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Biomedical subjects

J M Pemberton

Publications and source records attributed to J M Pemberton.

At least 73 records · Page 4Linked to original sources

Parasite-associated polymorphism in a cyclic ungulate population.

An unmanaged population of Soay sheep living on Hirta, St Kilda, Scotland is persistently unstable, fluctuating between about 600 and 1600 individuals. Population crashes occurring approximately every 3 years are primarily due to winter food shortage. In this paper we show that sheep experimentally relieved of their gastrointestinal nematodes (predominantly Teladorsagia spp.) survived a crash better than matched controls, showing that nematode parasites contribute to the probability that a sheep dies in a crash. We also show that over three successive crashes mortality was significantly different between individuals of the three different genotypes at the diallelic adenosine deaminase locus (Ada). FF animals were most likely to die, SS animals had an intermediate probability of dying, and FS animals were least likely to die. Finally, three independent lines of evidence suggest that nematode burdens differ between the three Ada genotypes. First, in August, heterozygous females are less likely to have nematode eggs in their faeces than homozygous females. Second, at lambing, the periparturient rise in faecal egg count was highest in homozygous FF individuals. Finally, during the Autumn mating season, heterozygous males has lower faecal egg counts than homozygotes, although this relation was complicated by interactions with year and age of male. These results are consistent with the idea that Ada allele frequencies are maintained in the sheep population by parasite-associated selection.

Adenosine Deaminase↗

Characterization of an endo-1,3(4)-beta-D-glucanase gene from Cellvibrio mixtus.

An endo-1,3(4)-beta-D-glucanase gene (cwd2) of Cellvibrio mixtus encoding laminarinase activity was cloned on a 3.9-kb PstI fragment. The Cwd2 enzyme, extracted from recombinant Escherichia coli, degraded both beta-1,3 glucans and beta-1,3-1,4 mixed-linkage glucans, was endohydrolytic and so conformed to the enzyme class 3.2.1.6. The pH and temperature optima of the enzyme were approximately 7 and 40 degrees C respectively. The M(r) of specifically labelled Cwd2 was approximately 34,000. This gene was quite distinct from two other C. mixtus beta-1,3 glucanases previously described.

Chromatography, Thin Layer↗

Detection of the genes encoding botulinum neurotoxin types A to E by the polymerase chain reaction.

The polymerase chain reaction (PCR) was used as the basis for the development of highly sensitive and specific diagnostic tests for organisms harboring botulinum neurotoxin type A through E genes. Synthetic DNA primers were selected from nucleic acid sequence data for Clostridium botulinum neurotoxins. Individual components of the PCR for each serotype (serotypes A through E) were adjusted for optimal amplification of the target fragment. Each PCR assay was tested with organisms expressing each of the botulinum neurotoxin types (types A through G), Clostridium tetani, genetically related nontoxigenic organisms, and unrelated strains. Each assay was specific for the intended target. The PCR reliably identified multiple strains having the same neurotoxin type. The sensitivity of the test was determined with different concentrations of genomic DNA from strains producing each toxin type. As little as 10 fg of DNA (approximately three clostridial cells) was detected. C. botulinum neurotoxin types A, B, and E, which are most commonly associated with human botulism, could be amplified from crude DNA extracts, from vegetative cells, and from spore preparations. This suggests that there is great potential for the PCR in the identification and detection of botulinum neurotoxin-producing strains.

Base Sequence↗

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↗

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↗

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↗

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↗

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↗

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↗

Cloning and characterization of an albicidin resistance gene from Klebsiella oxytoca.

A DNA fragment containing a gene for resistance to the antibiotic albicidin was isolated from Klebsiella oxytoca and shown to be expressed in Escherichia coli, where it also protected bacteriophage T7 replication from inhibition by albicidin. In vivo translation analysis demonstrated that the cloned 2.2kb DNA fragment coded for a 36 kiloDalton (kD) protein and a 25kD protein. The DNA sequence was determined for a 654-base-pair open reading frame contained within a 1.2kb subcloned DNA fragment encoding albicidin resistance. The predicted molecular weight of the polypeptide translated from the open reading frame was 25.8kD. A putative Shine-Dalgarno sequence precedes the open reading frame but a potential promoter sequence was not detected. A possible rho-independent transcription termination signal was found directly following the stop codon. The functional protein for albicidin resistance was isolated and purified. Both the molecular weight and NH2-terminal amino acid sequence of this protein correspond with that predicted from the DNA sequence of the open reading frame. The cloned albicidin resistance gene had no effect on the tsx (nupA) nucleoside uptake gene associated with spontaneous albicidin resistance in E. coli; also, it did not complement any of a range of E. coli DNAts mutants at restrictive temperatures. The cloned resistance gene product remained intracellular in exponential cultures of K. oxytoca and E. coli. Cell-free extracts from E. coli containing the resistance gene protected a sensitive strain of E. coli from inhibition by albicidin, as did the purified albicidin resistance protein. The mechanism of this albicidin resistance protein involved binding to albicidin to form a complex without antibiotic activity, but without catalysing further chemical modification of the antibiotic.

Amino Acid Sequence↗

Molecular cloning, characterization, and nucleotide sequence of an extracellular amylase gene from Aeromonas hydrophila.

The structural gene for excreted amylase from Aeromonas hydrophila JMP636 has been cloned within a 2.1-kilobase SmaI fragment of DNA. The amylase gene is transcribed from its own promoter in Escherichia coli, producing a gene product of Mr 49,000. The amylase gene product is secreted to the periplasm of E. coli; however, it is not excreted. Nucleotide sequencing revealed an open reading frame of 1,392 base pairs corresponding to a protein of 464 amino acid residues. A potential signal peptide of 21 amino acid residues is present at the NH2 terminal of the predicted protein. Three regions of homology with other procaryotic and eucaryotic alpha-amylases were detected within the predicted amino acid sequence.

Aeromonas↗

Construction of a transposon containing a gene for polygalacturonate trans-eliminase from Klebsiella oxytoca.

A DNA fragment containing a Klebsiella oxytoca gene for polygalacturonate trans-eliminase was cloned into the kanamycin resistance transposon Tn5. This new transposon, designated Tn5-Pga+, had a transposition frequency of 1 X 10(-6). The broad host range plasmid pR751::Tn5-Pga+ was conjugally transferred to a variety of genetic backgrounds. The ability to degrade polygalacturonate was expressed in Aeromonas hydrophila, Alcaligenes eutrophus, Azotomonas insolita, Escherichia coli, Pseudomonas putida and Rhodopseudomonas sphaeroides, but not in Zymomonas mobilis.

Cloning, Molecular↗

Cloning of a Gene Cluster from Cellvibrio mixtus which Codes for Cellulase, Chitinase, Amylase, and Pectinase.

The soil isolate Cellvibrio mixtus UQM2294 degraded a variety of polysaccharides including microcrystalline cellulose. Among 6,000 cosmid clones carrying C. mixtus DNA, constructed in Escherichia coli with pHC79, 50 expressed the ability to degrade one or more of the following substrates: carboxymethyl cellulose, chitin, pectin (polygalacturonic acid), cellobiose, and starch. These degradative genes are encoded in a single 94.1-kilobase segment of the C. mixtus genome; a preliminary order of the genes is starch hydrolysis, esculin hydrolysis, cellobiose utilization, chitin hydrolysis, carboxymethyl cellulose hydrolysis, and polygalacturonic acid hydrolysis. A restriction endonuclease cleavage map was constructed, and the genes for starch, carboxymethyl cellulose, cellobiose, chitin, and pectin hydrolysis were subcloned.

Journal Article↗

Lack of biochemical polymorphism in British fallow deer.

Seven-hundred and ninety-four samples of fallow deer (Dama dama L.) blood or tissue were collected from 37 sites in England and Wales. A selection of these samples was screened for electrophoretic variation at each of 30 loci (minimum of 88 samples per locus). No genetic variation was found. Possible explanations for the lack of polymorphism are discussed. It is suggested that European fallow deer experienced a genetic bottleneck during a period of captivity in Mesolithic or Neolithic times.

Animals↗

Genetic and physical map of the 2,4-dichlorophenoxyacetic acid-degradative plasmid pJP4.

Plasmid pJP4 is an 80-kilobase, IncP1, broad-host-range conjugative plasmid of Alcaligenes eutrophus encoding resistance to mercuric chloride and phenyl mercury acetate and degradation of 2,4-dichlorophenoxyacetic acid, 2-methyl-4-chlorophenoxyacetic acid, and 3-chlorobenzoate. By the use of cloning, transposon mutagenesis, and restriction endonuclease analysis, a biophysical and genetic map of pJP4 was generated.

2,4-Dichlorophenoxyacetic Acid↗