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The genetic system on Trypanosoma brucei has been analysed by generating large numbers of independent progeny clones from two crosses, one between two cloned isolates of Trypanosoma brucei brucei and one between cloned isolates of T. b. brucei and Trypanosoma brucei gambiense, Type 2. Micro and minisatellite markers (located on each of the 11 megabase housekeeping chromosomes) were identified, that are heterozygous in one or more of the parental strains and the segregation of alleles at each locus was then determined in each of the progeny clones. The results unequivocally show that alleles segregate in the predicted ratios and that alleles at loci on different chromosomes segregate independently. These data provide statistically robust proof that the genetic system is Mendelian and that meiosis occurs. Segregation distortion is observed with the minisatellite locus located on chromosome I of T. b. gambiense Type 2 and neighboring markers, but analysis of markers further along this chromosome did not show distortion leading to the conclusion that this is due to selection acting on one part of this chromosome. The results obtained are discussed in relation to previously proposed models of mating and support the occurrence of meiosis to form haploid gametes that then fuse to form the diploid progeny in a single round of mating.

The first genome survey sequencing of the rodent malaria parasite Plasmodium chabaudi is presented here. In 766 sequences, 131 putative gene sequences have been identified by sequence similarity database searches. Further, 7 potential gene families, four of which have not previously been described, were discovered. These genes may be important in understanding the biology of malaria, as well as offering potential new drug targets. We have also identified a number of candidate minisatellite sequences that could be helpful in genetic studies. Genome survey sequencing in P. chabaudi is a productive strategy in further developing this in vivo model of malaria, in the context of the malaria genome projects.

Trypanosoma brucei TREU 927/4 has been chosen as the reference strain targeted for complete sequencing of the genome of the African trypanosome. This line is pleomorphic in mammalian hosts and is fly transmissible; however it is relatively unstable with respect to variable surface glycoprotein (VSG) expression. Therefore, we subjected TREU 927/4 to 27 rapid syringe passages through mice, and derived a cloned line which expressed Glasgow University Trypanozoon antigen type (GUTat) 10.1 with relative stability. This line also retained pleomorphism in the bloodstream, being able to generate homogeneous populations of stumpy forms in mice. Furthermore, these parasites remain able to transform to procyclic forms synchronously in vitro and can complete their life cycle in tsetse flies. The passaged cell line was also adapted to in vitro bloodstream-form culture and transfected with a construct encoding the tetracycline repressor (TETR) protein. The resulting TETR subline no longer expressed the GUTat 10.1 VSG but remained able to generate uniform populations of stumpy form cells in mice immunocompromised with cyclophosphamide. They could also differentiate to procyclic forms synchronously in vitro. The generated lines and analyses of their growth and differentiation will provide a basic resource for the analysis and interpretation of gene function in the T. brucei genome reference strain.

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.

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.

We have investigated whether Trypanosoma brucei can undergo self-fertilisation. A group of 27 metacyclic clones derived from the tsetse transmission of a mixture of two genetically marked stocks was analysed and 22 clones were observed to be of non-hybrid phenotype. A group of 10 clones from this non-hybrid subset were then analysed for one isoenzyme, one restriction fragment length polymorphism and three karyotype markers potentially informative for the detection of self-fertilisation. Five of the 10 clones were found to be recombinant for at least one marker and we interpret these recombination events as indicating the clones to be products of self-fertilisation. We have also analysed a limited number of metacyclic clones from stocks of T. brucei each singly transmitted through tsetse flies but, so far, no evidence of recombination has been detected. We conclude that T. brucei is able to self-fertilise but there may be a requirement for the presence of dissimilar stocks to initiate such an event.

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.

The existence of a system of genetic exchange in Trypanosoma brucei is now established, but the frequency with which mating occurs and the mechanisms by which genes are exchanged are still unknown. This paper presents the results of a study of one pair of trypanosome stocks, which show that mating is a non-obligatory but frequent event in a life-cycle stage within the insect vector. Analysis of ten progeny clones using a total of eleven markers (iso-enzymes and DNA probes detecting restriction fragment length polymorphisms) has indicated that segregation of alleles occurs at five of these loci. The segregation patterns of a polymorphic EcoRI site in the maxi-circle of the kinetoplast DNA (kDNA) show that the progeny inherit one or other of the parental kDNA types. These results demonstrate that segregation of alleles occurs and that new combinations of alleles at different loci are generated in the progeny clones. The implications of these findings for defining the mechanism of gene exchange are discussed in relation to a simple mendelian genetic system involving meiosis and syngamy.