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Biomedical subjects

J M Pemberton

Publications and source records attributed to J M Pemberton.

12 recordsLinked to original sources
PubMed1992

An improved suicide vector for construction of chromosomal insertion mutations in bacteria.

We have constructed an R6K-based suicide vector (pJP5603) that requires a trans supply of the pir-encoded pi protein of plasmid R6K for replication. Therefore, efficient plasmid suicide results upon transfer to bacteria not harbouring pir. The 3.1-kb vector encodes kanamycin resistance and is mobilizable. When used in conjunction with a JM109 strain carrying pir, it has nine unique restriction sites available for alpha-complementation cloning. Vector functionality was demonstrated in Rhodobacter sphaeroides.

Bacterial Proteins↗
PubMed1992

DNA fingerprinting: parentage studies in natural populations and the importance of linkage analysis.

It has been suggested that a full linkage analysis is a prerequisite for confident paternity testing, by using DNA fingerprinting, in natural populations. These fears are based on a confusion between linkage and linkage disequilibrium and a misplaced assumption that linkage between bands will necessarily reduce the effective number of paternal-specific bands. Several methods for detecting linkage without resorting to the analysis of large sibships are considered, for example, by analysing half-sibships, by band-association, and by altering the experimental conditions used. Even if linkage is present, the magnitude of its effects are unlikely to undermine the accuracy of the technique, given the average levels of variability being detected. We conclude that the effects of linkage are only likely to present a problem when sample sizes are very small or when closely related individuals are being tested together.

Animals↗
PubMed1992

High-frequency electroporation and maintenance of pUC- and pBR-based cloning vectors in Pseudomonas stutzeri.

A number of Escherichia coli cloning vectors, based on ColE1-like replicons, were shown to be maintained in Pseudomonas stutzeri ATCC 17588. A restrictionless mutant of P. stutzeri was isolated, and this strain was used to develop an efficient electroporation system. With the E. coli cloning vector pHSG298, transformation frequencies of up to 2 x 10(7) transformants/micrograms DNA were achieved. This frequency is comparable to that obtained for CaCl2-mediated transformation of E. coli; thus, direct cloning of DNA into P. stutzeri is feasible. As will be discussed, this may prove useful for cloning DNA from high mol% G + C genera in cases in which E. coli is not a suitable heterologous cloning host.

Bacteriocin Plasmids↗
PubMed1992

Specific detection of Clostridium botulinum type B by using the polymerase chain reaction.

The polymerase chain reaction (PCR) and a radiolabeled oligonucleotide probe were used to specifically detect proteolytic and nonproteolytic Clostridium botulinum type B. Two synthetic primers deduced from the amino acid sequence data of type B neurotoxin were used to amplify a 1.5-kbp fragment corresponding to the light chain of the toxin. Although, nonspecific priming was observed when the PCR protocol was tested with other clostridial species, only the PCR product from C. botulinum type B isolates reacted with the radiolabeled internal probe. As little as 100 fg of DNA (approximately 35 clostridial cells) could be detected after only 25 amplification cycles.

Base Sequence↗
PubMed1990

Stable albicidin resistance in Escherichia coli involves an altered outer-membrane nucleoside uptake system.

Albicidin blocked DNA synthesis in intact cells of a PolA- EndA- Escherichia coli strain, and in permeabilized cells supplied with all necessary precursor nucleotides, indicating a direct effect on prokaryote DNA replication. Replication of phages T4 and T7 was also blocked by albicidin in albicidin-sensitive (Albs) but not in albicidin-resistant (Albr) E. coli host-cells. All stable spontaneous Albr mutants of E. coli simultaneously became resistant to phage T6. The locus determining albicidin sensitivity mapped at tsx, the structural gene for an outer-membrane protein used as a receptor by phage T6 and involved in transport through the outer membrane of nucleosides present at submicromolar extracellular concentrations. Albicidin does not closely resemble a nucleoside in structure. However, Albs E. coli strains rapidly accumulated both nucleosides and albicidin from the surrounding medium whereas the Albr mutants were defective in uptake of nucleosides and albicidin at low extracellular concentrations. An insertion mutation blocking Tsx protein production also blocked albicidin uptake and conveyed albicidin resistance. Albicidin supplied at approximately 0.1 microM blocked DNA replication within seconds in intact Albs E. coli cells, but a 100-fold higher albicidin concentration was necessary for a rapid inhibition of DNA replication in permeabilized cells. We conclude that albicidin is effective at very low concentrations against E. coli because it is rapidly concentrated within cells by illicit transport through the tsx-encoded outer-membrane channel normally involved in nucleoside uptake. Albicidin resistance results from loss of the mechanism of albicidin transport through the outer membrane.

Anti-Bacterial Agents↗
PubMed1990

Genes from Cellvibrio mixtus encoding beta-1,3 endoglucanase.

Two genes encoding beta-1,3 glucanase activity were cloned from the gram-negative soil bacterium Cellvibrio mixtus. The two clones, designated cwd (cell wall degradation) and lam (laminarin degradation), had distinct endonuclease restriction patterns and encoded enzymes with distinct substrate specificities. The 3.7-kilobase cwd insert encoded an enzyme which degraded yeast cell walls as well as the soluble beta-1,3 glucan laminarin and the insoluble beta-1,3 glucans zymosan and pachyman. The 1.8-kilobase lam insert encoded an enzyme which degraded laminarin only. Both enzymes degraded laminarin in an endohydrolytic manner to yield laminarobiose, laminarotriose, and laminarotetraose as major end products. Radiolabeled translation products of the cwd and lam transcripts were identified.

Bacterial Proteins↗
PubMed1978

Viral R plasmid Rphi6P: properties of the penicillinase plasmid prophage and the supercoiled, circular encapsidated genome.

Properties of the viral R plasmid Rphi6P are described. As a temperate bacteriophage, it plaques on the facultative phototroph Rhodopseudomonas sphaeroides. Under aerobic conditions the phage had a latent period of 180 min, a burst time of 200 min, and a burst size of 15 to 20 particles per infective center. The encapsidated viral genome occurred as a supercoiled, circular DNA duplex with a mean contour length of 16.5 +/- 10 micron. Percent guanine plus cytosine, as calculated from thermal denaturation profiles, was 63.5. Mitomycin C-induced loss of the prophage suggested an extrachromosomal location in the host cell. Use of this curing agent enabled the isolation of a plasmid-free strain of R. sphaeroides. Biophysical analysis of the plasmid-free strain lysogenized with Rphi6P confirmed that the prophage occurred as a plasmid in the host cell.

Adsorption↗