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PubMed1996

Intramolecular recombination of a mitochondrial minicircular plasmid-like DNA of date-palm mediated by a set of short direct-repeat sequences.

A molecular clone containing the complete sequence of a mitochondrial circular plasmid-like DNA (the R plasmid) isolated from the date-palm variety V3DP was used as a probe in Southern analyses of mitochondrial DNA prepared from other varieties. Another circular structure (the S plasmid) was detected in some of these varieties, and sequenced from variety V2DP. It appears that the R plasmid could have arisen from the S plasmid by an intermolecular recombination event at a set of 26-bp imperfect short direct repeats.

Base Sequence↗

PubMed1993

Integration of repeated sequences (pBR322) in the Bacillus subtilis 168 chromosome without affecting the genome structure.

The Escherichia coli plasmid pBR322 sequence (4363 bp) was integrated at the met, pro, or leuB locus of the Bacillus subtilis chromosome without duplication of the flanking chromosomal regions. The integrated pBR322 was stably maintained as part of the chromosome regardless of its orientation or location. It was found that a DNA segment as large as 17 kb cloned in pBR322 can be readily transferred to the B. subtilis chromosome by transformation. It was demonstrated that a second pBR322 sequence could be effectively introduced at different regions of the chromosome by sequential transformation using chromosomal DNA isolated from a strain that had already acquired a pBR322 sequence at a different locus. Similarly, a third pBR322 sequence could be introduced. By this method, two or three pBR322 sequences can be incorporated at unlinked loci without affecting the overall structure of the B. subtilis genome.

Bacillus subtilis↗

PubMed1982

Chromosomal genes for ColV plasmid-determined iron(III)-aerobactin transport in Escherichia coli.

Four chromosomal genes, tonA (fhuA), fhuB, tonB, and exbB, were required for the transport of iron(III)-aerobactin specified by the plasmids ColV-K311, ColV-K229, ColV-K328, and ColV-K30. These genes also determine the transport system in Escherichia coli for the iron ionophore ferrichrome. Aerobactin and ferrichrome are both iron ligands of the hydroxamate type, but they are of different structure. The ColV plasmids determine an outer membrane protein that serves as a receptor for cloacin. Cloacin-resistant mutants were devoid of iron(III)-aerobactin transport but were unimpaired in ferrichrome transport. We conclude that for iron(III)-aerobactin transport two outer membrane proteins, the TonA and the cloacin receptor protein, have to interact functionally or structurally or both.

Bacterial Outer Membrane Proteins↗

PubMed1983

Thermal regulation of membrane fluidity in Escherichia coli. Effects of overproduction of beta-ketoacyl-acyl carrier protein synthase I.

Multicopy plasmids bearing the structural gene (fabB) for beta-ketoacyl-acyl carrier protein (ACP) synthase I were constructed in vitro and transformed into various Escherichia coli strains. Introduction of these plasmids into fabB strains resulted in a fabB+ phenotype and a large (8- to 10-fold) overproduction of synthase I activity. Strains carrying these plasmids were also unusually resistant to cerulenin (an antibiotic that specifically inhibits beta-ketoacyl-ACP synthase activity) and overproduced cis-vaccenic acid. Strains (fabF-) lacking beta-ketoacyl-ACP synthase II are deficient in both cis-vaccenic acid synthesis and thermal regulation. Introduction of the fabB plasmids into these strains resulted in the restoration of cis-vaccenic acid synthesis. However, the plasmid-engendered cis-vaccenic acid synthesis of these strains was unaffected by temperature. These results demonstrate that synthase II, the product of the fabF gene, is the sole enzyme regulating the temperature-dependent composition of the membrane phospholipid acyl chains.

3-Oxoacyl-(Acyl-Carrier-Protein) Synthase↗

PubMed1996

Atomic force microscopic study on topological structures of pBR322 DNA.

Plasmid pBR322 DNA (0.5 mg/mL) isolated from Escherichia coli HB101 was suspended in Tris-HCI-EDTA (1 mol/L-0.1 mol/L, pH 8.5); then a drop of the above solution was deposited on freshly cleaved mica substrate. After adsorption for about 1 mm, the sample was stained with phosphotungstic acid. The residual solution was removed with a piece of filter paper. Afterwards the sample was imaged with a home-made atomic force microscope (AFM) in air. The AFM images of pBR322 DNA with a molecular resolution have been obtained. These images show that pBR322 DNA exists in several different topological structures: (i) relaxed circular DNA with a different diameter; (ii) supercondensed DNA with different particle sizes; (iii) dimeric catenane connected by one relaxed circular molecule and another close-compacted molecule which might be either supercoiled or intramolecular knotted form; (iv) oligomeric catenane with multiple irregular molecules in which DNA is interlocked into a complex oligomer; (v) possibly-existing intermediates formed from monomer to oligomer.

DNA, Bacterial↗

PubMed2003

Effect of poly(beta-hydroxybutyrate) accumulation on the stability of a recombinant plasmid in Escherichia coli.

Poly(beta-hydroxybutyrate) (PHB) production using a recombinant Escherichia coli VG1 (pTU14) was carried out. PHB granules were gradually biosynthesized in recombinant cells as inclusion bodies and the morphology of cells became linearly inflated with PHB accumulation from 6 h to 36 h of culture. The stability of plasmid pTU14 was inevitably affected by in-cell PHB accumulation. During repeated subcultures under both PHB-accumulating and non-PHB-accumulating conditions, plasmid DNAs of pTU14 were extracted and analyzed after more than 60 generations. The results showed that plasmid pTU14 is structurally stable but segregationally unstable, and that the segregational instability worsened with increasing accumulation of PHB granules. This is due to not only the serious metabolic burden of plasmid replication and gene expression on the host cell, but also the volume effects of PHB granules on the natural random distribution of plasmids during the binary fission of cells.

Journal Article↗

PubMed1986

Characterisation of chloramphenicol resistance plasmids of Staphylococcus aureus and S. epidermidis by restriction enzyme mapping techniques.

Chloramphenicol resistance (Cmr) plasmids pSK2 and pSK5 from Staphylococcus aureus and pSK102 and pSK103 from S. epidermidis have been characterised and detailed restriction endonuclease cleavage maps constructed. TaqI digestion profiles illustrated the identity of pSK5 and pSK102 and also revealed a high degree of similarity between these four Cmr plasmids from Australian staphylococci and three Cmr plasmids from S. aureus strains of geographically unrelated origin. DNA-DNA hybridisation indicated that the chloramphenicol acetyltransferase determinant carried by pSK5/pSK102 could be found on other structurally-distinct Cmr plasmids. The role of S. epidermidis as a reservoir for Cmr plasmids found in S. aureus is discussed.

Base Sequence↗

PubMed1990

Regulation of tfdCDEF by tfdR of the 2,4-dichlorophenoxyacetic acid degradation plasmid pJP4.

The closely linked structural genes tfdCDEF borne on the 2,4-dichlorophenoxyacetic acid (TFD) catabolic plasmid, pRO101, were cloned into vector pRO2321 as a 12.6-kilobase-pair BamHI C fragment and designated pRO2334. The first gene in this cluster, tfdC, encodes chlorocatechol 1,2-dioxygenase and was expressed constitutively. Chlorocatechol 1,2-dioxygenase expression by pRO2334 was repressed in trans by the negative regulatory element, tfdR, on plasmid pRO1949. Derepression of tfdC was achieved when Pseudomonas aeruginosa PAO4032 containing both plasmids pRO2334 and pRO1949 was grown in minimal glucose medium containing TFD, 2,4-dichlorophenol, or 4-chlorocatechol, suggesting that TFD and other pathway intermediates can act as inducing compounds. Genetic organization of the tfdCDEF cluster was established by deletion of the tfdC gene, which resulted in the loss of tfdD and tfdE activity, suggesting that genes tfdCDEF are organized in an operon transcribed from the negatively regulated promoter of tfdC. Deletion subcloning of pRO1949 was used to localize tfdR to a 1.2-kilobase-pair BamHI-XhoI region of the BamHI E fragment of plasmid pRO101. The tfdR gene product was shown not to regulate the expression of tfdB, which encodes 2,4-dichlorophenol hydroxylase.

2,4-Dichlorophenoxyacetic Acid↗

PubMed1982

Nitrogen fixation (nif) genes and large plasmids of Rhizobium japonicum.

The location of structural nitrogen-fixation genes was determined for the slow- and fast-growing types of Rhizobium japonicum. Slow-growing R. japonicum strains do not harbor structural nif genes, homologous to nifD and nifH, on large plasmids (100 to 200 megadaltons). In contrast, all fast-growing R. japonicum strains, except PRC194, contain structural nif genes on large plasmids.

DNA, Recombinant↗

PubMed1985

Construction of a series of ompF-ompC chimeric genes by in vivo homologous recombination in Escherichia coli and characterization of the translational products.

OmpF and OmpC are major outer membrane proteins. Although they are homologous proteins, they function differently in several respects. As an approach to elucidate the submolecular structures that determine the difference, a method was developed to construct a series of ompF-ompC chimeric genes by in vivo homologous recombination between these two genes, which are adjacent on a plasmid. The genomic structures of these chimeric genes were determined by restriction endonuclease analysis and nucleotide sequence determination. In almost all cases, recombination took place between the corresponding homologous regions of the ompF and ompC genes. Many of the chimeric genes produced proteins that migrated to various positions between the OmpF and OmpC proteins on polyacrylamide gel. On the basis of the results, a domain contributing to the mobility difference the OmpF and OmpC proteins was identified. Some chimeric genes did not accumulate outer membrane proteins, despite the fact that the fusion of the ompF and ompC genes was in frame. Bacterial cells possessing the chimeric proteins were also tested as to their sensitivity to phages which require either OmpF or OmpC as a receptor component. The chimeric proteins were either of the OmpF or OmpC type with respect to receptor activity. Based on the observations, the roles of submolecular domains in the structure, function, and biogenesis of the OmpF and OmpC proteins are discussed.

Bacterial Outer Membrane Proteins↗

PubMed1998

Protection of mice against lethal coxsackievirus B3 infection by using DNA immunization.

Vaccination with DNA and recombinant vaccinia viruses (rec.VV) has been studied with the coxsackievirus B3 (CVB3) model system. Plasmids encoding all structural proteins of CVB3, when injected intramuscularly, induced only low levels of virus-specific antibodies. However, DNA vaccination with the major structural protein VP1 protected 72.2% of mice from lethal challenge, whereas VP1 expressed by rec.VV was much less efficient.

Animals↗

PubMed2005

Two-cistronic expression plasmids for high-level gene expression in Escherichia coli preventing translational initiation inhibition caused by the intramolecular local secondary structure of mRNA.

Two-cistronic expression plasmids for the wild-type solubilized domain of porcine NADPH-cytochrome P450 reductase (PsCPR) gene in Escherichia coli were systematically constructed using a solubilized domain of porcine cytochrome b5 gene (Psb5 gene) or a derivative of it as the first cistron to examine their utility for second gene expression preventing the translational inhibition caused by the intramolecular local secondary structure of mRNA at the ribosome-binding site (RBS). The mRNAs from the plasmids lacking an RBS for the second cistron (SD2) accumulated very low levels of PsCPR, while those from the plasmids having an SD2 accumulated higher levels of PsCPR. The level of accumulation of PsCPR by the mRNA from plasmid pCbSD-T-CPR-3, which has an SD2 upstream of the termination codon of the first cistron, was higher than for those with an SD2 in the intercistronic region. The predicted intramolecular local secondary structures at the SD2 of mRNAs from these plasmids were stable enough to cause translational initiation inhibition. These results indicate that the use of a two-cistronic expression plasmid is an effective way to overcome translational initiation inhibition. Improved plasmids, pCP1 and pCP2P, were constructed from pCbSD-T-CPR-3. Using these plasmids, the solubilized donain of porcine NADH-cytochrome b5 reductase was also highly accumulated on prevention of the translational initiation inhibition. These plasmids are expected to be useful tools for the comprehensive high-level expression of heterologous genes in E. coli cells.

Amino Acid Sequence↗

PubMed1975

Electron microscope heteroduplex studies of sequence relations among bacterial plasmids: identification and mapping of the insertion sequences IS1 and IS2 in F and R plasmids.

Heteroduplex experiments between the plasmid R6 and one strand of the deoxyribonucleic acid (DNA) of a lambda phage carrying the insertion sequence IS1 show that IS1 occurs on R6 at the two previously mapped junctions of resistance transfer factor (RTF) DNA with R-determinant DNA. From previous heteroduplex experiments, it then follows that IS1 occurs at the same junctions in R6-5, R100-1, and R1 plasmids. Heteroduplex experiments with the DNA from a lambda phage carrying the insertion sequence IS2 show that one copy of IS2 occurs in R6, R6-5, and R100-1 (but not R1) at a point within the RTF with coordinates 67.5 TO 68.9 kilobase units (kb). In an accompanying paper, Ptashne and Cohen (1975) show that the insertion sequence IS3 occurs on R6 and R6-5. R100-25, a traC mutant, differs from its parent R100-1 only in that it contains an additional copy of IS1 inserted within the tra gene region of 82.1 kb. R100-31, atraX, TC-s mutant of R100-1, is deleted in R100-1 sequences starting at one of the IS3 termini (46.9 kb) and extending with RTF to 61.0 kb. Heteroduplex studies of F plasmids with the DNA of a lambda phage bearing insertion sequence IS2 show that the sequence of F with coordinates 16.3-17.6F is IS2. The occurrence of IS1 at the two junctions of R-determinant DNA and RTF DNA in R plasmids provides a structural basis to explain the mechanism of the previously observed formation of molecules containing one RTF unit and several tandem copies of the R-determinant unit, when R plasmids in Proteus mirabilis are grown in the presence of antibiotics, and the segregation of an R plasmid into an RTF unit and an R-determinant unit. In general, correlation of our results with previous studies shows that insertion sequences play a role in a variety of F- and R-related intra- and intermolecular recombination phenomena.

Base Sequence↗

PubMed1976

The arrangement of 18-S and 28-S ribosomal ribonucleic acids within the 40-S precursor molecule of Xenopus laevis.

The arrangement of 18-S rRNA and 28-S rRNA within their 40-S common precursor molecule (pre-rRNA) of Xenopus laevis was investigated by electron microscopic analysis of secondary structure of nascent pre-rRNA chains of oocytes, and by 5'-end analysis of 18-S rRNA and 28-S rRNA hybridized to the EcoRI fragment of rDNA cloned as plasmid pCD42. Secondary structure mapping of phenol-extracted RNA from nucleolar cores revealed complete pre-rRNA chains or molecules at various stages of processing and pre-rRNA molecules apparently lacking one end. In this latter group, which was regarded as representing nascent chains, more than 90% of the molecules had no 28-S rRNA REGION. This shows that the 28-S rRNA sequence is transcribed after the 18-S rRNA region and hence must be located nearer to the 3' end of the pre-rRNA molecule. For 5' end-group determination [3H]uridine-labelled 18-S rRNA and 28-S rRNA were hybridized, as fragments of about 200 nucleotides, to the plasmid pCD42 containing coding sequences for four-fifths of the 18-S rRNA sequence, the external transcribed spacer, the non-transcribed spacer and a tenth of the 28-S rRNA sequence. The RNA was recovered from the hybrids and analyzed for uridine 3',5'-bisphosphate (pUp) after alkaline hydrolysis. The pUp content of the hybridized 18-S rRNA fragments was 20-fold higher than in those of 28-S rRNA, THUS DEMONSTRATING THAT THE 5' END OF THE 18-S rRNA is located next to the external spacer region. From these results it is concluded that the 18-S rRNA is located close to the 5' end of the 40-S pre-rRNA molecule.

Animals↗

PubMedDate not supplied

[Visualization of the cruciform structure of superhelical DNA by use of atomic force microscopy].

Atomic force microscopy was used to visualize the cruciform structure in supercoiled plasmid pUC8 DNA immobilized on aminomodified mica. The cruciform hairpin was 14 base pairs in size, as determined from atomic force microscopy images of pUC8 DNA in air. Molecular modeling confirmed that the cruciform structure is formed by hairpins with self-complementary homopyrimidine-homopurine sequences (dT)8(dA)6 and a loop 4 nucleotides long.

Base Sequence↗

PubMed1984

Mutagenesis of the metJBLF gene cluster with transposon Tn5: localization of the metF transcription unit.

Mutants of a specialized lambda dmet transducing phage bearing the metJBLF gene cluster of Escherichia coli K12 were constructed using transposon Tn5. Two of these mutants, lambda dmet128::Tn5, MW77 and lambda dmet128::Tn5, 3-1, were used to locate precisely as well as confirm the existence of the metF transcription unit (approximately 1,000 base pairs in size). The introduction of new restriction sites within the metJBLF gene cluster due to the Tn5 insertion events allowed the metF transcription unit to be cloned into the high copy number plasmid pBR322. Analyses of the structures of two of these recombinant plasmids, pTJ77H and pTJ13-1H, are presented. Expression of the plasmid borne metF allele in cells grown in the absence, or presence, of exogenous L methionine (0.2 mM) demonstrates that the amplification of the metF copy number does not abolish met regulon mediated control of the gene's activity.

Coliphages↗

PubMed2000

Characterization of a new class of DNA delivery complexes formed by the local anesthetic bupivacaine.

Bupivacaine, a local anesthetic and cationic amphiphile, forms stable liposomal-like structures upon direct mixing with plasmid DNA in aqueous solutions. These structures are on the order of 50-70 nm as determined by scanning electron microscopy, and are homogeneous populations as analyzed by density gradient centrifugation. The DNA within these structures is protected from nuclease degradation and UV-induced damage in vitro. Bupivacaine:DNA complexes have a negative zeta potential (surface charge), homogeneous nature, and an ability to rapidly assemble in aqueous solutions. Bupivacaine:DNA complexes, as well as similar complexes of DNA with other local anesthetics, have the potential to be a novel class of DNA delivery agents for gene therapy and DNA vaccines.

1-Octanol↗

PubMed1985

Regulation of expression from the glnA promoter of Bacillus subtilis requires the glnA gene product.

Expression of the cloned glnA gene [coding for glutamine synthetase (EC 6.3.1.2)] of Bacillus subtilis was 10-fold higher in an Escherichia coli strain grown under nitrogen-limiting conditions than in the same strain under nitrogen-excess conditions. Mutations in the E. coli glnA, glnB, glnD, glnE, glnF, glnG, and glnL genes had no effect on the observed regulation. To test whether sequences within the B. subtilis DNA (3.2 kilobase pairs) were responsible for the observed regulation, a plasmid carrying a transcriptional fusion of the B. subtilis glnA promoter with E. coli lacZ was constructed. beta-Galactosidase levels coded for by this plasmid were found to be negatively regulated in trans by a plasmid carrying the entire B. subtilis glnA gene. Analysis of various deletion plasmids showed that the 1.4-kilobase-pair region encoding glutamine synthetase was necessary for the observed regulation of beta-galactosidase. Plasmids coding for 67% or more of the glutamine synthetase polypeptide gave at least partial repression, but a plasmid carrying 30% of the structural gene, as well as a plasmid carrying a deletion internal to glnA, gave no repression. DNA downstream from glnA (to within 130 base pairs of the end of the gene) was not required for the observed regulation. These results suggest that the glnA gene of B. subtilis is autoregulated, supporting the model for glnA control proposed by Dean et al. [Dean, D. R., Hoch, J. A. & Aronson, A. I. (1977) J. Bacteriol. 131, 981-987].

Bacillus subtilis↗