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PubMed1983

Tn1525, a kanamycin R determinant flanked by two direct copies of IS15.

We have isolated plasmid pIP112 (IncI1) from Salmonella panama and characterized by restriction endonucleases analysis and by recombinant DNA techniques a transposable element designated Tn1525. This 4.44 kilobase (kb) transposon confers resistance to kanamycin by synthesis of an aminoglycoside phosphotransferase (3') (5") type I and contains two copies of IS15 (1.5 kb) in direct orientation. The modular organisation of Tn1525 offers the possibility for intramolecular homologous recombination between the two terminal direct repeats and thus accounts for the in vivo structural lability of plasmid pIP112: instability of kanamycin resistance and tandem amplification of the kanamycin determinant. Other transposons mediating resistance to kanamycin by the same enzymatic mechanism were analysed by agarose and polyacrylamide gel electrophoresis, following digestion with restriction endonucleases, and by Southern hybridizations. These comparisons indicate that, although the structural genes for the phosphotransferases are homologous, Tn1525 differs from Tn903 and Tn2350 and is closely related but distinct from Tn6. Using the same techniques Tn1525 was detected on plasmids belonging to different incompatibility groups and originating from various species of Gram-negative clinical isolates. These results indicate that Tn1525 is representative of a new family of class I composite transposons already spread in diverse pathogenic bacterial genera.

Chromosome Mapping↗
PubMed2003

Completed sequence of plasmid pIP501 and origin of spontaneous deletion derivatives.

The sequence of plasmid pIP501 (30,603 bp) was completed using previously published and newly acquired data. The sites at which two spontaneous deletions had occurred were identified. One was between tracts of repeated heptamers and the other between regions of secondary structure associated with plasmid replication. A high level of identity ( >95%) between plasmid pIP501 and part of plasmid pRE25, which had been isolated from Enterococcus faecalis associated with a food source, was confirmed.

Base Sequence↗
PubMed1979

Transaminase B from Escherichia coli: quaternary structure, amino-terminal sequence, substrate specificity, and absence of a separate valine-alpha-ketoglutarate activity.

Transaminase B (branched-chain amino acid aminotransferase, EC 2.6.1.42), the ilvE gene product, was purified to apparent homogeneity from an Escherichia coli K-12 strain which carries the ilvE gene both on the host chromosome and on a plasmid. The oligomeric structure of the enzyme, as determined by analytical ultracentrifugation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was confirmed to be that of a hexamer with a molecular weight of about 182,000 and apparently identical subunits. Cross-linking with dimethylsuberimidate yielded trimers, dimers, and monomers, but essentially no species of higher molecular weight. These results are consistent with a double-trimer arrangement of the subunits in native enzyme. The amino-terminal sequence was found to be: Gly Thr Lys Lys Ala Asp Tyr Ile (Trp) Phe Asn Gly (Thr) (Met) Val. Purified transaminase B catalyzed transamination between alpha-ketoglutarate and l-isoleucine, l-leucine, l-valine, and, to a lesser extent, l-phenylalanine and l-tyrosine, the latter reacting very sluggishly. The enzyme was free of aspartate transaminase and of transaminase C. The apparent K(m) values for the branched-chain alpha-ketoacids were smaller than those for the corresponding amino acids. The lowest K(m) was recorded for dl-alpha-keto-beta-methyl-n-valerate, and the highest was recorded for l-valine. The ratio of the valine- and isoleucine-alpha-ketoglutarate activities did not change significantly during purification, and both activities were quantitatively removed from crude extract by antibody raised against purified transaminase B. These observations argue against the existence of a separate valine-alpha-ketoglutarate transaminase. Anti-E. coli transaminase B antibody cross-reacted with crude extract from Salmonella typhimurium, but not with extract obtained from Pseudomonas aeruginosa.

Amino Acid Sequence↗
PubMed1986

An extrachromosomal plasmid is the etiological precursor of kalDNA insertion sequences in the mitochondrial chromosome of senescent neurospora.

In the kalilo strains of N. intermedia, senescence is initiated by insertion of a 9.0 kb foreign nucleotide sequence, kalDNA, into mitochondrial DNA. A 9.0 kb linear DNA plasmid that is structurally homologous to the mitochondrial kalDNA insertion sequences exists in high copy numbers in close association with the nuclei of presenescent and senescent kalilo cells, but is not present in cells of long-lived normal strains. The free kalilo plasmid has not been detected in mitochondria, suggesting that the element does not contain a mitochondrial origin of replication. Unexpectedly, the nuclear plasmid, like the mitochondrial insertion element, follows a strict pattern of maternal inheritance. We surmise that the extramitochondrial plasmid is the etiological precursor of the kalDNA insertion sequences that appear in the mtDNAs of senescent cell lines and conclude that the kalilo element induces senescence because it is a mutator of mitochondrial genes.

DNA Replication↗
PubMed2005

Nucleotide sequence, structural organization and host range of pRS4, a small cryptic Pediococcus pentosaceus plasmid that contains two cassettes commonly found in other lactic acid bacteria.

The complete nucleotide sequence of the cryptic plasmid pRS4 (3550 bp) from Pediococcus pentosaceus RS4 was determined. Sequence analysis revealed the presence of three open reading frames (ORFs). The putative protein coded by ORF 1 showed 93% identity with the mobilization protein of Lactobacillus casei plasmid pLC88 and 94% identity with a sequenced fragment of the mobilization protein of P. damnosus plasmid pF8801, suggesting a common origin for these three mobilization proteins. The putative protein coded by ORF 2 showed 92% identity with the replication protein of L. plantarum plasmid pWCFS101, a plasmid that replicates via the rolling circle (RC) mechanism, suggesting a similar replication mechanism for pRS4. Supporting this hypothesis, a putative double strand origin (dso) and a region with palindromic sequences that could function as single strand origin (sso), were detected in pRS4. A function could not be assigned to ORF 3. Since ORF 1 exhibits high identity with L. casei plasmid pLC88 but lower identity (58%) with other Lactobacillus plasmids, and ORF 2 exhibits high identity with the L. plantarum plasmid pWCFS101 but lower identity (55-58%) with other Lactobacillus plasmids (including pLC88), two independent cassettes, from different sources, seem to be involved in the structure of pRS4. Plasmids derived from pRS4 containing the chloramphenicol resistance gene were successfully electrotransformed in L. plantarum, L. casei, P. pentosaceus, and Pediococcus acidilactici, suggesting that pRS4 could be used as a cloning vector for lactic acid bacteria. To our knowledge pRS4 is the first RC-replicating plasmid of P. pentosaceus that has been completely sequenced and used as cloning vector.

Base Sequence↗
PubMed1989

Characterization of mutant mitochondrial plasmids of Neurospora spp. that have incorporated tRNAs by reverse transcription.

The Mauriceville and Varkud mitochondrial plasmids of Neurospora spp. are closely related, closed-circular DNAs (3.6 and 3.7 kilobases, respectively) whose nucleotide sequences and genetic organization suggest relationships to mitochondrial introns and retroelements. We have characterized nine suppressive mutants of these plasmids that outcompete mitochondrial DNA and lead to impaired growth. All nine suppressive plasmids contain small insertions, corresponding to or including a mitochondrial tRNA (tRNATrp, tRNAGly, or tRNAVal) or a tRNA-like sequence. The insertions are located at the position corresponding to the 5' end of the major plasmid transcript or 24 nucleotides downstream near a cognate of the sequence at the major 5' RNA end. The structure of the suppressive plasmids suggests that the tRNAs were inserted via an RNA intermediate. The 3' end of the wild-type plasmid transcript can itself be folded into a secondary structure which has tRNA-like characteristics, similar to the tRNA-like structures at the 3' ends of plant viral RNAs. This structure may play a role in replication of the plasmids by reverse transcription. Major transcripts of the suppressive plasmids begin at the 5' end of the inserted mitochondrial tRNA sequence and are present in 25- to 100-fold-higher concentrations than are transcripts of wild-type plasmids. Mapping of 5' RNA ends within the inserted mtDNA sequences identifies a short consensus sequence (PuNPuAG) which is present at the 5' ends of a subset of mitochondrial tRNA genes. This sequence, together with sequences immediately upstream in the plasmids, forms a longer consensus sequence, which is similar to sequences at transcription initiation sites in Neurospora mitochondrial DNA. The suppressive behavior of the plasmids is likely to be directly related to the insertion of tRNAs leading to overproduction of plasmid transcripts.

DNA Mutational Analysis↗
PubMed1985

Generation of deletions through a cis-acting mutation in plasmid pC194.

When plasmid pC194-1 is ligated to pBR322 to generate plasmid pHV15-1, deletions occur with high frequency within the joined pBR322 DNA. Generation of deletions is recE4 independent, and occurs in B. subtilis with a 1,000-fold higher frequency than in Escherichia coli. In the hybrid plasmid pVH15-1, deletion end-points are not at random, but at defined locations within pBR322. We propose that the base alteration, characterizing pC194-1, has stabilized within the plasmid a stem/loop structure, which acts as a deletion generator.

Bacillus subtilis↗
PubMed1987

Enzyme systems initiating replication at the origin of the Escherichia coli chromosome.

More than ten proteins are known to participate in replication of plasmids bearing the unique origin of the Escherichia coli chromosome (oriC). Initiation of replication of oriC plasmids has been resolved into five separable stages. An initial complex formation (Stage I) requires an oriC plasmid, dnaA protein and HU protein. In the presence of ATP at a temperature of greater than 28 degrees C, a dnaB-C protein complex interacts to form a prepriming complex (Stage II). This is followed by extensive unwinding of the template that depends on the further addition of gyrase and single-strand binding protein (SSB) (Stage III). Hydrolysis of an rNTP by dnaB protein (a helicase action) and of ATP by gyrase (a swivelling action) drives the extreme unwinding of the template. This unwound template-protein complex is the substrate for priming by primase (Stage IV) and elongation by DNA polymerase III holoenzyme (Stage V). Priming of all DNA chains is done by primase; RNA polymerase functions in template activation rather than priming. DNA polymerase III holoenzyme, composed of at least seven subunits, synthesizes the DNA chains. The alpha subunit is the polymerase, the epsilon subunit is the 3'----5' exonuclease; alpha + epsilon is the proofreading activity. Following the synthesis of new DNA chains, DNA polymerase I and ribonuclease H remove the RNA primers, polymerase I fills the gaps, and ligase seals the daughter strands (Stage VI). Replication produces plasmids identical in structure and sequence to the initial template.

Base Sequence↗
PubMedDate not supplied

Preparation and in vitro transfection efficiency of chitosan microspheres containing plasmid DNA:poly(L-lysine) complexes.

PURPOSE: Studies on DNA complexes with cationic polymers are prompted by the search for nonviral DNA carriers for gene therapy. Among them, poly(L-Lysine) (PLL) has been extensively studied. On the other hand, these systems deliver DNA as a bolus without long-term release. The aims of this study were to encapsulate plasmid DNA:poly(L-lysine) (pDNA:PLL) complexes into chitosan microspheres as an alternative to the PLL based gene delivery and investigate its in vitro release and transfection characteristics as well as plasmid DNA integrity and stability against serum and DNase I challenge. METHODS: pUC18 plasmid DNA that encoded beta-galactosidase was used as a model. The microspheres were prepared by complex coacervation method and the release and in vitro transfection properties were investigated. pDNA:PLL complexes were prepared at two different mass ratios. In vitro release studies were performed at 37 +/- 0.5 degrees C and drug release was monitored both spectrophotometrically and fluorometrically. Structural integrity of the pDNA:PLL complexes were determined by Southern blotting analysis. Protective effect of encapsulation of pDNA:PLL complexes against DNase I and serum treatment were also studied. In vitro transfection studies were performed by using 3T3 cell line. RESULTS: According to our in vitro release data, the mass ratio of pDNA:PLL significantly affected the release of pDNA:PLL complexes from chitosan microspheres, and the structure of the plasmid DNA did not change during the experiments. pDNA:PLL-loaded chitosan microspheres indicated high stability against fetal bovine serum and DNase I treatment for a week. In vitro transfection data showed that pDNA:PLL-loaded chitosan microspheres could be effectively transfected 3T3 cells in vitro. CONCLUSION: As a conclusion, pDNA:PLL complexes could be encapsulated into chitosan microspheres with maintaining their structural and functional integrity and this system may be a good alternative for polycation based gene carriers.

3T3 Cells↗
PubMed2005

Metallo-beta-lactamase IMP-1 in Providencia rettgeri from two different hospitals in Japan.

In 2002, 495 indole-positive proteae strains were isolated from patients at 60 hospitals in Japan. Nine indole-positive proteae strains had reduced susceptibility to imipenem (MIC > or = 8 microg ml(-1)) and were identified as Providencia rettgeri by BD Phoenix. Eight of the nine Prov. rettgeri isolates were confirmed as metallo-beta-lactamase producers by the double-disc synergy test. All the metallo-beta-lactamases were classified as IMP-1 by PCR and DNA sequence analysis. These bla(IMP-1) genes were encoded in the integron structure on conjugative plasmids. These plasmids could transfer from Prov. rettgeri clinical isolates to Escherichia coli ML4903 at a frequency between 1.5 x 10(-5) and 5.5 x 10(-7). The eight bla(IMP)-positive strains were isolated from two hospitals, and showed two different PFGE patterns, two different integron structures and two different incompatibility groups, which corresponded to the two hospitals. These results strongly suggest the possibility of nosocomial infections by bla(IMP-1)-producing Prov. rettgeri isolates.

Anti-Bacterial Agents↗
PubMed1996

Co-induction of DNA relaxation and synthesis of DnaK and GroEL proteins in Escherichia coli by expression of LetD (CcdB) protein, an inhibitor of DNA gyrase encoded by the F factor.

We examined the influence of overexpression of LetD (CcdB) protein, an inhibitor of DNA gyrase encoded by the F factor of Escherichia coli, on DNA supercoiling and induction of heat shock proteins. Cells were transformed with a plasmid carrying the structural gene for LetD protein under control of the tac promoter, and LetD protein was induced by adding isopropyl beta-D-thiogalactopyranoside (IPTG) to the culture medium. Analysis by agarose gel electrophoresis in the presence of chloroquine revealed relaxation of plasmid DNA in cells depending on the concentration of IPTG employed for induction. Protein pulse-labeling experiments with [35S]methionine and cysteine revealed that synthesis of DnaK and GroEL proteins was also induced by IPTG, and concentrations necessary for DNA relaxation and induction of the heat shock proteins were much the same. Expression of mutant LetD protein lacking two amino acid residues at the C-terminus induced neither DNA relaxation nor the synthesis of DnaK and GroEL proteins. Induction of wild-type LetD protein but not mutant LetD protein markedly enhanced synthesis of sigma32. We interpret these results to mean that DNA relaxation in cells caused by the expression of LetD protein induces heat shock proteins via increased synthesis of sigma32.

Bacterial Proteins↗
PubMed1991

Secondary structure of Escherichia coli glucosamine-6-phosphate deaminase from amino acid sequence and circular dichroism spectroscopy.

The secondary structure of the purified glucosamine-6-phosphate deaminase from Escherichia coli K12 was investigated by both circular dichroism (CD) spectroscopy and empirical prediction methods. The enzyme was obtained by allosteric-site affinity chromatography from an overproducing strain bearing a pUC18 plasmid carrying the structural gene for the enzyme. From CD analysis, 34% of alpha-helix, 9% of parallel beta-sheet, 11% of antiparallel beta-sheet, 15% turns and 35% of non-repetitive structures, were estimated. A joint prediction scheme, combining six prediction methods with defined rules using several physicochemical indices, gave the following values: alpha-helix, 37%; beta-sheet, 22%; turns, 18% and coil, 23%. The structure predicted showed also a considerable degree of alternacy of alpha and beta structures; 64% of helices are amphipathic and 90% of beta-sheets are hydrophobic. Overall, the data suggest that deaminase has as dominant motif, an alpha/beta structure.

Aldose-Ketose Isomerases↗
PubMed1992

Sex pheromone plasmid pAD1-encoded surface exclusion protein of Enterococcus faecalis.

During conjugative transfer of sex pheromone plasmids of Enterococcus faecalis a so-called surface exclusion protein reduces the frequency with which these plasmids are transferred to cells already possessing the same plasmid. We report here the DNA sequence of a 3.8 kb fragment of the sex pheromone plasmid pAD1 containing the structural gene sea1 for surface exclusion protein and a small open reading frame (ORF) upstream of sea1. Surface exclusion protein Sea1 was found to be highly homologous to the surface exclusion protein Sec10 encoded by the sex pheromone plasmid pCF10. Hybridization studies with DNA probes derived from the structural gene sea1 demonstrated that, with the exception of pAM373, all known sex pheromone plasmids carry a homologous gene. These studies also indicated that the genetic organization is similar in these plasmids, with the structural gene for surface exclusion protein being located 5' to that for aggregation substance.

Amino Acid Sequence↗
PubMed1980

Immunoglobulin gamma 1 heavy chain gene: structural gene sequences cloned in a bacterial plasmid.

Immunoglobulin gamma 1 heavy chain cDNA was cloned into an Escherichia coli plasmid pCR1 and its nucleotide sequence was determined. The hybrid plasmid contained approx. 900-bases-long gamma 1 chain cDNA sequence, including the complete sequence of the CH2 and CH3 domains and the 3' untranslated region, and partial sequence of the CH1 domain. The nucleotide sequence predicts an extra lysine at the carboxyl-terminus of the gamma 1 chain. Comparison of the nucleotide sequence of 3' untranslated regions of the immunoglobulin gamma 1 chain and kappa light chain showed a significant homology although lengths are quite divergent.

Animals↗