Molecular variation of Trypanosoma brucei subspecies as revealed by AFLP fingerprinting

Genetic analysis of Trypanosoma spp. depends on the detection of variation between strains. We have used the amplified fragment length polymorphism (AFLP) technique to develop a convenient and reliable method for genetic characterization of Trypanosome (sub)species. AFLP accesses multiple independent sites within the genome and would allow a better definition of the relatedness of different Trypanosome (sub)species. Nine isolates (3 from each T. brucei subspecies) were tested with 40 AFLP primer combinations to identify the most appropriate pairs of restriction endonucleases and selective primers. Primers based on the recognition sequences of EcoRI and BglII were chosen and used to analyse 31 T. brucei isolates. Similarity levels calculated with the Pearson correlation coefficient ranged from 15 to 98%, and clusters were determined using the unweighted pair-group method using arithmetic averages (UPGMA). At the intraspecific level, AFLP fingerprints were grouped by numerical analysis in 2 main clusters, allowing a clear separation of T. b. gambiense (cluster I) from T. b. brucei and T. b. rhodesiense isolates (cluster II). Interspecies evaluation of this customized approach produced heterogeneous AFLP patterns, with unique genetic markers, except for T. evansi and T. equiperdum, which showed identical patterns and clustered together

DNA probe- and PCR-based methods for the detection of trypanosomes

Accurate identification of an infectious organism facilitates the study of epidemiology of the disease it causes and makes possible rational evaluation of the relative success of different control strategies. Highly repetitive, tandem or interspersed DNA sequences unique to different species, type or sub-type of the African trypanosomes have been cloned as recombinant plasmids for use in specific and sensitive identification of the trypanosomes. Protocols have been adapted for use with these DNA probes in the detection of trypanosomes in the blood or buffy coat samples from mammalian hosts and the saliva of live tsetse flies. The detection of parasite DNA relies upon hybridization with parasite type-specific DNA probe labelled with digoxigenin, followed by revealing the hybridized probe using anti-digoxigenin antibodies conjugated to alkaline phosphatase and the addition of enzyme substrates resulting in either visible colour or emission of light detectable by autoradiography. Combined with the polymerase chain reaction (PCR), the method detects trypanosomes in bufffy-coat samples from antigenaemic but aparasitaemic cattle, and in the saliva of live infected tsetse flies. The majority of these recombinant DNA probes are presently available from various sources

A study on comparative molecular properties of Trypanosoma (Nannomonas) Congolense.

he many diverse and phenotypically unrelated diseases that occur with high frequency in aging mammalian species may have only a few primary causes. There is ample experimental evidence indicating that some of the essential and beneficial cellular metabolic reactions spontaneously generate free radicals and other highly reactive agents which may diffuse from their place of origin throughout the cell and damage some of its components. Free radicals have been implicated by radiobiological data as one of the mechanisms by which the radiation damage to cellular components is effected. The cellular genome has been shown to be a very important target of free radical damage to cells. The effect of the free radicals would not be different whether the free radicals are spontaneously generated or are caused by radiation. The occurrence in the cell of free radical-dependent reactions and the potential damage these radicals can cause has led to the proposition that protein-DNA cross-links may be spontaneously caused as side effects of the essential cellular metabolic processes. Since protein-DNA cross-links as caused by irradiation of cells is very poorly reparable, it is proposed further that the naturally caused protein-DNA cross-links accumulate with age in mammalian species and that these cross-links may be an important primary cause of aging in mammals. The possible natural occurrence and accumulation of protein-DNA cross-links in mouse liver tissue was investigated as the problem of this thesis using several methods: the SDS-KC1, chloroform and nitrocellulose filter assays to detect protein-DNA complexes and the differential centrifugation and exclusion chromatography assays to determine extractability of chromosomal proteins. Results of the SDS-KCl and chloroform methods imply that protein-DNA cross-links occur in mouse liver chromatin and that chromatin prepared from old mice have more abundant protein-DNA cross-links than that prepared from young mice. This indication is further supported by results obtained using the more sensitive nitrocellulose membrane filter assay for protein-DNA cross-links. Data from the salt extractability assay also support the conclusion of a higher bonding stability of chromosomal proteins obtained from older animals. Collectively, these data strongly suggest that protein-DNA cross-link formation is one of the important processes that occur with age in mammalian chromatin. These results support our working hypothesis that protein-DNA cross-links may be an important primary aging process in mammalian species

Physical and transcriptional organization of the ribosomal RNA genes of the savannah-type Trypanosoma congolense

Ribosomal RNA genes have been cloned from the major species of African trypanosomes. Complete nucleotide sequence composition of the small subunit (SSU) and portions of the large subunit (LSU) ribosomal RNA genes was determined for each of these trypanosome species. In contrast to the situation in Trypanosoma brucei, in savannah-type T. congolense the LSU ribosomal RNA is cleaved twice, to generate two additional prominent fragments. This leads to the different profiles observed when the rRNA molecules from these two trypanosome species are resolved in agarose gels. From the nucleotide sequences of the 18S RNA, a phylogenetic tree was derived depicting the relationships among the T. congolense complex of trypanosomes and the other species of trypanosomes

Alterations in gene transcription patterns associated with the Development of isometamidium resistance in Trypanosoma congolense

In order to search for T. congolense genes whose transcription status may be influenced by response to isometamidium chloride, we applied the Representational Difference Analysis (RDA) (Lisitsyn et al., 1993; Hubank & Schatz, 1994) to the comparative analysis of cDNA synthesised from two isogenic trypanosome clones, the isometamidium chloride sensitive T. congolense ILI 180 (Nantulya et al., 1984) and its resistant derivative, IL3343 (Peregrine et al., 1997). Three major categories of genes were found: (i) those transcribed only in the trypanosome clone sensitive to isometamidium chloride, (ii) those transcribed more abundantly in only one of the two isogenic trypanosome clones, and (iii) those that are transcribed at approximately the same level, but which detect highly polymorphic loci in the isogenic trypanosome clones