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At least 883 records · Page 49Linked to original sources
PubMed1989

Characterization of the termini of linear plasmids from Nectria haematococca and their use in construction of an autonomously replicating transformation vector.

The mitochondria of isolate FS37 from Nectria haematococca mating population I (Fusarium solani f. sp. cucurbitae) contain two linear plasmids, pFSC1 and pFSC2, of 9.2 and 8.3 kbp, respectively. Evidence for a protein blocking the 5' termini of these plasmids was obtained from exonuclease digestion experiments. A single protein band with an apparent Mr of 80 K was labeled when the DNA-protein complex of either plasmid was reacted with [125I] Bolton-Hunter reagent and then digested with DNase I. DNA sequence analysis of the termini of both plasmids revealed long inverted repeats of 1,211 bp (pFSC1) and 1,027 bp (pFSC2). No sequence similarity was found between the terminal inverted repeats (TIRs) of pFSC1 and pFSC2, nor was any similarity identified between the TIRs of these plasmids and sequences of TIRs from other linear DNAs. A restriction fragment containing the TIR of pFSC1 conferred autonomous replication when incorporated into an integrative transformation vector of Ustilago maydis.

Base Sequence↗
PubMed1984

Thermophilic anaerobic bacteria which ferment hemicellulose: characterization of organisms and identification of plasmids.

Seven thermophilic anaerobic bacteria which ferment xylan were isolated from natural geothermal features in the western United States. Typically, these strains were Gram-negative non-sporeforming rods with an unusual double-layered cell wall which resembled that observed in Thermobacteroides acetoethylicus. The strains differed from known thermophilic anaerobes in their ability to utilize a very wide variety of carbohydrates and in their ability to grow in a chemically-defined medium and/or at pH 3.5. Four of the strains contained cryptic plasmids of 1.2 or 1.5 X 10(6) daltons. The taxonomic characteristics of the strains are discussed in terms of their relatedness to those of Thermoanaerobium, Thermoanaerobacter, and Thermobacteroides species.

Bacteria, Anaerobic↗
PubMed1985

Isolation and characterization of mutants of the RP4 plasmid coding for increased resistance to ampicillin.

E. coli strain J53(RP4) was mutagenized with ethyl methanesulfonate and N-methyl-N'-nitro-N-nitrosoguanidine. Clones showing a two-to threefold increase in resistance to ampicillin were produced. This increase was not due to an increased number of RP4 copies per chromosome. The level of penicillinase activity was twice higher in comparison with the parental strain. No detectable changes were found in the region coding for the resistance to ampicillin on the plasmid by restriction analysis.

Ampicillin↗
PubMed1984

Characterization of the bovine papilloma virus plasmid maintenance sequences.

Bovine Papilloma Virus (BPV-1) establishes itself as a multicopy nuclear plasmid in somatic mammalian cells in culture. We report here that two discontinuous regions within the viral genome can independently support extrachromosomal replication of the Tn5 neomycinr gene in cells that provide viral factors in trans. The viral plasmid maintenance sequences (PMS) act in cis and will integrate along with the marker gene in cell lines that do not provide BPV-1 gene products. PMS-1 is localized within a 521 bp region upstream of the BPV-1 early transcription unit; PMS-2 has been localized to a 140 bp region within the putative reading frame for the E1 protein of the viral genome. Recombinant plasmids carrying either of the PMS elements are unrearranged and stably maintained at a constant copy number supernumerary to the resident BPV-1 genomes even in the absence of selective pressure. Specific deletion mutants within the viral genome show that BPV-1 gene products required for morphological transformation are dispensable for plasmid maintenance. In mouse cells cotransformed with such deletion derivatives and an unlinked marker gene (neomycinr or Tk), the marker genes integrate into the host genome while the BPV molecules are nonselectively carried as nuclear plasmids. This result implies that the BPV-1 genome must have signals that specifically preclude integration in the presence of transacting factors.

Animals↗
PubMed1984

Characterization of a mini-F plasmid derived from an F mutant expressing incompatibility in the autonomous but not in the integrated state.

Plasmid DNA from Escherichia coli F' ser/MA219 harboring an altered F' factor, which expressed incompatibility in the autonomous but not in the integrated state (DeVries and Maas, 1973, J. Bacteriol. 115, 213-220), was digested with the restriction endonuclease EcoRI and ligated to a nonreplicating trpED fragment. A miniplasmid was obtained containing a 5.7-kb EcoRI fragment capable of self-replication. This plasmid, designated pRE300, was incompatible with mini-F as well as with ColE1 derivatives. It represents a cointegrate formed in vivo between a 2.2-kb segment of the F replication region and a ColE1-type replicon of unknown derivation. The F-derived component of pRE300 corresponds to a minimalized F replicon (43.85-46.05 kb F) retaining oriII and the incB locus but missing the incC and incD functions. It is postulated that the Inc- mutation resulted from the insertion of a transposable DNA sequence into the incC locus of the parent F plasmid.

Chromosome Mapping↗
PubMed1986

The characterization of a conjugative R-plasmid isolated from Aeromonas salmonicida.

We have analyzed two related R-plasmids isolated from Aeromonas salmonicida: pAr32 and pJA8102-1, which encode resistance to chloramphenicol, streptomycin, and sulfonamides (Su). The genetic map of pJA8102-1 indicated that this plasmid consists of at least three functionally different regions. One is the region conferring ability to transfer (tra), one is the region coding for drug resistance genes (r-det), and one is the region encoding the replication functions (rep). Both plasmids contain repeated sequences (RS); pJA8102-1 has four copies of this RS, while pAr-32 has three copies. The RS sequence of pJA8102-1 are located adjacent to each of the drug-resistance genes. These sequences might have been responsible for the duplication of the Su-resistance gene on this plasmid.

Aeromonas↗
PubMed1992

Characterization of transcription and processing from plasmids that use polIII and a yeast tRNA gene as promoter to transcribe promoter-deficient downstream DNA.

Transfer RNA (tRNA) with mutations affecting the internal promoters and thereby causing them to be nontranscribable by polIII (exemplified, e.g., by nematode tDNAPro with large insertions between the two internal promoters) could be transcribed by polIII both in vitro (yeast) and in vivo (oocytes) when cloned behind a yeast tRNAArg gene. PolIII initiated RNA synthesis could also proceed into a downstream structural gene normally read by polIII. the resultant yeast-nematode dimeric tRNA linked in front of mRNA sequences was recognized by processing enzymes to give mature tRNA. Thus, a yeast tRNA gene preceded by its 5' flank can function as a promoter for polIII transcription of any DNA, also of DNA coding for genes that otherwise could not have been expressed either in vitro or in vivo.

Animals↗
PubMed1991

Clonal variation of DNA repair in a human glioma cell line.

Clonal heterogeneity in response to ionizing radiation was found for a human glioma cell line, IN859. We have investigated the most sensitive clone, the most resistant clone and the parent line for differences in DNA repair fidelity using the method of plasmid reconstitution. Significant differences in repair fidelity were found between the two clones, and between the sensitive clone and the parent line. The resistant clone and the parent line showed the greater repair fidelity. A comparison of two different restriction enzymes, which cleave the plasmid with blunt or cohesive-ended double-strand breaks, did not reveal differences in repair fidelity. Equal numbers of plasmids were integrated in each cell line, but the sensitive clone showed a higher frequency of misrepair of cleaved plasmid. Misrepair was characterized by partial or complete loss of sequence at the site of plasmid cleavage. We conclude that the radiosensitive clone exhibits increased misrepair.

Blotting, Southern↗
PubMed1981

Construction and characterization of a novel two-plasmid system for accomplishing temperature-regulated, amplified expression of cloned adventitious genes in Escherichia coli.

We describe a two-plasmid system that utilizes the lacZ gene promoter and temperature-responsive plasmid replicons to accomplish closely regulated high-level expression of heterologous genes in Escherichia coli. One of the plasmids fails to replicate at 42 degrees C and contains a gene encoding the lac repressor; the second plasmid, which undergoes multicopy "runaway" replication at elevated temperatures, contains an adventitious gene under control of the operator-promoter system of the lacZ gene. Concurrent derepression of lac promoter function and amplification of copy number of the lac-controlled gene occurs when the temperature is elevated. We have used a structural gene encoding chloramphenicol acetyltransferase to demonstrate that the gene product under control of the lacZ promoter represents a major fraction of the total protein synthesized at 43 degrees C, whereas only minimal quantities of this enzyme are made at 30 degrees C. The system described allows the controlled expression of gene products that may have detrimental effects on cell growth, and provides a simple method for identifying radioactivity-labeled protein products of cloned genes in bacterial whole-cell extracts. The system also offers an alternative to intragenic temperature-sensitive mutations for studying the function of various enzymatic or regulatory proteins.

Acetyltransferases↗
PubMed1982

Construction and characterization of new cloning vehicles. VI. Plasmid pBR329, a new derivative of pBR328 lacking the 482-base-pair inverted duplication.

The 4150-bp plasmid pBR329 was constructed by the the insertion into pBR327 of an 877-bp DNA fragment carrying the Cmr gene from pBR328. This new cloning vector does not contain the 482-bp inverted duplication that has been reported to be present in pBR325 and pBR328 (Prentki et al., 1981). In pBR329 the Cmr gene lacks its original promoter but is transcribed counterclockwise toward the Apr gene by a promoter located to the right of the HindIII site in the Tcr gene.

Base Sequence↗
PubMed1985

Characterized full-length and truncated plasmid clones of the crystal protein of Bacillus thuringiensis subsp. kurstaki HD-73 and their toxicity to Manduca sexta.

Bacillus thuringiensis subsp. kurstaki HD-73 produces a crystal protein which is lethal to many lepidopteran larvae. The gene encoding this crystal protein has been isolated from a 75-kb plasmid and engineered into a recombinant Escherichia coli plasmid for analysis. The complete nucleotide sequences of the coding region and 387-bp 5' and 376-bp 3' to the coding region have been determined. The 3537-bp of the coding region specify a protein of Mr 133 330. The full-length gene and several 3' -truncated derivatives of the gene were examined in both E. coli and in an E. coli minicell-expression system to determine if the carboxy end of the protein is essential for toxicity. The results presented here provide the primary structure of the crystal protein gene and show that the N-terminal 68-kDal peptide is toxic, but at a lower level than the full-length gene product.

Amino Acid Sequence↗
PubMed1992

Characterization and properties of a novel plasmid vector for Bacillus thuringiensis displaying compatibility with host plasmids.

A novel plasmid vector, composed of a 1.7-kb Bacillus thuringiensis (B.t.) replicon, a multiple cloning site, and an erythromycin-resistance marker gene from Bacillus subtilis, was constructed for use in B.t. Unlike other vectors which have been reported to be acceptable for B.t., this new B.t. vector was stably maintained in the absence of Er and did not displace host plasmids, some of which carry crystal protein-encoding genes (cry genes). The compatibility of this B.t. vector with native plasmids is highly desirable when introducing new cry genes into a wild-type B.t. strain. When a cryIIIA gene of B.t. tenebrionis was cloned in this vector and introduced into B.t. kurstaki (kur) HD119, cryIIIA was highly expressed without affecting the level of expression of native cry genes. The stability of this vector and its compatibility with native B.t. plasmids were achieved by subcloning only nucleotide sequences required for the vector to replicate in B.t. The origin of replication was first cloned on a 9.6-kb Bg/II fragment from a 75-kb plasmid of B.t. kur HD73 and then localized to a 2.4-kb region within the 9.6-kb fragment. Sequencing of the 2.4-kb region revealed the presence of an open reading frame (ORF), encoding a putative 312-amino acid (aa) protein. The deduced aa sequence of the ORF showed no homology to any published aa sequences. Deletion analysis indicated that the B.t. vector required at least the ORF and up to 300 bp surrounding the ORF, in order to replicate.

Amino Acid Sequence↗
PubMed2005

Characterization of pES213, a small mobilizable plasmid from Vibrio fischeri.

Most Vibrio fischeri strains isolated from the Euprymna scolopes light organ carry plasmids, often including both a large (>40kb) plasmid, and one or more small (<12kb) plasmids. The large plasmids share homology with pES100, which is the lone plasmid in V. fischeri type strain ES114. pES100 appears to encode a conjugative system similar to that on plasmid R721. The small plasmids lack extensive similarity to pES100, but they almost always occur in cells that also harbor a large plasmid resembling pES100. We found that many or all of these small plasmids share homology with pES213, a plasmid in strain ES213. We determined the 5501-bp pES213 sequence and generated selectable antibiotic resistance encoding pES213 derivatives, which enabled us to examine replication, retention, and transfer in V. fischeri. An 863-bp fragment of pES213 with features characteristic of theta-type replicons conferred replication without requiring any pES213 open reading frame (ORF). We estimated that pES213 derivatives were maintained at 9.4 copies per genome, which corresponds well with a model of random plasmid segregation to daughter cells and the approximately 10(-4) per generation frequency of plasmid loss. pES213 derivatives mobilized between V. fischeri strains at frequencies up to approximately 10(-4) in culture and in the host, apparently by employing the pES100 conjugative apparatus. pES213 carries two homologs of the putative pES100 origin of transfer (oriT), and V. fischeri strains lacking the pES100 conjugative relaxase, including a relaxase mutant, failed to serve as donors for transmission of pES213 derivatives. In other systems, genes directing conjugative transfer can function in trans to oriT, so it was noteworthy that ORFs adjacent to oriT, VFB51 in pES100 and traYZ in pES213, enhanced transfer 100- to 1000-fold when provided in cis. We also identified and disrupted the V. fischeri recA gene. RecA was not required for stable pES213 replication but surprisingly was required in donors for efficient transfer of pES213 derivatives. These studies provide an explanation for the prevalence and co-occurrence of pES100- and pES213-type plasmids, illuminate novel elements of pES213 mobilization, and provide the foundation for new genetic tools in V. fischeri.

Aliivibrio fischeri↗
PubMed2005

Characterization of the Micromonospora rosaria pMR2 plasmid and development of a high G+C codon optimized integrase for site-specific integration.

pMR2, an 11.1 kb plasmid was isolated from Micromonospora rosaria SCC2095, NRRL3718, and its complete nucleotide sequence determined. Analysis revealed 13 ORFs including homologs of a KorSA regulatory protein and TraB plasmid transfer protein found on other actinomycete plasmids. pMR2 contains att/int functions consisting of an integrase, an excisionase, and a putative plasmid attachment site (attP). The integrase gene contained a high frequency of codons rarely used in high G+C actinomycete coding regions. The gene was codon optimized for actinomycete codon usage to create the synthetic gene int-OPT. pSPRX740, containing an rpsL promoter and the att/int-OPT region, was introduced into Micromonospora halophytica var. nigra ATCC33088. Analysis of DNA flanking the pSPRX740 integration site confirmed site-specific integration into a tRNA(Phe) gene in the M. halopytica var. nigra chromosome. The pMR2 attP element and chromosomal attachment (attB) site contain a 63 bp region of sequence identity overlapping the 3' end of the tRNA(Phe) gene. Plasmids comprising the site-specific att/int-OPT functions of pMR2 can be used to integrate genes into the chromosome of actinomycetes with an appropriate tRNA gene. The development of an integrative system for Micromonospora will expand our ability to study antibiotic biosynthesis in this important actinomycete genus.

Attachment Sites, Microbiological↗
PubMed2006

Characterization of broad host range cryptic plasmid pCR1 from Corynebacterium renale.

Plasmid pCR1 is a cryptic plasmid harboured by Corynebacterium renale. It is the smallest corynebacterial plasmid known to date. Although its natural host is animal corynebacteria, it can replicate in several strains of soil corynebacteria. It can also replicate in Escherichia coli, in which it is stably maintained. The copy number of pCR1 in this host is higher than that of pUC19, with which it shows unidirectional incompatibility. It is also incompatible with pBK2, a plasmid bearing the common corynebacterial replicon pBL1. Its size is 1488bp, as revealed by DNA sequencing. A total of eight open reading frames (ORF) were detected in this plasmid, the largest of which codes for a putative Rep protein of predicted molecular mass of 21kDa. The plasmid pCR1 can be mobilized by the plasmid R6K from E. coli to other corynebacteria. Sequence analysis revealed the presence of an oriT homologous to that of R64. An E. coli plasmid pKL1 shows more than 90% identity with pCR1. Like many coryenbacterial plasmids, pCR1 also replicates by rolling circle mode.

Amino Acid Sequence↗