomous rolling circle replication protein (Rep), an ORF predicted to encode a protein homologous to members of the FtsK/SpoIIIE cell division-DNA segregation protein family, and an operon encoding a putative toxin-antitoxin plasmid addiction system (txf-axf). Deletion analysis localized the pKH9 replication region in a 0.96-kbp fragment. The pKH9 rep gene is not present in this fragment, suggesting that pKH9 can replicate in fusobacteria independently of the Rep protein. A pKH9-based, compact Escherichia coli-F. nucleatum shuttle plasmid was constructed and found to be compatible with a previously described pFN1-based fusobacterial shuttle plasmid. Deletion of the pKH9 putative addiction system (txf-axf) reduced plasmid stability in fusobacteria, indicating its addiction properties and suggesting it to be the first plasmid addiction system described for fusobacteria. pKH9, its genetic elements, and its shuttle plasmid derivatives can serve as useful tools for investigating fusobacterial properties important in biofilm ecology and pathogenesis.

Amino Acid Motifs↗
PubMed2004

Characterization of a theta-replicating plasmid from Streptococcus thermophilus.

Plasmids of Streptococcus thermophilus were previously classified, based on DNA homology, into at least four groups (A-D). Here, we report the characterization of plasmids of group B and D. The sequence analysis of pSMQ173b (group D) indicates that this plasmid contains 4449 bp, five open reading frames (ORFs) and replicates via the rolling-circle mechanism of the pGI3 family. The plasmid pSMQ308 (group B) contains 8144 bp and six ORFs. Two ORFs likely encode a primase/helicase and an integrase. Northern blot experiments demonstrate that these two orfs are transcribed within the three strains containing plasmids of group B. Two-dimensional agarose gel electrophoresis shows that pSMQ308 replicates via a theta mechanism. To our knowledge, this is the first report of a plasmid replicating via a theta mode in S. thermophilus. Finally, a classification of 20 sequenced S. thermophilus plasmids into six groups based on their mode of replication is proposed.

Amino Acid Sequence↗
PubMed1990

Physical map and properties of a 90-MDa plasmid of Azospirillum brasilense Sp7.

Homology was previously detected between the DNA restriction fragments containing Rhizobium meliloti nodulation genes and the 90-MDa plasmid, p90, of Azospirillum brasilense Sp7. Two DNA loci from Sp7 genome that complement mutations in the exopolysaccharide synthesis genes, exoB and exoC, of R. meliloti were also shown to be present on the plasmid. A more detailed characterization of the plasmid was undertaken to establish its physical map and to localize the nod homologies and other specific regions. Six loci were mapped, the region homologous to the nodulation genes, nodPQ, of R. meliloti, the exoB and exoC mutation-correcting loci, a locus for Ap resistance, a bla homology region different from the Ap resistance locus, and a region necessary for the maintenance of p90 as an independent replicon. Mobilization into Agrobacterium tumefaciens of p90-Tn5-Mob was obtained at a frequency of 10(-4), with the plasmid helper pJB3JI. Self-transfer of p90 was not demonstrated. Fragments of p90 hybridized with a plasmid of 90 MDa present in most A. brasilense and some A. lipoferum strains, suggesting a plasmid family in Azospirillum.

Ampicillin Resistance↗
PubMed2001

The TOL plasmid pWW0 xylN gene product from Pseudomonas putida is involved in m-xylene uptake.

The upper operon of the TOL plasmid pWW0 of Pseudomonas putida encodes a set of enzymes involved in the conversion of toluene and xylenes to their carboxylic acid derivatives. The last gene of the upper operon, xylN, encodes a 465-amino-acid polypeptide which exhibits significant sequence similarity to FadL, an outer membrane protein involved in fatty acid transport in Escherichia coli. To analyze the role of the xylN gene product, xylN on TOL plasmid pWW0 was disrupted by inserting a kanamycin resistance gene, and the phenotypes of P. putida harboring the wild-type and xylN mutant TOL plasmids were characterized. The growth of P. putida harboring the wild-type TOL plasmid was inhibited by a high concentration of m-xylene, while that of P. putida harboring the xylN mutant TOL plasmid was not. The apparent K(s) value for the oxidation of m-xylene in intact cells of the xylN mutant was fourfold higher than that of the wild-type strain, although the TOL catabolic enzyme activities in cell extracts from the two strains were almost identical. We therefore presume that the xylN gene product is a porin involved in the transport of m-xylene and its analogues across the outer membrane. Western blot analysis confirmed the localization of XylN in the outer membrane.

Bacterial Outer Membrane Proteins↗
PubMed1985

Role of the b subunit of the Escherichia coli proton-translocating ATPase. A mutagenic analysis.

A plasmid that carries the uncF gene of Escherichia coli, which codes for the b subunit of the proton-translocating ATPase (F1F0), was mutagenized with hydroxylamine and ethyl methanesulfonate. Mutated plasmids were characterized by complementation analysis and by in vitro transcription/translation. Eleven of the plasmids mutated specifically in uncF were studied in detail: the nucleotide sequences of their uncF genes were determined and their effects on the F1F0 were measured. The results suggest that a C-terminal portion of the b subunit that involves the glycine residue at position 131, is required for the formation of a functional proton pore as well as for the binding of F1 to F0. A mutation in the N-terminal portion of the b subunit, in the glycine residue at position 9, also prevented the formation of a functional proton pore, but had only a small effect on the binding of F1 to F0.

Amino Acid Sequence↗
PubMed1989

Involvement of integration host factor (IHF) in maintenance of plasmid pSC101 in Escherichia coli: characterization of pSC101 mutants that replicate in the absence of IHF.

Escherichia coli mutants defective in the stable maintenance of plasmid pSC101 have been isolated following Tn10 insertion mutagenesis. One class of mutations affecting pSC101 replication was located in the genes himA and himD (hip), which encode the two subunits of integration host factor (IHF), a small histonelike DNA-binding protein that has multiple cellular functions. Mutants of pSC101 that could replicate in the absence of IHF were isolated and characterized; four independent mutational alterations were found to affect the third codon of the pSC101 rep gene, resulting in the replacement of glutamic acid by lysine. The compensating alteration appears to function by altering the activity of the pSC101 rep protein in him mutants.

Bacterial Proteins↗
PubMed1995

Cloning and characterization of a novel, plasmid-encoded trimethoprim-resistant dihydrofolate reductase from Staphylococcus haemolyticus MUR313.

In recent years resistance to the antibacterial agent trimethoprim (Tmp) has become more widespread, and several trimethoprim-resistant (Tmpr) dihydrofolate reductases (DHFRs) have been described from gram-negative bacteria. In staphylococci, only one Tmpr DHFR has been described, the type S1 DHFR, which is encoded by the dfrA gene found on transposon Tn4003. In order to investigate the coincidence of high-level Tmp resistance and the presence of dfrA, we analyzed the DNAs from various Tmpr staphylococci for the presence of dfrA sequences by PCR with primers specific for the thyE-dfrA genes from Tn4003. We found that 30 or 33 isolates highly resistant to Tmp (MICs, > or = 512 micrograms/ml) contained dfrA sequences, whereas among the Tmpr (MICs, < or = 256 micrograms/ml) and Tmps isolates only the Staphylococcus epidermidis isolates (both Tmpr and Tmps) seemed to contain the dfrA gene. Furthermore, we have cloned and characterized a novel, plasmid-encoded Tmpr DHFR from Staphylococcus haemolyticus MUR313. The dfrD gene of plasmid pABU17 is preceded by two putative Shine-Dalgarno sequences potentially allowing for the start of translation at two triplets separated by nine nucleotides. The predicted protein of 166 amino acids, designated S2DHFR, encoded by the longer open reading frame was overproduced in Escherichia coli, purified, and characterized. The molecular size of the recombinant S2DHFR was determined by ion spray mass spectrometry to be 19,821.2 +/- 2 Da, which is in agreement with the theoretical value of 19,822 Da. In addition, the recombinant S2DHFR was shown to exhibit DHFR activity and to be highly resistant to Tmp.

Amino Acid Sequence↗