Biomedical subjects

J M Pemberton

Publications and source records attributed to J M Pemberton.

At least 55 records · Page 3Linked to original sources

PubMed1998

Major histocompatibility complex variation associated with juvenile survival and parasite resistance in a large unmanaged ungulate population.

Antagonistic coevolution between hosts and parasites has been proposed as a mechanism maintaining genetic diversity in both host and parasite populations. In particular, the high levels of genetic diversity widely observed at the major histocompatibility complex (MHC) of vertebrate hosts are consistent with the hypothesis of parasite-driven balancing selection acting to maintain MHC genetic diversity. To date, however, empirical evidence in support of this hypothesis, especially from natural populations, has been lacking. A large unmanaged population of Soay sheep (Ovis aries L.) is used to investigate associations between MHC variation, juvenile survival, and parasite resistance. We show in an unmanaged, nonhuman population that allelic variation within the MHC is significantly associated with differences in both juvenile survival and resistance to intestinal nematodes. Certain MHC alleles are associated with low survivorship probabilities and high levels of parasitism or vice versa. We conclude that parasites are likely to play a major role in the maintenance of MHC diversity in this population.

Alleles↗

PubMed1998

Microsatellites reveal heterosis in red deer.

The fitness consequences of inbreeding and outbreeding are poorly understood in natural populations. We explore two microsatellite-based variables, individual heterozygosity (likely to correlate with recent inbreeding) and a new individual-specific internal distance measure, mean d2 (focusing on events deeper in the pedigree), in relation to two measures of fitness expressed early in life, birth weight and neonatal survival, in 670 red deer calves (Cervus elaphus) born on the Isle of Rum between 1982 and 1996. For comparison, we also analyse inbreeding coefficients derived from pedigrees in which paternity was inferred by molecular methods. Only 14 out of 231 calves (6.1%) had non-zero inbreeding coefficients, and neither inbreeding coefficient nor individual heterozygosity was consistently related to birth weight or neonatal survival. However, mean d2 was consistently related to both fitness measures. Low mean d2 was associated with low birth weight, especially following cold Aprils, in which foetal growth is reduced. Low mean d2 was also associated with low neonatal survival, but this effect was probably mediated by birth weight because fitting birth weight to the neonatal survival model displaced mean d2 as an explanatory variable. We conclude that in the deer population fitness measures expressed early in life do not show evidence of inbreeding depression, but they do show evidence of heterosis, possibly as a result of population mixing. We also demonstrate the practical problems of estimating inbreeding via pedigrees compared with a direct marker-based estimate of individual heterozygosity. We suggest that, together, individual heterozygosity and mean d2, estimated using microsatellites, are useful tools for exploring inbreeding and outbreeding in natural population.

Age Factors↗

PubMed1998

Bovine microsatellite loci are highly conserved in red deer (Cervus elaphus), sika deer (Cervus nippon) and Soay sheep (Ovis aries).

We tested 174 bovine microsatellite primer pairs for use in a primitive breed of sheep and two species of deer. Of 173 markers, 127 (73.4%) gave a product in Soay sheep (Ovis aries) of which 54 (42.5%) were polymorphic. One hundred and twenty-nine of 174 (74.1%) markers gave a product in red deer (Cervus elaphus) of which 72 (55.8%) were polymorphic. In sika deer (Cervus nippon) 126 of 171 (73.7%) microsatellite primers gave a product with 47 (37.3%) polymorphic. The proportion of bovine microsatellite loci conserved across artiodactyl species was significantly greater in this study than previously reported. Reasons for this high degree of microsatellite conservation are discussed. We suggest that a high resolution comparative map of the artiodactyls can be constructed using microsatellites.

Alleles↗

PubMed1998

Statistical confidence for likelihood-based paternity inference in natural populations.

Paternity inference using highly polymorphic codominant markers is becoming common in the study of natural populations. However, multiple males are often found to be genetically compatible with each offspring tested, even when the probability of excluding an unrelated male is high. While various methods exist for evaluating the likelihood of paternity of each nonexcluded male, interpreting these likelihoods has hitherto been difficult, and no method takes account of the incomplete sampling and error-prone genetic data typical of large-scale studies of natural systems. We derive likelihood ratios for paternity inference with codominant markers taking account of typing error, and define a statistic delta for resolving paternity. Using allele frequencies from the study population in question, a simulation program generates criteria for delta that permit assignment of paternity to the most likely male with a known level of statistical confidence. The simulation takes account of the number of candidate males, the proportion of males that are sampled and gaps and errors in genetic data. We explore the potentially confounding effect of relatives and show that the method is robust to their presence under commonly encountered conditions. The method is demonstrated using genetic data from the intensively studied red deer (Cervus elaphus) population on the island of Rum, Scotland. The Windows-based computer program, CERVUS, described in this study is available from the authors. CERVUS can be used to calculate allele frequencies, run simulations and perform parentage analysis using data from all types of codominant markers.

Animals↗

PubMed1997

No evidence for major histocompatibility complex-dependent mating patterns in a free-living ruminant population.

Conventionally, the extraordinary diversity of the vertebrate major histocompatibility complex (MHC is thought to have evolved in response to parasites and pathogens affecting fitness. More recently, reproductive mechanisms such as disassortative mating have been suggested as alternative mechanisms maintaining MHC diversity. A large unmanaged population of Soay sheep (Ovis aries L.) was used to investigate reproductive mechanisms in the maintenance of MHC diversity. Animals were sampled as new-born lambs and between 887 and 1209 individuals were typed at each of five microsatellite markers located either within or flanking the ovine MHC. All loci were in Hardy-Weinberg proportions. A novel likelihood-based approach was developed to analyse mating patterns using paternity data. No evidence for non-random mating with respect to MHC markers was found using this technique. We conclude that MHC diversity in the St Kildan Soay sheep population is unlikely to be maintained by mating preferences and that, in contrast with evidence from experimental mice populations, MHC variation plays no role in the mating structure of this population.

Animals↗

PubMed1997

Secreted enzymes of Aeromonas.

A hallmark characteristic of species of Aeromonas is their ability to secrete a wide variety of enzymes associated with pathogenicity and environmental adaptability. Among the most intensively studied are beta-lactamases, lipases, hemolytic enterotoxins, proteases, chitinases, nucleases and amylases. Multiple copies of genes encoding each type of enzyme provide additional biological diversity. Except for the chitinases, these multiple copies show little evolutionary relatedness at the DNA level and only limited similarity at the protein level. Indeed a number of the genes, such as nuclease H of A. hydrophila, have no similarity to known prokaryotic or eukaryotic sequences. The challenge is to determine how these genes evolved, where they originated and why Aeromonas possesses them in such abundance and variety.

Aeromonas↗

PubMed1997

Genetic and behavioural evidence of monogamy in a mammal, Kirk's dik-dik (Madoqua kirkii).

Little is known about the mating behaviour of monogamous mammals. Here, we present behavioural and genetic evidence of fidelity in a socially monogamous dwarf antelope, Kirk's dik-dik. DNA microsatellite analysis revealed no evidence of extra-pair paternity (EPP) in dik-diks: mothers' partners matched the paternal genotype in all 12 juveniles tested. One likely reason for the absence of EPP is that males guard their mates closely during oestrus and over-mark all female scent, thereby reducing the likelihood of other males attempting to mate. In addition, males may be limited in their ability to search for extra-pair populations (EPCs) by activities associated with pair-bond maintenance. Year-round, males maintained proximity within pairs, followed their females' activity patterns, and spent approximately 64% of their time with their partners. However, males did attempt to obtain EPCs when the opportunity arose, and genetic monogamy in dik-diks is probably best explained by the behaviour of females: in contrast to many monogamous female birds, female dik-diks do not appear to seek EPC partners. We propose that females avoid extra-pair males because they are unable to mate with them without instigating a potentially dangerous conflict.

Animals↗

PubMed1997

A gene encoding an exo-beta-glucosidase from Cellvibrio mixtus.

Cellvibrio mixtus produces an array of endohydrolytic enzymes involved in the initial phases of beta-glycan polysaccharide degradation in the soil. These enzymes convert complex, high-molecular-weight, insoluble polysaccharides into low-molecular-weight, soluble oligosaccharides which must be further degraded for cellular uptake and catabolism. Little is known about the enzymes involved in this latter process in C. mixtus. In this paper we report the cloning of the lam2 gene, which encodes an exohydrolase of low-molecular-weight, soluble beta-glucans and which may be involved in the latter stages of beta-glucan degradation in C. mixtus. The Lam2 enzyme has a broad substrate specificity including beta-1,3-, beta-1,4-, beta-1,6-, and beta-1,3-1,4-linked glucans. Analysis of the end-products of laminarin hydrolysis showed that the Lam2 enzyme is an exohydrolase that removes terminal glucose residues from its substrate. The substrate specificity and action pattern of this enzyme indicate that it belongs to EC3.2.1.21 (beta-glucosidase). The 2.5-kb lam2 insert encodes a protein of approximately 91 kDa as determined by in vivo transcription/translation and deletion analyses. Southern analysis showed that a single copy of the gene exists on the C. mixtus chromosome.

Biodegradation, Environmental↗

PubMed1996

The maintenance of genetic polymorphism in small island populations: large mammals in the Hebrides.

Conventionally, small populations living on islands are expected to lose genetic variation by drift. Fluctuations in population size, combined with polygynous mating systems, are expected to contribute to the process by increasing sampling effects on genetic variation. However, in individually monitored populations of Red deer on Rum and Soay sheep on St. Kilda, which experience fluctuations in population size, two processes have been identified which mitigate loss of genetic variation. First, in a number of examples, population reductions are associated with selection. Selection may be in favour of heterozygotes, or, as we have documented in several cases, it may fluctuate in direction temporally. Second, in Soay sheep, in which mortality over population crashes is male-biased, ostensibly leading to low effective numbers of males, molecular studies show that there are systematic changes in the reproductive success of young males, and in variance in male success, that broaden genetic representation compared with expectation.

Adenosine Deaminase↗

PubMed1996

Density-dependent selection in a fluctuating ungulate population.

Despite considerable theoretical interest no direct examples of density-dependent natural selection acting on simple polymorphic variation have been documented in a natural population. Here we show that the magnitude of selective differences in survival between phenotypes in two conspicuous polymorphisms of coat colour and horn type in Soay sheep Ovis aries living on St Kilda, Scotland are associated with marked changes in population density. Selection is strongest in years of high density but weak in years of low density. In addition to direct observations of density-dependent 'soft' selection in a natural population, the analysis revealed that the level of overcompensatory mortality (responsible for promoting population instability) was higher after accounting for genetic variation in the coat and horn morph traits. The results emphasize the importance of understanding the interaction between selection and population demography for both genetic and ecological studies of natural populations.

Animals↗

PubMed1996

Cloning, sequencing, and characterization of the nucH gene encoding an extracellular nuclease from Aeromonas hydrophila JMP636.

An Escherichia coli clone expressing activity on DNase agar was obtained by cloning chromosomal DNA of Aeromonas hydrophila JMP636 into plasmid pUC19. Examination (of the clone's nuclease activity on a sodium dodecyl sulfate (SDS)-polyacrylamide gel containing DNA as a substrate revealed an activity band at approximately 100 kDa. Subsequently, subcloning localized the gene, designated nucH, to a 3.6-kb DNA fragment (pJP9521). Southern blotting of the nucH gene against chromosomal DNA of JMP636 confirmed that it had originated from this strain and demonstrated that it was present in a single copy, although additional faint bands were also detected. Analysis of the subclone using in vivo transcription and translation revealed only a single polypeptide of approximately 110 kDa. Sequencing of pJP9521 predicted an open reading frame of 3,213 bp encoding a protein of 1,070 amino acids and having a molecular mass of 114 kDa. Comparison of the deduced nucleotide sequence and the NucH predicted protein sequence with relevant databases indicated that no known homologs have previously been identified. A signal sequence was predicted from these data, and cellular fractionation of a nucH clone in E. coli indicated that the protein was able to be processed to the periplasm. An activity similar in size was detected in an extracellular protein sample of JMP636, while inactivation of the nucH gene resulted in loss of this activity band. By native SDS-polyacrylamide gel electrophoresis, NucH substrate specificity, cofactor requirements, and sensitivity to denaturing agents were assessed.

Aeromonas hydrophila↗

PubMed1995

Molecular genetic variation and individual survival during population crashes of an unmanaged ungulate population.

Theoretical models of the effect of population bottlenecks on genetic variation assume that individuals are removed at random from the population. We investigated this assumption in a naturally regulated, unstable population of Soay sheep (Ovis aries). During rapid population declines or 'crashes', individuals were not removed at random with respect to genotype: we found associations between individual survival and certain genotypes at five polymorphic protein or microsatellite DNA loci (Ada, Got, Tf, MAF18 and OPACAP). Some loci appeared to show simple associations with survival whereas others had more complex interactions with crash year or age: all displayed different patterns of association between the sexes. Simple overdominance was not a general feature of our data; it seems likely that fluctuating selecting, countervailing selection in different fitness components or frequency-dependent selection may explain the pattern and complexity of the associations shown at different loci. Our study cannot distinguish between selection acting at these loci or at other, closely linked loci. However, our empirical study implies that the molecular genetic outcome of population bottlenecks in natural populations does not always follow theoretical expectations based on the random removal of genotypes. Bottlenecks in which individuals are removed at random are distinct from bottlenecks in which there is scope for selection via non-random survival of individuals.

Alleles↗

PubMed1995

A lipase of Aeromonas hydrophila showing nonhemolytic phospholipase C activity.

Extracellular lipase activity detected on tributyrin agar has been identified in a cosmid clone, JM3084, constructed from the chromosome of Aeromonas hydrophila and vector pHC79. This lipase, named apl-1, also exhibits nonhemolytic phospholipase C activity on lecithin and p-nitrophenylphosphorylcholine. Subcloning of the cosmid JMP3084 with partial Sau3a1 digestion localized the lipase gene to a 3.4-kb DNA fragment. Southern blot analysis shows the gene apl-1 to exist in single copy on the A. hydrophila chromosome. Expression of apl-1 in the pT7 system identified a single protein of molecular weight 70 kDa. Nucleotide sequencing of apl-1 has identified an open reading frame of 2055 bases predicting a protein of 73 kDa. The presence of an amino terminal signal sequence of 18 amino acids accounts for this molecular weight disparity. Further analysis of the lipase amino acid sequence revealed the presence of a classical serine active lipase site (Gly-X-Ser-X-Gly) located between residues 561 and 570. The A. hydrophila chromosomal copy of apl-1 has been inactivated by use of the mutagenesis vector pJP5603, resulting in the complete removal of phospholipase C activity and lowered levels of lipase activity detected on tributyrin agar.

Aeromonas hydrophila↗

PubMed1995

Extensive protein and microsatellite variability in an isolated, cyclic ungulate population.

We investigated polymorphism at protein and microsatellite DNA loci in an isolated, unmanaged and cyclic population of Soay sheep on the island of Hirta, St. Kilda. Extensive molecular variation was revealed at both protein loci (mean heterozygosity 7.78 per cent) and microsatellite loci (mean heterozygosity 50.93 per cent). Typically, large mammals possess limited protein variation and we were surprised to observe such a level of protein heterozygosity, particularly considering the genetic history of the Hirta population. Indeed, compared to other mammals, Soay sheep lie within the top 17 per cent of the distribution of average protein heterozygosities. We discuss the level of protein heterozygosity in the context of other mammalian species, other breeds of sheep and the genetic history of Soay sheep. Possible explanations for a large average and interlocus variance in protein heterozygosity are proposed. Although little data are available from other studies to compare with microsatellite DNA variability in this population, we discuss the potential application of microsatellite markers to interpopulation and interspecific genetic studies.

Animals↗

PubMed1995

Nonamplifying alleles at microsatellite loci: a caution for parentage and population studies.

While genotyping wild red deer (Cervus elaphus) at microsatellite loci for paternity assignment, we found three loci (MAF65, BOVIRBP and CelJP23) with segregating nonamplifying alleles. Nonamplifying alleles were detected through mismatches between known mother-offspring pairs and by significant deviations from Hardy-Weinberg equilibria. In a wide range of molecular ecology application, and especially in parentage assignment, the possible existence of undetectable alleles must be taken into account; this may be particularly important for microsatellite data.

Alleles↗

PubMed1994

Polymerase chain reaction for detection of Clostridium botulinum types A, B and E in food, soil and infant faeces.

The application of the polymerase chain reaction (PCR) for detection of Clostridium botulinum types A, B and E in foods, environmental and clinical samples was evaluated and compared to the mouse bioassay. Samples inoculated with 10, 100 and 1000 spores of Cl. botulinum types A and B included pasteurized milk, UHT milk, infant formula, infant faeces, meat juice, canned tuna, mushrooms, blood sausage and soil. Clostridium botulinum type E spores were inoculated into fish eggs, canned tuna, picked herring, raw fish and soil at similar levels. Spores were added to 2.5 g of each sample with the exception of soil which was inoculated in 10 g samples. The presence of Cl. botulinum in sample enrichments was determined by both PCR and the bioassay. An overall correlation of 95.6% was observed between PCR results and the mouse bioassay. Of the total of 114 samples tested there was disparity between the mouse bioassay and the PCR in three samples of soil inoculated with 100 type A or E spores and 10 type B spores per 10 g, respectively, and two samples of infant faeces inoculated with 10 type A or B spores per 2.5 g. All of these samples gave negative animal results and positive PCR results.

Animals↗

PubMed1994

Sequencing, chromosomal inactivation, and functional expression in Escherichia coli of ppsR, a gene which represses carotenoid and bacteriochlorophyll synthesis in Rhodobacter sphaeroides.

Sequencing of a DNA fragment that causes trans suppression of bacteriochlorophyll and carotenoid levels in Rhodobacter sphaeroides revealed two genes: orf-192 and ppsR. The ppsR gene alone is sufficient for photopigment suppression. Inactivation of the R. sphaeroides chromosomal copy of ppsR results in overproduction of both bacteriochlorophyll and carotenoid pigments. The deduced 464-amino-acid protein product of ppsR is homologous to the CrtJ protein of Rhodobacter capsulatus and contains a helix-turn-helix domain that is found in various DNA-binding proteins. Removal of the helix-turn-helix domain renders PpsR nonfunctional. The promoter of ppsR is located within the coding region of the upstream orf-192 gene. When this promoter is replaced by a lacZ promoter, ppsR is expressed in Escherichia coli. An R. sphaeroides DNA fragment carrying crtD', -E, and -F and bchC, -X, -Y, and -Z' exhibited putative promoter activity in E. coli. This putative promoter activity could be suppressed by PpsR in both E. coli and R. sphaeroides. These results suggest that PpsR is a transcriptional repressor. It could potentially act by binding to a putative regulatory palindrome found in the 5' flanking regions of a number of R. sphaeroides and R. capsulatus photosynthesis genes.

Aerobiosis↗

PubMed1994

Application of PCR to a clinical and environmental investigation of a case of equine botulism.

PCR for the detection of botulinum neurotoxin gene types A to E was used in the investigation of a case of equine botulism. Samples from a foal diagnosed with toxicoinfectious botulism in 1985 were reanalyzed by PCR and the mouse bioassay in conjunction with an environmental survey. Neurotoxin B was detected by mouse bioassay in culture enrichments of serum, spleen, feces, and intestinal contents. PCR results compared well with mouse bioassay results, detecting type B neurotoxin genes in these samples and also in a liver sample. Other neurotoxin types were not detected by either test. Clostridium botulinum type B was shown to be prevalent in soils collected from the area in which the foal was raised. Four methods were used to test for the presence of botulinum neurotoxin-producing organisms in 66 soil samples taken within a 5-km radius: PCR and agarose gel electrophoresis (types A to E), PCR and an enzyme-linked assay (type B), hybridization of crude alkaline cell lysates with a type B-specific probe, and the mouse bioassay (all types). Fewer soil samples were positive for C. botulinum type B by the mouse bioassay (15%) than by any of the DNA-based detection systems. Hybridization of a type B-specific probe to DNA dot blots (26% of the samples were positive) and PCR-enzyme-linked assay (77% of the samples were positive) were used for the rapid analysis of large numbers of samples, with sensitivity limits of 3 x 10(6) and 3,000 cells, respectively. Conventional detection of PCR products by gel electrophoresis was the most sensitive method (300-cell limit), and in the present environmental survey, neurotoxin B genes only were detected in 94% of the samples.

Animals↗