The system of genetic exchange in <i>Trypanosoma brucei</i> and other trypanosomatids

In this chapter, we discuss our current understanding of the systems of genetic exchange in trypanosomatids and the im-pact the recent genome projects have had on this area of research. We focus mainly on the details of Trypanosoma brucei as it is the most extensively studied of the “trityps”, but will also refer to a recently discovered novel mechanism of genetic exchange in T. cruzi and the apparent rarity of genetic ex-change in Leishmania sp.The system of genetic exchange in Trypanosoma brucei has been known to exist since the late eighties when a genetic cross between different strains was carried out by co-transmission through the tsetse fly. We discuss the segregation of nuclear, chromosomal and kDNA markers and outline the two current models for the mechanism of genetic exchange. We also present how the completion of the genome project has allowed the identification of polymorphic micro and minisatel-lite markers distributed throughout the genome, which have been used to prove formally that meiosis, independent assortment and crossing over occur in this para-site, as would be predicted in a conventional Mendelian system. Such data have been used to construct the first genetic map of T. brucei, which opens up the use of genetic analysis, coupled with positional cloning and the genome sequence, as a tool to identify the genes involved in a range of traits relevant to the disease

Human infectivity trait in <i>Trypanosoma brucei</i>: stability, heritability and relationship to sra expression

Some Trypanosoma brucei lines infect humans whereas others do not because the parasites are lysed by human serum. We have developed a robust, quantitative in vitro assay based on differential uptake of fluorescent dyes by live and dead trypanosomes to quantify the extent and kinetics of killing by human serum. This method has been used to discriminate between 3 classes of human serum resistance; sensitive, resistant and intermediate. TREU 927/4, the parasite used for the T. brucei genome project, is intermediate. The phenotype is expressed in both bloodstream and metacyclic forms, is stably expressed during chromic infections and on cyclical transmission through tsetse flies. Trypanosomes of intermediate phenotype are distinguished from sensitive populations of cells by the slower rate of lysis and by the potential to become fully resistant to killing by human serum as a result of selection or long-term serial passaging in mice, and to pass on full resistance phenotype to its progeny in a genetic cross. The sra gene has been shown previously to determine human serum resistance in T. brucei but screening for the presence and expression of this gene indicated that it is not responsible for the human serum resistance phenotype in the trypanosome lines that we have examined, indicating that an alternative mechanism for HSR exists in these stocks. Examination of the inheritance of the phenotype in F1 hybrids for both bloodstream and metacyclic stages from 2 genetic crosses demonstrated that the phenotype is co-inherited in both life-cycle stages in a manner consistent with being a Mendelian trait, determined by only one or a few genes

The use of Multiple Displacement Amplification to increase the detection and genotyping of <i>Trypanosoma</i> samples immobilised on FTA filters.

Whole genome amplification methods are a recently developed tool for amplifying DNA from limited template. We report its application in trypanosome infections, characterized by low parasitemias. Multiple displacement amplification (MDA) amplifies DNA with a simple in vitro step and was evaluated on mouse blood samples on FTA filter cards with known numbers of Trypanosoma brucei parasites. The data showed a 20-fold increase in the number of PCRs possible per sample, using primers diagnostic for the multicopy ribosomal ITS region or 177-bp repeats, and a 20-fold increase in sensitivity over nested PCR against a single-copy microsatellite. Using MDA for microsatellite genotyping caused allele dropout at low DNA concentrations, which was overcome by pooling multiple MDA reactions. The validity of using MDA was established with samples from Human African Trypanosomiasis patients. The use of MDA allows maximal use of finite DNA samples and may prove a valuable tool in studies where multiple reactions are necessary, such as population genetic analyses

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

Sub-lethal effects of an oil pollution incident on breeding kittiwakes Rissa tridactyla

Kittiwakes from the colony closest to the site of the wreck of the 'Braer' oil tanker in Shetland, Scotland, in 1993 were studied in the breeding season following the spill. Sub-lethal effects of the spill on breeding kittiwakes were assessed by comparison with an extensive data set collected at the same site (Sumburgh Head) during the 3 yr prior to the spill. Breeding performance in 1993 was very similar to that in 1992, with relatively good breeding success and short, frequent foraging trips. However, haematological data revealed a significant level of anaemia in the breeding kittiwakes at Sumburgh in 1993 compared to birds sampled in the same year at control colonies elsewhere in Scotland. The return rate of adults to the Sumburgh colony between 1992 and 1993 was exceptionally low, and nest-site and mate fidelity also appeared low. This was not a consequence of reduced survival, but was due to some adults not breeding in 1 or 2 yr following the spill, and subsequently returning to breed at the colony in later years. Such behavioural disruption may have considerable consequences for the dynamics and structure of the colony

Genetic evidence that metacyclic forms of Trypanosoma brucei are diploid

The hypothesis that metacyclic trypanosomes are haploid has been tested genetically. Five cloned stocks of Trypanosoma brucei (each having four known isoenzyme markers and six known restriction fragment length polymorphisms) have been independently transmitted through tsetse flies. Fifteen individual metacyclic organisms were taken from flies with mature cyclical infections and used to establish fresh clones. All the sub-clones from all the flies proved to be identical to the starting (parental) stocks, with respect to all the markers examined, including those markers which were heterozygous in the parental stocks. We conclude that metacyclic trypanosomes are diploid, and are not the product of an obligatory meiosis.</p

Visualisation and analysis of proteomic data from the procyclic form of <i>Trypanosoma brucei</i>

We have undertaken a large scale study of the proteins expressed in the procyclic form of the parasite Trypanosoma brucei, which causes African sleeping sickness, using 2-DE and MS. The complete data set encompasses over 2000 identifications, of which 770 are distinct proteins. We have discovered that multiple protein isoforms appear to be common in T. brucei, as most proteins have been matched to more than one gel spot. We have developed visualisation software to investigate the differences between isoforms, based on the information from the results of database searches with MS data. We are able to highlight instances where PTMs are the most likely cause of variant forms. In other cases, spots that appear reproducibly across replicates contain fragments of proteins, arising either as experimental artefacts or as part of protein degradation. We are also able to classify clusters of gel spots into different groups based on the pattern of peptides that have been matched from MS data. The entire data set is stored within a relational database system that allows complex queries (http://www.gla.ac.uk/functionalgenomics). Using specific proteins as examples, we demonstrate how the visualisation software and the database query facilities can be used

Genetic analysis of the human infective trypanosome <i>Trypanosoma brucei gambiense</i>: chromosomal segregation, crossing over, and the construction of a genetic map

&lt;b&gt;Background&lt;/b&gt; Trypanosoma brucei is the causative agent of human sleeping sickness and animal trypanosomiasis in sub-Saharan Africa, and it has been subdivided into three subspecies: Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense, which cause sleeping sickness in humans, and the nonhuman infective Trypanosoma brucei brucei. T. b. gambiense is the most clinically relevant subspecies, being responsible for more than 90% of all trypanosomal disease in humans. The genome sequence is now available, and a Mendelian genetic system has been demonstrated in T. brucei, facilitating genetic analysis in this diploid protozoan parasite. As an essential step toward identifying loci that determine important traits in the human-infective subspecies, we report the construction of a high-resolution genetic map of the STIB 386 strain of T. b. gambiense. &lt;b&gt;Results&lt;/b&gt; The genetic map was determined using 119 microsatellite markers assigned to the 11 megabase chromosomes. The total genetic map length of the linkage groups was 733.1 cM, covering a physical distance of 17.9 megabases with an average map unit size of 24 kilobases/cM. Forty-seven markers in this map were also used in a genetic map of the nonhuman infective T. b. brucei subspecies, permitting comparison of the two maps and showing that synteny is conserved between the two subspecies. &lt;b&gt;Conclusion&lt;/b&gt; The genetic linkage map presented here is the first available for the human-infective trypanosome T. b. gambiense. In combination with the genome sequence, this opens up the possibility of using genetic analysis to identify the loci responsible for T. b. gambiense specific traits such as human infectivity as well as comparative studies of parasite field populations