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Biomedical subjects

A Tait

Publications and source records attributed to A Tait.

At least 37 records · Page 2Linked to original sources
PubMed2001

Hydrogen isotope analysis of natural abundance and deuterium-enriched waters by reduction over chromium on-line to a dynamic dual inlet isotope-ratio mass spectrometer.

This paper describes the application of a simple chromium reduction furnace which can be interfaced with a dual inlet isotope-ratio mass spectrometer thus providing the capacity for cheap, fast, accurate and precise measurement of deltaD(V-SMOW) by dynamic mass spectrometry. Measurements are precise to the order of < or =0.5 per thousand. Mean 95% confidence intervals for the Vienna Standard Mean Ocean Water (V-SMOW) to Standard Light Antarctic Precipitation (SLAP) range are in the order of 2.5 per thousand and the system is linear over the range -428 to 23,000 per thousand. Memory effects do exist, but are small for natural abundance samples and can be minimised by careful planning of the analytical load.

Chromium↗
PubMed2001

Theileria annulata: identification, by differential mRNA display, of modulated host and parasite gene expression in cell lines that are competent or attenuated for differentiation to the merozoite.

To identify both host and parasite genes that show altered expression during differentiation of Theileria annulata from the macroschizont to the merozoite stage of the life cycle, the RNA profiles of two T. annulata-infected clonal cell lines (D7 and D7B12) with the same genetic background have been compared by RNA display. In the cloned cell line D7, T. annulata differentiates from the macroschizont to the merozoite at 41 degrees C, whereas in the cell line D7B12, which was derived by recloning D7, the parasite does not differentiate. Therefore, genes that show altered expression levels in either clone could be modulated by the differentiation event and are possible candidates for regulators of this process. Differential display was carried out initially on RNA extracted from D7 and D7B12 macroschizont-infected cells cultured at 37 degrees C and secondly on RNA extracted from the two cell lines incubated at 41 degrees C to induce differentiation to the merozoite. The first procedure identified 29 cDNA fragments that displayed altered levels between D7 and D7B12, 9 of which were confirmed to be differentially expressed by Northern blot analysis. Of these 9 gene fragments, 8 were found to be of host origin, while 1 was parasite derived. The second RNA display analysis identified 14 transcripts that showed altered levels during a differentiation time course, of which 6 were confirmed to be differentially expressed between D7B12 cells and differentiating D7 cells by Northern blot analysis. Of these 6 gene fragments, 1 was of host and 5 were of parasite origin. The parasite genes either showed levels of RNA consistent with constitutive gene expression or, in one case, a genuine upregulation of mRNA associated with the differentiation process.

Animals↗
PubMed2001

Evidence for multiple origins of human infectivity in Trypanosoma brucei revealed by minisatellite variant repeat mapping.

In recent years a wide variety of biochemical and molecular typing systems has been employed in the study of parasite diversity aimed at investigating the level of genetic diversity and delineating the relationship between different species and subspecies. However, such methods have failed to differentiate between two of the classically defined subspecies of the protozoan parasite Trypanosoma brucei: the human infective, T. b. rhodesiense, which causes African sleeping sickness, and the non-human infective T. b. brucei. This has led to the hypothesis that T. b. rhodesiense is a host range variant of T. b. brucei. In this paper we test this hypothesis by examining highly polymorphic tandemly repeated regions of the trypanosome genome, i.e., minisatellite loci. We have employed the technique of minisatellite variant repeat mapping by PCR (MVR-PCR), which determines the distribution of variant repeat units along the tandem array of one minisatellite, MS42. The maps generated by this technique not only allow unequivocal allele identification but also contain within them cladistic information which we used to determine the possible genetic relationship between the different subspecies of T. brucei. Our findings revealed that human infective (T. b. rhodesiense) isolates from Uganda are more closely related to the local non-human infective isolates (T. b. brucei) than they are to other human infective stocks from different regions, suggesting that human infectivity has originated independently in these different geographical regions. This would infer that the separate classification of all human infective stocks from East Africa into the subspecies T. b. rhodesiense is genetically inappropriate and it would be better to consider geographically separate populations as host range variants of T. brucei brucei or perhaps as a series of different subspecies. Based on these data, it is clear that MVR mapping is a very useful tool for the analysis of zoonotic eukaryotic pathogens where delineation of the origins of outbreaks of disease and definition of human infective strains are key questions.

Africa↗
PubMed2001

The detection of geographical substructuring of Trypanosoma brucei populations by the analysis of minisatellite polymorphisms.

Analysis of natural populations of Trypanosoma brucei has shown that there is linkage disequilibrium between alleles at pairs of loci in isolates taken from the field. This disequilibrium can occur as a result of a low frequency of genetic exchange, the masking of frequent genetic exchange by the rapid expansion of a few genotypes or by the treatment of 2 (or more) genetically isolated populations as a single population. We have analysed stocks from 2 geographically separate locations using 3 minisatellite markers to determine the frequencies of the alleles in each area and the frequency and nature of the multilocus genotypes. The results show that many alleles and multilocus genotypes are unique to each geographical location, supporting the conclusion that these populations are genetically isolated with limited or no gene flow between them. This geographical substructuring needs to be taken into account in considering the origins of the linkage disequilibrium in a number of populations.

Alleles↗
PubMed2000

Minisatellite marker analysis of Trypanosoma brucei: reconciliation of clonal, panmictic, and epidemic population genetic structures.

The African trypanosome, Trypanosoma brucei, has been shown to undergo genetic exchange in the laboratory, but controversy exists as to the role of genetic exchange in natural populations. Much of the analysis to date has been derived from isoenzyme or randomly amplified polymorphic DNA data with parasite material from a range of hosts and geographical locations. These markers fail to distinguish between the human infective (T. b. rhodesiense) and nonhuman infective (T. b. brucei) "subspecies" so that parasites derived from hosts other than humans potentially contain both subspecies. To overcome some of the inherent problems with the use of such markers and diverse populations, we have analyzed a well-defined population from a discrete geographical location (Busoga, Uganda) using three recently described minisatellite markers. The parasites were primarily isolated from humans and cattle with the latter isolates further characterized by their ability to resist lysis by human serum (equivalent to human infectivity). The minisatellite markers show high levels of polymorphism, and from the data obtained we conclude that T. b. rhodesiense is genetically isolated from T. b. brucei and can be unambiguously identified by its multilocus genotype. Analysis of the genotype frequencies in the separated T. b. brucei and T. b. rhodesiense populations shows the former has an epidemic population structure whereas the latter is clonal. This finding suggests that the strong linkage disequilibrium observed in previous analyses, where human and nonhuman infective trypanosomes were not distinguished, results from the treatment of two genetically isolated populations as a single population.

Animals↗
PubMed2000

Trypanosoma brucei: identification of trypanosomes with genotypic similarity to human infective isolates in tsetse isolated from a region free of human sleeping sickness.

In previous work, we have developed a molecular method that defines genotypes of Trypanosoma brucei and allows distinction of the human-infective subspecies T. b. rhodesiense from the non-human-infective T. b. brucei without recourse to measurement of resistance to lysis by human serum. Using this approach, we are also able to determine the geographical range of specific genotypes associated with a particular focus. In this study, we have characterised T. brucei isolates collected from tsetse in a region where human sleeping sickness has never been reported and which is some 500 km from the Busoga sleeping sickness focus of Uganda. We show that some of the trypanosome isolates taken from tsetse in this region have considerable genotypic similarity to trypanosomes from the Busoga focus, demonstrating a surprisingly wide dispersal of these trypanosome genotypes. Furthermore, the similarity of these genotypes to human-infective trypanosomes in the Busoga focus suggest the possible circulation of human-infective trypanosomes in this location. We also demonstrate that the genetic diversity in trypanosomes isolated from tsetse is significantly higher than that in those isolated from humans, confirming other studies that show that there exists a significant restriction in the range of genotypes that can be transmitted to humans.

Animals↗
PubMed2000

Amplified restriction fragment length polymorphism in parasite genetics.

The amplified restriction fragment length polymorphism (AFLP) technique is a relatively new method for the analysis of polymorphism that has not yet been widely used in parasitology. In this article, Dan Masiga, Andy Tait and Mike Turner provide a brief introduction to AFLP and illustrate how it can be used in the investigation of marker inheritance in genetic crosses and in the analysis of polymorphism of field populations. They also briefly highlight the strengths and weaknesses of AFLP in comparison with other methods for detecting polymorphism and conclude that AFLP is a very useful addition to the range of techniques available.

Animals↗
PubMed2000

Phylogenetic analysis of partial mitochondrial cytochrome oxidase c subunit I and large ribosomal RNA sequences and nuclear internal transcribed spacer I sequences from species of Cyathostominae and Strongylinae (Nematoda, Order Strongylida), parasites of the horse.

Three nucleotide data sets, one nuclear (ITS-2) and two mitochondrial (COI and l-rRNA), have been investigated in order to determine relationships among species of Strongylinae and Cyathostominae, intestinal parasites of the horse. The data exhibited a strong mutational bias towards A and T and in the COI gene, silent sites appeared to saturate rapidly partly due to this substitution bias. Thus, the COI gene was found to be less phylogenetically informative than the l-rRNA and ITS-2 genes. Combined analysis of the l-rRNA and ITS-2 genes supported a monophyletic clade of the cyathostomes with Tridentoinfundibulum gobi, which had previously been classified as a nematode of' uncertain origin'. The Strongylinae grouped consistently outside the clade containing the cyathostomes and T. gobi. Molecular analysis failed to provide strong evidence for the separation of cyathostomes into classical genera, as previously defined by morphological classification.

Animals↗
PubMed1999

Analysis of ploidy (in megabase chromosomes) in Trypanosoma brucei after genetic exchange.

The megabase chromosomes of Trypanosoma brucei are normally diploid, but the extent to which this ploidy is maintained when parasites undergo genetic exchange is not known. To investigate this issue, a panel of 30 recombinant clones resulting from the co-transmission through tsetse flies of three different parental T. brucei lines in all pair-wise combinations (STIB 247, STIB 386 and TREU 927/4) were examined. These clones are products of 28 different mating events; four of them result from self-fertilisation and the others are F1 hybrids. DNA contents of the three parental lines were determined by flow cytometry and shown to differ only slightly with DNA content increasing in the order 927/4 < 247 < 386. Flow cytometry of the recombinant clones indicated DNA contents were similar to the parents in 28 clones and raised approximately 1.5 times the parental values in only two. The two F1 hybrid progeny with raised DNA contents were shown by marker analysis to be trisomic for seven independent loci indicating that they were probably triploid whereas progeny with DNA contents similar to parental values inherited a single allele from each parent for four independent loci indicating that they were diploid.

Animals↗
PubMed1999

A high level of mixed Trypanosoma brucei infections in tsetse flies detected by three hypervariable minisatellites.

The issue of whether genetic exchange occurs at a significant frequency in natural populations of Trypanosoma brucei is controversial and one of the arguments against a high frequency has been the apparent lack of host infections with mixtures of trypanosome genotypes. Three minisatellite markers (MS42, CRAM, 292) within the coding regions of three genes have been identified and PCR based methods developed for detecting variation at these loci using crude lysates of infected blood as templates. Initial PCR analysis, using primers flanking the repeats, of DNA from two cloned stocks of the parasite has shown that two DNA fragments of different size were amplified from each stock. Analysis of the inheritance of these fragments into the F1 progeny of crosses demonstrated that the different size fragments were alleles that segregated in a Mendelian manner. The alleles at each of the three loci segregated independently consistent with their localisation on three different chromosomes. Analysis of a series of cloned isolates from tsetse flies showed that these loci were highly variable giving heterozygosities of 94% and the identification of 12 distinct alleles in a sample of 17 cloned isolates. In order to determine whether isolates are heterogeneous in terms of trypanosome genotype, the allelic variation at these three loci was examined in uncloned samples from tsetse flies isolated in Kiboko, Kenya and Lugala, Uganda. A significant proportion of the isolates (36% in Lugala and 47% in Kiboko) contained more than two alleles at one or more of the loci thus demonstrating that a high proportion of tsetse flies were infected with more than one genotype of trypanosomes. This was established, unequivocally, for two isolates by generating a series of cloned trypanosome lines from each and determining the genotype of each clone; one isolate (927) contained seven different genotypes with a high proportion of the possible combinations of alleles at each locus. These results indicate the possibility of frequent genetic exchange in the field, they imply that a significant proportion of mammalian hosts must contain mixtures of different trypanosome genotypes and they demonstrate the advantages of using minisatellite markers for the analysis of the population structure of T. brucei.

Alleles↗
PubMed1999

Evidence for localisation of a Theileria parasite AT hook DNA-binding protein to the nucleus of immortalised bovine host cells.

Immortalisation of bovine leukocytes by the macroschizont stage of the tick transmitted protozoan parasite, Theileria annulata, results in the clonal expansion of infected cells and dissemination throughout the bovine host. The parasite-encoded factors which induce this unique transformation event have not been defined to date. In this study, a gene family (TashAT) has been characterised that encodes polypeptides with homology to known DNA-binding proteins. Expression of TashAT genes occurs at the intracellular macroschizont stage of the parasite life cycle and during differentiation to the merozoite, negative regulation of TashAT genes is detected early relative to other macroschizont genes. Interestingly, the early reduction in TashAT expression coincides with the initial decrease in host cell proliferation. One member of the family, TashAT2, was characterised in detail and the predicted polypeptide sequence was found to harbor three AT hook DNA-binding domains. Antisera generated against two distinct regions of TashAT2 both located the antigen to the host cell nucleus and, combined with protein translation inhibition and immunoprecipitation studies, provide evidence that this polypeptide could be transported from the parasite to this location. Further evidence for this postulation was provided by transfection studies which demonstrated that TashAT2 does encode the structural information required for localisation to the nucleus of a mammalian cell. Thus, TashAT2 is a potential candidate for a parasite-encoded factor that modulates host cell gene expression and may be involved in the control of host cell proliferation.

Amino Acid Sequence↗
PubMed1999

A novel gene encoding a ras-like GTP-binding protein from Trypanosoma brucei: an evolutionary ancestor of the ras and rap genes of higher eukaryotes?

The ras superfamily of GTP binding proteins encompasses a wide range of family members, related by conserved amino-acid motifs, and act as molecular binary switches that play key roles in cellular processes. Gene duplication and divergence has been postulated as the mechanism by which such family members have evolved their specific functions. We have cloned and sequenced a ras-like gene, tbrlp, from the primitive eukaryote Trypanosoma brucei. The gene encodes a protein of 227 amino acids and contains the six conserved subdomains that designate it as a ras/rap subfamily member. However, the presence of key diagnostic residues characteristic of both the ras and rap families of GTP confuse the familial classification of this gene. Phylogenetic analysis of the GTP binding domain places its origins at the divergence point of the ras/rap families and suggests that tbrlp is an ancestral gene to the ras/rap genes of higher eukaryotes.

Amino Acid Sequence↗
PubMed1998

The molecular karyotype of the megabase chromosomes of Trypanosoma brucei and the assignment of chromosome markers.

We present the molecular karyotype of the megabase chromosomes of Trypanosoma brucei stock TREU927/4 (927). We have identified 11 diploid chromosomes ranging in size from 1 to 5.2 Mb approximately and pairs of homologues differ in size by up to 15%. A total of 401 cDNA probes were hybridised to T. brucei stock 927 chromosomes and 168 chromosome-specific markers were defined. Most of these markers were hybridised to the separated chromosomal DNA of two other cloned field isolates and four F1 progeny clones from a laboratory cross. The chromosomes vary in size by up to two and a half times between stocks and the DNA content of the 11 pairs of homologues varies by up to 33% in different stocks. Stock 927 contains the smallest chromosomes and the least nuclear genomic DNA. Nevertheless, all 11 syntenic groups of cDNA probes are maintained in all stocks. In the F1 hybrids only we have identified one extra PFG band to which none of our probes hybridise. We have shown that probes thought to be specific for the bloodstream-form variant surface glycoprotein expression sites hybridise to different chromosomes in different stocks and may hybridise to either one or both of a homologous pair of chromosomes. We have also determined the chromosomal location of the ribosomal RNA gene arrays.

Animals↗
PubMed1998

Characterisation of a polo-like protein kinase gene homologue from an evolutionary divergent eukaryote, Trypanosoma brucei.

The polo-like protein kinase gene family (PLKs) encodes proteins which are involved in the control of exit from mitosis in higher eukaryotes. We have cloned and analysed a polo-like kinase, tbplk, from an evolutionary divergent eukaryote, Trypanosoma brucei. The gene encodes a 767 amino acid protein of predicted size 86.8 kDa with 50.4% identity to mammalian PLKs over the protein kinase catalytic domain and it possesses a conserved motif, the 'polo-box', which is found in all PLKs. Phylogenetic analysis demonstrates that this gene is clearly a member of the PLK family, although it has some distinctive features such as a large C-terminal insertion when compared with mammalian PLKs. The gene is single copy and expressed in both bloodstream and procyclic stage trypanosomes. Sequencing of tbplk from a number of trypanosome isolates reveals a length polymorphism in a run of asparagine residues within the coding region. The presence of PLKs in a wide range of organisms, including such a primitive organism as T. brucei, suggests that PLKs may have a key role in the function of the cell cycle.

Amino Acid Sequence↗
PubMed1998

Identification of a Theileria annulata antigen expressed in multiple stages of the parasite life cycle.

In order to identify sporozoite surface molecules which may be involved in invasion and could act as potential vaccine candidates, a number of Mabs were raised in mice against T. annulata sporozoites. These were assayed for their ability to block sporozoite invasion of bovine peripheral blood mononuclear (PBM) cells in vitro. One of these, Mab 4B11, was found to neutralize sporozoite invasion to a high degree and to recognize a group of sporozoite antigens on Western blots. A T. annulata lambdagt11 genomic expression library was screened with Mab 4B11 and a positive clone containing a 900-bp insert (KP8) analysed further. Data from Southern and Northern blotting indicated that the gene containing the KP8 sequence, termed sporozoite and macroschizont gene 2 (spm2), was expressed both in T. annulata sporozoites and in later parasite life-cycle stages, macroschizont-infected leucocytes and piroplasms. The KP8 sequence was expressed in E. coli as a fusion protein with glutathione-S-transferase (GST) using the vector pGEX1lambdaT. Bovine antiserum raised against GST-KP8 recognised a single high molecular weight molecule on Western blots corresponding to one of the antigens recognised by Mab 4B11, expressed in sporozoites, macroschizont-infected leucocytes, and piroplasms. While our evidence suggests that the spm2 molecule alone is not responsible for sporozoite neutralization, it is a multistage antigen likely to function both in T. annulata sporozoites and in subsequent parasite life-cycle stages.

Amino Acid Sequence↗