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At least 703 records · Page 39Linked to original sources
PubMed1997

Characterization of a linear DNA plasmid from the filamentous fungal plant pathogen Glomerella musae [Anamorph: Colltotrichum musae (Berk. & Curt.) Arx.].

A 7.4-kilobase (kb) DNA plasmid was isolated from Glomerella musae isolate 927 and designated pGML1. Exonuclease treatments indicated that pGML1 was a linear plasmid with blocked 5' termini. Cell-fractionation experiments combined with sequence-specific PCR amplification revealed that pGML1 resided in mitochondria. The pGML1 plasmid hybridized to cesium chloride-fractionated nuclear DNA but not to A + T-rich mitochondrial DNA. An internal 7.0-kb section of pGML1 was cloned and did not hybridize with either nuclear or mitochondrial DNA from G. musae. Sequence analysis revealed identical terminal inverted repeats (TIR) of 520 bp at the ends of the cloned 7.0-kb section of pGML1. The occurrence of pGML1 did not correspond with the pathogenicity of G. musae on banana fruit. Four additional isolates of G. musae possessed extrachromosomal DNA fragments similar in size and sequence to pGML1.

Ascomycota↗
PubMed1997

The complete nucleotide sequence and characterization of the linear DNA plasmid pRS64-2 from the plant pathogenic fungus Rhizoctonia solani.

The complete nucleotide sequence of one of three linear DNA plasmids (pRS64-2) from the plant pathogenic fungus Rhizoctonia solani was determined. The pRS64-2 DNA consisted of 2877 nucleotides. The nucleotide sequences of the middle 2.2-kb regions of the other two plasmids (pRS64-1 and pRS64-3) were also determined. Comparison of the nucleotide sequences among the three plasmid DNAs indicated the presence of four regions with more than 86% sequence homology, suggesting the development of three plasmid DNAs from a common ancestor. A computer-based study of the pRS64-2 DNA-folding at both termini predicted hairpin loop structures. The hairpin loops consisted of the left- and right-hand terminal 113 and 105 nucleotides, respectively, and had no sequence homology. They had not undergone flip-flop inversion. The hairpins form cruciform base-paired structures.

Base Sequence↗
PubMed2001

Isolation and characterization of novel F-plasmid sopB mutants defective in gene silencing.

The product of the sopB gene on the Escherichia coli F-plasmid has been shown to silence genes in the vicinity of its binding region, sopC, when overexpressed. We searched for mutants defective in SopB-dependent silencing by screening for a plasmid incompatibility phenotype, in order to examine the relationship between gene silencing and the intracellular localization of SopB, as revealed by a green fluorescent protein (GFP)-SopB fusion. Nine new mutants were isolated. One of them, in which leucine 92 is replaced by proline, was completely compatible with a sopC-carrying plasmid and was defective in other silencing activities. When expressed as a GFP fusion protein, the L92P mutant was found to be uniformly distributed in the cell. This implies a link between silencing and SopB localization, supporting the view that a high local concentration of SopB drives non-specific DNA binding in segments of the plasmid adjacent to sopC. Despite the lack of apparent localization of GFP fluorescence, the mutant protein, like the wild-type SopB, was found mostly in the inner membrane fraction, indicating that the association with the inner membrane was retained.

Bacterial Proteins↗
PubMed1989

The F1F0-ATPase of Escherichia coli. The substitution of alanine by tyrosine at position 25 in the c-subunit affects function but not assembly.

A site-directed mutation in the gene which codes for the c-subunit of the F1F0-ATPase, resulting in the substitution of Ala-25 by Tyr, has been constructed and characterized. A plasmid carrying the mutation was used to transform strain AN943 (uncE429). The resulting strain is unable to grow on succinate as sole carbon source and possesses an uncoupled growth yield. Membranes prepared from the mutant possess low levels of ATPase activity and are proton-impermeable. The F1-ATPase activity was found to be inhibited by 80% when bound to the membrane. When carried on a plasmid, the mutation is dominant in complementation tests with all mutant unc alleles tested and when transformed into wild-type strain AN346, the mutation results in an uncoupled phenotype. A mutant which overcomes this dominance was isolated and found to possess an 11-amino-acid deletion extending from Ile-55 to Met-65 within the c-subunit. These results are discussed in relation to the previously isolated Ala-25 to Thr mutant (Fimmel, A.L., Jans, D.A., Hatch, L., James, L.B., Gibson, F. and Cox, G.B. (1985) Biochim. Biophys. Acta 808, 252-258) and in relation to a previously proposed model for the F0 (Cox, G.B., Fimmel, A.L., Gibson, F. and Hatch, L. (1986) Biochim. Biophys. Acta 849, 62-69).

Alanine↗
PubMed1987

Isolation and partial characterization of 2-microns yeast plasmid as a transcriptionally active minichromosome.

Yeast cell extracts from 2-microns-containing strains (cir+) showed higher transcriptional activity than their corresponding isogenic sets (cir0). These extracts were used to purify transcriptionally active 2-microns minichromosomes in a sucrose gradient. Minichromosomes were transcribed in vitro and, employing hybridization techniques, the RNA synthesized was shown to present 2-microns-specific sequences. This model system should permit the direct study of transcriptionally active eucaryotic chromatin.

Chromosomes↗
PubMed1994

Shuttle-vector mutagenesis by aflatoxin B1 in human cells: effects of sequence context on the supF mutational spectrum.

Rat-liver microsomes were used to activate aflatoxin B1 for in vitro modification of the pS189 shuttle vector and the related signature vector pSP189, both of which carry the Escherichia coli supF gene as a mutational target. Plasmid degradation was minimized by carrying out the in vitro incubations in the absence of Mg2+ ions. Modified plasmids were transfected into human Ad293 cells, then recovered and electroporated into E. coli MBM7070 for mutant identification. Point mutation frequencies for in vitro modified plasmids were dramatically increased over the spontaneous background level. Mutant plasmids were characterized by DNA-sequence analysis. The vast majority of aflatoxin B1-induced mutations were base substitutions, mostly G:C to T:A transversions. The spectrum of aflatoxin B1-induced mutations in the pS189 supF gene was very similar to that observed previously for the pS189 supF gene with aflatoxin B1 activated by cytochrome P450 1A2 (CYP1A2) synthesized from a cDNA expression vector within transfected Ad293 cells. However, the spectrum for pSP189, which carries the same supF gene as pS189, but with different surrounding sequences, exhibited some notable differences from that of pS189; this suggests that sequence context effects on mutagenic specificity can operate over distances of tens of base pairs.

Aflatoxin B1↗
PubMed1987

Characterization of the drug resistance plasmid NTP16.

A functional and physical analysis of the multicopy plasmid NTP16 is presented. The plasmid-encoded drug resistance determinants are located, as are regions encoding the origin of replication, incompatibility functions, copy number determinants, and mobility functions. It is demonstrated that NTP16 probably arose from the closely related plasmid NTP1 by the acquisition of a novel kanamycin resistance transposon, Tn4352, followed by deletion of some NTP1 sequences. The incompatibility behavior of NTP16 derivatives indicates a system of control rather more complex than that which operates in ColE1. In addition to the RNA I/primer RNA system, the production of a further trans-acting product is demonstrated and its site of action located. A series of derivative plasmids have been created which may prove useful as vectors for genetic engineering.

Ampicillin↗
PubMed1987

Characterization and sequence of a plasmid from the trachoma biovar of Chlamydia trachomatis.

The 7.5-kb plasmid of Chlamydia trachomatis, trachoma biovar, was mapped for restriction enzyme sites and sequenced. The complete nucleotide sequence, the first reported for any chlamydial plasmid, revealed nine open reading frames which could code for polypeptides greater than 10 kDa. These putative polypeptides contain 35-47% hydrophobic amino acid residues. Two putative polypeptides of 30 kDa are highly basic and one of 23 kDa is acidic. A region composed of four 22-bp repeats, AT-rich clusters, an inverted repeat, and a 30-kDa basic protein is similar in organization to the origins of replication in a number of Escherichia coli plasmids. Northern blot analysis of the plasmid RNA showed transcripts longer than the length of the plasmid, suggesting a single transcription initiation signal.

Base Sequence↗
PubMed1988

Cloning and partial characterization of three small cryptic plasmids from Bacillus thuringiensis.

The strain H1.1 of Bacillus thuringiensis var. thuringiensis harbors three small cryptic plasmids: pGI1, pGI2, and pGI3 (8.2, 9.2, and 10.6 kb, respectively). Two of these plasmids (i.e., pGI2 and pGI3) were successfully cloned in their entirety into the vector pBR322, whereas only overlapping DNA fragments covering pGI1 were obtained in Escherichia coli. A curing-hybridization technique was used to obtain isolates of B. thuringiensis missing one or another small cryptic plasmid. These derivatives were examined for any change in a phenotypic trait, but no specific function could be assigned to one of these plasmids. Hybridization and restriction mapping data revealed that the transposon Tn4430 accounts for 45% of the pGI2 plasmid DNA.

Bacillus thuringiensis↗
PubMed1984

Klebsiella infection in a neonatal intensive care unit: role of bacteriological surveillance.

An outbreak of 12 cases of infection occurred over a 9-month period in a Regional Referral Neonatal Intensive Care Unit. The pathogen was a gentamicin- and multiply-resistant Klebsiella oxytoca (K55), of high virulence. Seven of 10 neonates with septicaemia died, the majority within 24 h of the onset of infection. Screening for the resistant klebsiella revealed that 64 (39 per cent) of 164 neonates became carriers and remained so throughout their stay. Hand carriage appeared to be a mode of cross-contamination and infection. Cohorting colonized neonates together with rigorous cross-infection control measures eliminated this organism from the unit after 29 weeks. There is evidence to suggest that in one case the infecting organism was acquired from a contaminated blood gas analyser. It is necessary to use incompatibility grouping and restriction endonuclease digestion for complete characterization of plasmids and their molecular weights. However, the finding that each isolate examined carried the same five plasmids as judged by co-electrophoresis on agarose gels, and expressed the same extent and degree of transferable antibiotic resistance provides evidence to suggest that this outbreak was due to spread of a resistant clone of K. oxytoca (K55).

Carrier State↗
PubMed1990

Characterization of a small cryptic plasmid isolated from a methicillin-resistant strain of Staphylococcus aureus.

The nucleotide sequence of a small (1613 bp) plasmid, pOX2000, isolated from a methicillin-resistant strain of Staphylococcus aureus has been determined. The sequence contains only one large ORF and the predicted amino acid sequence shows homology to the REP proteins of some other small staphylococcal plasmids. In addition there are two palindromic sequences, palA and palJ, that are similar to but not identical with the palindromes known from other staphylococcal plasmids to be involved in lagging strand initiation and possibly leading strand termination, respectively. Preliminary functional analysis of pOX2000 has been carried out by assessing the effect of interrupting the sequence at three unique restriction endonuclease sites. The plasmid pOX2000, and its relationship to other small staphylococcal plasmids, is discussed.

Amino Acid Sequence↗
PubMed1992

Sequencing and characterization of pST1, a cryptic plasmid from Streptococcus thermophilus.

Eighty-six strains of S. thermophilus were examined for their plasmid content. Thirteen strains were found to contain one or two plasmids ranging in size from 2.1 to 7.4 kb. DNA-DNA hybridization analysis revealed the presence of five distinct groups of DNA homology. The complete nucleotide sequence of plasmid pST1 (Accession number X65856), which belongs to the major homology group, was determined. It has a molecular size of 2093 bp, a GC content of 35% and contains one major open reading frame of 945 bp (ORF A). The predicted protein, designated Rep A, showed sequence homology with replication proteins from a group of plasmids which are known to replicate via single-stranded DNA intermediates (ssDNA plasmids).

Amino Acid Sequence↗
PubMed1983

Isolation and characterization of pock-forming plasmids for Streptomyces griseus from soil actinomycetes.

Thirty independent actinomycetes strains carrying plasmids were isolated from soil. These plasmids were purified as cccDNA by CsCl-EtBr equilibrium density-gradient centrifugation. Plasmids that induce "pocks", namely formation of circular zones of sporulation-inhibition, were selected by protoplast transformation of streptomycin-producing strain, Streptomyces griseus ATCC10137. Six pock-forming plasmids, pOA7, pOA11, pOA15, pOA23, pOA29 and pOA30, were obtained, and their cleavage maps, transformation frequencies, and copy numbers, as well as their stability, are described.

Actinomycetales↗
PubMed1986

Construction and characterization of pBR322-derived plasmids with deletions of the RNA I region.

A region upstream from the origin of replication in ColE1-type plasmids has been shown to be necessary for replication. Two RNA transcripts are produced from this area, RNA II, which yields the primer for DNA polymerase initiation at the origin and RNA I, which is complementary to the 5' end of RNA II and acts to inhibit primer formation. We have constructed plasmids which do not possess the nucleotide sequence for RNA I, or the normal 5' terminus and promoter of RNA II. The RNA II analog, in these plasmids, is believed to be synthesized by the readthrough transcription of the upstream trimethoprim-resistant dihydrofolate reductase (DHFR) gene at a level comparable to that produced by the tryptophan promoter. These plasmids have a copy number of about tenfold higher than that of pBR322 during logarithmic growth and are compatible with other ColE1-type plasmids. These plasmids are stably maintained in several strains when selective pressure is present and the plasmids are stably maintained during exponential growth in W3110 strains without selective pressure. In all strains examined, the dimeric form of the plasmid was lost from cells much more rapidly than those containing the monomeric form.

Base Sequence↗