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D A Ritchie

Publications and source records attributed to D A Ritchie.

At least 73 records · Page 4Linked to original sources
PubMed1991

Biochemical and biophysical analysis of plasmid pMJ600-encoded tellurite [TeO2(3-)] resistance.

Escherichia coli AB1157, and the transconjugant AB1157 (pMJ600), were used to study the mechanism of tellurite resistance conferred by pMJ600, which contains the tellurite resistance determinant cloned from the IncHI-2 conjugative plasmid pMER610. The transconjugant can tolerate a 100-fold higher concentration of potassium tellurite [K2TeO3] than the plasmid-free strain. Both strains were found to accumulate tellurite irreversibly at equivalent rates, with elemental tellurium being deposited intracellularly, direct efflux of tellurite was not found to contribute to the resistance mechanism. However, under these conditions, growth, protein synthesis and oxygen uptake ceased in AB1157, but-were unaffected in the transconjugant. No NADH- or NADPH-linked reduction of tellurite was detected in crude cell extracts of either strain; however, cell extracts of both reduced tellurite at alkaline pH in the absence of any co-factors.

Drug Resistance, Microbial↗
PubMed1991

Novel mercury resistance determinants carried by IncJ plasmids pMERPH and R391.

HgCl2 resistance (Hgr) in a strain of Pseudomonas putrefaciens isolated from the River Mersey was identified as plasmid-borne by its transfer to Escherichia coli in conjugative matings. This plasmid, pMERPH, could not be isolated and was incompatible with the chromosomally integrated IncJ Hgr plasmid R391. pMERPH and R391 both express inducible, narrow-spectrum mercury resistance and detoxify HgCl2 by volatilization. The cloned mer determinants from pMERPH (pSP100) and R391 (pSP200) have very similar restriction maps and express identical polypeptide products. However, these features show distinct differences from those of the Tn501 family of mer determinants. pSP100 and pSP200 failed to hybridize at moderate stringency to merRTPA and merC probes from Tn501 and Tn21, respectively. We conclude that the IncJ mer determinants are only distantly related to that from Tn501 and its closely homologous relatives and that it identifies a novel sequence which is relatively rare in bacteria isolated from natural environments.

Cloning, Molecular↗
PubMed1991

High frequency transformation of Streptomyces niveus protoplasts by plasmid DNA.

A procedure has been developed for transforming protoplasts of the novobiocin producing strain Streptomyces niveus at high frequency. This required the isolation of strains LH13 and LH20 defective in DNA restriction from the wild type (ATCC 19793) which is transformed at very low frequencies. The LH13 and LH20 derivatives were obtained by curing pIJ702 DNA from the few S. niveus transformed protoplasts obtained by transformation of the wild type with high concentrations of pIJ702 DNA. Protoplasts of S. niveus strains LH13 and LH20 produced about 10(6) transformants/micrograms DNA with modified pIJ702 DNA derived by replication in S. niveus. Unmodified DNA (derived from replication in S: lividans) from a series of pIJ101, SCP2 and pSN2-based derivatives, gave transformation frequencies in the range of 10(2)-10(3) transformants/micrograms DNA. Optimal conditions for the formation and transformation of S. niveus protoplasts are described.

DNA, Bacterial↗
PubMed1991

Accidental golf club injuries.

Over a 2 month period, 33 patients with injuries caused by golf clubs were identified among attenders at the accident and emergency department at Glasgow Royal Infirmary. The vast majority of these were to the face and head, including three compound skull fractures. Attention is drawn to the average age of those injured (8.1 years) and to the fact that only one of the injuries occurred on a golf course. The dangers of unsupervised experimentation with golf clubs by young children are emphasized.

Accidents↗
PubMed1990

Genetic instability in Streptomyces niveus plasmid pSN2: in vivo formation of deletion derivatives.

A plasmid designated pSN2 (molecular size 32.0 kb) was isolated from the wild type of Streptomyces niveus ATCC 19793. To permit phenotypic identification of pSN2 the 1.9 kb BclI fragment was replaced in vitro by the 1.1 kb BclI fragment of pIJ702 carrying the thiostrepton resistance (tsr) gene to form the plasmid pSN3. pSN3 transforms S. lividans to thiostrepton resistance at high frequency and is stably maintained. However, when used to transform S. niveus pSN3 was unstable and produced a 5.5 kb thiostrepton resistant deletion derivative pLG5. pLG5 is also stable and expresses thiostrepton resistance in S. lividans but on transformation of S. niveus was unstable and produced a further thiostrepton resistant derivative, pLG10, of 6.5 kb. pLG5 and pLG10 like pSN3 transform S. lividans at high frequency and produce pocks. DNA hybridizations with a probe derived from pLG5 confirm that pLG5 is derived from DNA sequences present on pSN2 and pSN3.

Chromosome Deletion↗
PubMed1990

Novobiocin-resistance sequences from the novobiocin-producing strain Streptomyces niveus.

Two distinct DNA sequences expressing novobiocin resistance in Streptomyces lividans were cloned from the novobiocin-producing species Streptomyces niveus. Clone pGL101 (5kb) conferred resistance to 50 micrograms ml-1 novobiocin, whereas clones pGL102 and pGL103, which carry the same 6.5kb insert but in opposite orientations, expressed resistance to 150 micrograms ml-1. The cloned inserts from pGL101 and pGL103 failed to hybridize with each other or with the cloned novobiocin-resistant gyrB sequence from Streptomyces sphaeroides. Both probes hybridized strongly with DNA from the novobiocin-producing species S. niveus and S. sphaeroides but no hybridization (pGL103) or very weak hybridization (pGL101) was detected with DNA from the non-producing species S. lividans, Streptomyces griseus and Streptomyces antibioticus. S. niveus contains at least three novobiocin-resistance determinants with the pGL101 and pGL103 cloned sequences specific for novobiocin-producing strains of Streptomyces.

Blotting, Southern↗
PubMed1990

Cloning vectors derived from Streptomyces niveus plasmid pSN2.

Two high copy number, broad host range, general purpose cloning vectors, pLG5 and pLG10, derived from the unstable Streptomyces niveus plasmid pSN2 are described. pLG5 (5.5 kb) and pLG10 (6.5 kb) both carry the thiostrepton resistance (TsrR) and lethal zygosis (Ltz+) markers and have single cloning sites within a non-essential region and the tsr gene. pLG505 (7.4 kb) was constructed by cloning the viomycin resistance (vph) gene into the single BamHI site of pLG5 to give a further vector with insertion and replacement sites which inactivate either the TsrR or VioR functions.

Cloning, Molecular↗
PubMed1988

The nucleotide sequence of a plasmid determinant for resistance to tellurium anions.

The plasmid pMJ606 contains a 5-kb insert specifying resistance to tellurium salts (TeR) which was cloned from the large conjugative plasmid pMER610. Nucleotide sequence analysis of this insert has identified five open reading frames (ORFs). ORFs 1, 2, 4 and 5 correspond in terms of their predicted polypeptide products to the 41, 15.5, 22 and 23-kDa polypeptides, respectively, which are synthesised by the resistance determinant in maxicells. An additional ORF, ORF3, whose product has not been identified is predicted by the sequence data. The sequence of the presumptive polypeptide specified by ORF3 indicates a membrane location. The nucleotide and predicted amino acid sequences of ORF4 and ORF5 show considerable homology which is consistent with the duplication and minor divergence of an ancestral gene. ORFs 4 and 5 appear to retain the same function and while each can function separately and contribute to the resistance mechanism, the full level of wild-type resistance requires both genes to function. The transcriptional signals for the TeR determinant may be located beyond and 5' of the sequences cloned in pMJ606.

Amino Acid Sequence↗
PubMed1988

Restriction pattern and polypeptide homology among plasmid-borne mercury resistance determinants.

The structural and functional properties of mercury resistance determinants cloned from a series of independently isolated conjugative plasmids were compared with those of the prototype HgR determinants from Tn501 and plasmid R100 (containing Tn21). Restriction endonuclease mapping classified the HgR determinants into at least three different but related structural groups which are distantly related to those from Tn501 and R100. These relationships were confirmed by the functional analysis of sub-clones and gamma delta insertion mutations and from the polypeptides specified by the cloned HgR determinants. Each mercury resistance clone synthesized polypeptides equivalent in size to the merA, merT, and merP gene products. However, those for merA and merT showed considerable size variation. No polypeptide equivalent to merD or merC of R100 was detected.

Bacterial Proteins↗
PubMed1987

Genetic and physical analysis of plasmid genes expressing inducible resistance of tellurite in Escherichia coli.

A large (greater than 250 kb) conjugative plasmid, pMER610, specifying resistance to tellurium and mercury was isolated from an Alcaligenes strain and transferred by conjugation to Escherichia coli AB1157. The acquisition of pMER610 by AB1157 increased the resistance to both telurite and tellurate by 100-fold. Expression of tellurite resistance by pMER610 and the cloned Ter determinant was inducible by prior exposure to tellurite at levels sub-toxic to the sensitive AB1157. Physical analysis of the cloned Ter fragment located the resistance determinant to a 3.55 kb region. Insertion of Tn 1000 (gamma delta) into this region produced two classes of sensitive mutations, fully sensitive and intermediate or hyposensitive, which map in adjacent regions and form two complementation groups. Maxicell analysis identified four polypeptides (15.5, 22, 23 and 41 kDa) expressed by the Ter clone. The 23 kDa polypeptide may not be required for resistance since tellurium-sensitive gamma delta insertion mutations were not detected in the 23 kDa coding region.

Alcaligenes↗
PubMed1987

Further characterization of phage T1 DNA clones.

A series of phage T1 DNA clones containing Sau3A fragments of 1 kb or more were tested for their ability to rescue the defects of amber mutations representing all known T1 genes. Rescue was detected by either complementation or recombination. These data have extended the correlation of the T1 genetic and physical maps and provided more conclusive proof that the large map distances at the left end of the genetic map reflect both increased physical separation of the known genetic markers and enhanced recombination and that the right and left chromosomal ends are recombinogenic to the same extent.

DNA, Recombinant↗
PubMed1986

Phage T1-mediated transduction of a plasmid containing the T1 pac site.

The T1 pac site has been cloned into a plasmid vector. This recombinant plasmid was tested for T1-mediated transduction efficiency in comparison with a plasmid containing the phage lambda T1-pac-like site esp-lambda, plasmids containing T1 sequences other than the pac site, and plasmids containing neither T1 sequences nor known pac sites. The data obtained indicate that there are at least two distinct mechanisms of T1-mediated plasmid transduction. One requires the presence of any T1 sequence on the plasmid and probably takes place via cointegrate formation with the homologous region of an infecting T1 genome. The other is specifically dependent on the presence of a pac site on the plasmid. Plasmids are packaged as head-to-tail multimers that have one heterogeneous molecular end and the other terminated at pac, and the direction of packaging with respect to the pac site is the same for plasmids as for T1. Possible roles of pac in plasmid packaging and their implications with regard to the packaging of phage DNA are discussed.

Cloning, Molecular↗
PubMed1986

In vitro packaging of foreign DNA into heads of bacteriophage T1.

The isolation of a collection of 44 morphologically T1-like phages is described. It is shown that these phages share some similarity with T1 in terms of cross-inactivation with anti-T1 serum, particle proteins and DNA packaging in vitro by the headful process. Virion DNA extracted from these phages was treated with T1 in vitro packaging extracts and the reaction mixtures were tested for the formation of infectious phage particles. The packaging efficiencies observed varied from about 1 to 100% of that of virion T1 DNA. Phage lambda virion DNA was packaged with an efficiency of between 0.01 and 2% (5 X 10(1) to 3 X 10(3) p.f.u./micrograms DNA), the shorter deleted derivative lambda L47 being packaged more efficiently than normal length lambda C1857 DNA. Virion DNA from phages T3 and T7 was also packaged at an efficiency similar to that for lambda. The in vitro packaging of T1 DNA requires the presence of the pac sequence which initiates headful packaging from a concatemeric precursor. The high efficiency of packaging DNA from some of the T1-like phages may indicate the presence of similar packaging sequences. However, in the case of lambda L47, which is known not to contain such a sequence, the in vitro DNA packaging reaction must occur by a secondary pathway unrelated to the headful mechanism.

Bacteriophage lambda↗
PubMed1985

A coupled in vitro system for the formation and packaging of concatemeric phage T1 DNA.

Extracts derived from E. coli cells infected non-permissively with phage T1 amber mutants were used in an in vitro system to investigate the packaging of T1 DNA into phage heads. The standard extract used infections with amber mutants in genes 1 and 2 (g1- g2-) which are defective in T1 DNA synthesis but can synthesis the proteins required for particle morphogenesis. g1- g2- extracts packaged T1+ virion DNA molecules with an efficiency of 3 X 10(5) pfu/micrograms DNA. Extracts from cells infected with phage also defective in DNA synthesis but carrying additional mutations in genes 3.5 or 4 which are required for concatemer formation in vivo (g1- g3.5- and g1- g4- extracts) package T1 virion DNA at substantially lower efficiencies. Analysis of the DNA products from these in vitro reaction showed that concatemeric DNA is formed very efficiently by g1- g2- extracts but not by g1- g3.5- or g1- g4- extracts. These results are interpreted as evidence that the T1 in vitro DNA packaging system primarily operates in a similar manner to the in vivo headful mechanism. This is achieved in vitro by the highly efficient conversion of T1 virion DNA into concatemers which are then packaged with a much lower efficiency into heads to form infectious particles. A secondary pathway for packaging T1 DNA into heads and unrelated to the headful mechanism may also exist.

DNA Replication↗
PubMed1985

Correlation of the genetic and physical maps of phage T1.

A series of BglII- and Sau3A-derived fragments of phage T1 DNA were cloned into the plasmid vector pLV59 and the clones were tested for their ability to complement and recombine with infecting genomes of am mutants representing all known T1 essential genes. The resulting data were used to correlate the T1 genetic and physical maps. This analysis led to the following conclusions: the large map distances observed at the left (early) end of the genetic map arise partly from increased genetic recombination at the chromosomal left end and partly from increased physical distances between identified genetic markers in this region, the right chromosomal end is also recombinogenic, genes 3.5 to 18, which occupy the right two-thirds of the DNA molecule, are tightly compacted with little space for additional protein-specifying sequences in this region.

Base Sequence↗