Different allele frequencies in Trypanosoma brucei brucei and Trypanosoma brucei gambiense populations

Restriction fragment length polymorphism (RFLP) has been analysed in Trypanosoma brucei DNA following hybridization with different DNA probes. This polymorphism seems to be due to allelic variation, and not to variation between sequence duplicates, since the genomic environment of the probed polymorphic fragments is conserved over considerable distances. In an analysis of 35 non-gambiense stocks, we found different combinations of homozygotes and heterozygotes for the four RFLP probes used, in keeping with previous observations that genetic reassortment occurs in T. b. brucei. Moreover, the non-gambiense populations from West and East Africa can be differentiated according to their characteristic allele frequencies. In sharp contrast, we found that the 49 T. b. gambiense stocks, analysed with the same probes, share the same single allelic combination and are all homozygous for each one of the four markers. This characteristic gambiense allele combination is very common among Western non-gambiense isolates, but rare or absent among Eastern ones. Two stocks isolated from man in West Africa turned out to be non-gambiense by all molecular criteria examined, including total nuclear DNA content. Taken together, these observations suggest that human serum-resistant variants may appear among the West African T. b. brucei population, and that T. b. gambiense evolved from one of these resistant variants as a man-adapted subspecies that became genetically isolated from the rest of the West African trypanosome population.     

Different trans RNA splicing events in bloodstream and procyclic Trypanosoma brucei

Most trypanosomatid genes are transcribed into polycistronic precursor RNAs that are processed into monocistronic mRNAs possessing a 39-nucleotide spliced leader (SL) at their 5′-ends and polyadenylation at their 3′-ends. We show here that precursor RNA derived from a luciferase gene integrated in reverse orientation at the rDNA locus of Trypanosoma brucei is processed into three major SL-containing RNAs in bloodstream cells and a single SL-containing RNA in procyclic RNAs. This difference in trans RNA splicing between bloodstream and procyclic cells is independent of the 5′- and 3′-UTRs flanking the luciferase coding region. Thus, bloodstream cells can recognize some sequences in precursor RNA as a SL addition site that procyclic cells do not. These alternative SL addition sites may be aberrant or they might be utilized to expand the number of gene products from individual genes. Future experiments on endogenous genes will be necessary to examine the latter possibility.     

Transport of methionine in Trypanosoma brucei brucei

African trypanosomes live free in the bloodstream and central nervous system of mammalian hosts and also within the midgut of the tsetse fly vectors which transmit them. The parasite plasma membrane represents the interface between both hosts and parasite, and trypanosomes accumulate many essential metabolites via specific transport processes. L-Methionine uptake by procyclic and bloodstream forms of Trypanosoma brucei has been measured and shown to be mediated by a transporter presenting similar characteristics in both forms of the parasite. The carrier shows, in both forms, a relatively high affinity for methionine (Km ca. 30 microM). The effect of inhibitors of ion gradients across the membrane indicated that the uptake process is likely to be dependent upon a proton motive force. Various methionine analogues were tested against the transporter and these have demonstrated that the recognition depends on the motif common to all amino acids, and an electronegative group at the position of the sulphur atom separated from the alpha-carbon atom by a two carbon spacer.     

The architecture of variant surface glycoprotein gene expression sites in Trypanosoma brucei

Trypanosoma brucei evades the immune system by switching between Variant Surface Glycoprotein (VSG) genes. The active VSG gene is transcribed in one of approximately 20 telomeric expression sites (ESs). It has been postulated that ES polymorphism plays a role in host adaptation. To gain more insight into ES architecture, we have determined the complete sequence of Bacterial Artificial Chromosomes (BACs) containing DNA from three ESs and their flanking regions. There was variation in the order and number of ES-associated genes (ESAGs). ESAGs 6 and 7, encoding transferrin receptor subunits, are the only ESAGs with functional copies in every ES that has been sequenced until now. A BAC clone containing the VO2 ES sequences comprised approximately half of a 330 kb 'intermediate' chromosome. The extensive similarity between this intermediate chromosome and the left telomere of T. brucei 927 chromosome I, suggests that this previously uncharacterised intermediate size class of chromosomes could have arisen from breakage of megabase chromosomes. Unexpected conservation of sequences, including pseudogenes, indicates that the multiple ESs could have arisen through a relatively recent amplification of a single ES.     

Characterization of subunits of the RNA polymerase I complex in Trypanosoma brucei

The Trypanosoma brucei homologue of the RNA polymerase I (RNA Pol I) subunit Rpa12p of Saccharomyces cerevisiae was cloned and characterized. This protein did not appear to be essential for growth in either bloodstream or procyclic forms of the parasite. Trypanosomes expressing a C-terminal tagged version of TbRPA12 were generated in order to purify RNA Pol I from both developmental stages. Tandem affinity purification (TAP) revealed a number of proteins associating with TbRPA12, some of which appeared to be stage-specific. Mass spectrometry allowed the identification of four subunits in addition to TbRPA12, namely TbRPA1, TbRPA2, TbRPC40 and one isoform of TbRPB5 (Tb1RPB5), as well as an unknown 30kDa protein and histones H2A and H3. Whereas these studies demonstrated that TbRPA1 was phosphorylated, no evidence for phosphorylation of TbRPA2 was found.     

Purification of an eight subunit RNA polymerase I complex in Trypanosoma brucei

Trypanosoma brucei harbors a unique multifunctional RNA polymerase (pol) I which transcribes, in addition to ribosomal RNA genes, the gene units encoding the major cell surface antigens variant surface glycoprotein and procyclin. In consequence, this RNA pol I is recruited to three structurally different types of promoters and sequestered to two distinct nuclear locations, namely the nucleolus and the expression site body. This versatility may require parasite-specific protein-protein interactions, subunits or subunit domains. Thus far, data mining of trypanosomatid genomes have revealed 13 potential RNA pol I subunits which include two paralogous sets of RPB5, RPB6, and RPB10. Here, we analyzed a cDNA library prepared from procyclic insect form T. brucei and found that all 13 candidate subunits are co-expressed. Moreover, we PTP-tagged the largest subunit TbRPA1, tandem affinity-purified the enzyme complex to homogeneity, and determined its subunit composition. In addition to the already known subunits RPA1, RPA2, RPC40, 1RPB5, and RPA12, the complex contained RPC19, RPB8, and 1RPB10. Finally, to evaluate the absence of RPB6 in our purifications, we used a combination of epitope-tagging and reciprocal coimmunoprecipitation to demonstrate that 1RPB6 but not 2RPB6 binds to RNA pol I albeit in an unstable manner. Collectively, our data strongly suggest that T. brucei RNA pol I binds a distinct set of the RPB5, RPB6, and RPB10 paralogs.     

Histone modifications in Trypanosoma brucei

Several biological processes in Trypanosoma brucei are affected by chromatin structure, including gene expression, cell cycle regulation, and life-cycle stage differentiation. In Saccharomyces cerevisiae and other organisms, chromatin structure is dependent upon posttranslational modifications of histones, which have been mapped in detail. The tails of the four core histones of T. brucei are highly diverged from those of mammals and yeasts, so sites of potential modification cannot be reliably inferred, and no cross-species antibodies are available to map the modifications. We therefore undertook an extensive survey to identify posttranslational modifications by Edman degradation and mass spectrometry. Edman analysis showed that the N-terminal alanine of H2A, H2B, and H4 could be monomethylated. We found that the histone H4 N-terminus is heavily modified, while, in contrast to other organisms, the histone H2A and H2B N-termini have relatively few modifications. Histone H3 appears to have a number of modifications at the N-terminus, but we were unable to assign many of these to a specific amino acid. Therefore, we focused our efforts on uncovering modification states of H4. We discuss the potential relevance of these modifications.     

Serum lipoprotein and Trypanosoma brucei brucei interactions in vitro

Trypanosoma brucei brucei IL3201 and IL3202, which are dependent on serum high or low-density lipoproteins to multiply under axenic culture conditions, acquired lipoprotein-associated 3H-lipids without binding, accumulating or degrading apolipoproteins. Uptake by the T. b. brucei of lipoprotein-associated [1 alpha, 2 alpha(n)-3H]cholesterol, [1 alpha, 2 alpha(n)-3H]cholesteryl linoleate, [1 alpha, 2 alpha(n)-3H]cholesteryl oleoyl ether and L-3-phosphatidyl [N-methyl-3H]choline, 1,2-dipalmitoyl, occurred at 37 degrees C but not at 0 degree C, and tended towards saturation with increasing concentrations of 3H-lipid-labelled lipoproteins in the incubation mixture. The uptake processes did not discriminate between high- or low-density lipoproteins, did not require exogenous divalent ions and were not inhibited by the presence of acidotropic agents (chloroquine, ammonium chloride) in the incubation mixture. Uptake by T. b. brucei of lipoprotein cholesterol was likely to result mainly from desorption and diffusion processes, whereas specific binding sites were probably involved in the uptake by T. b. brucei of lipoprotein cholesteryl linoleate, cholesteryl oleoyl ether and possibly phosphatidylcholine. Exponentially growing T. b. brucei hydrolysed cholesteryl linoleate to cholesterol and had only a small capacity to reesterify cholesterol, whereas committed non-dividing stumpy form T. b. brucei had a large capacity to esterify cholesterol. Conversion products of phosphatidylcholine were generated during or after uptake of this phospholipid by exponentially growing T. b. brucei.