In Kinetoplastids, protein-coding genes are transcribed polycistronically by RNA polymerase II. Individual mature mRNAs are generated from polycistronic precursors by 5' trans splicing of a 39-nt capped leader RNA and 3' polyadenylation. It was previously known that trans splicing generally occurs at an AG dinucleotide downstream of a polypyrimidine tract, and that polyadenylation is coupled to downstream trans splicing. The few polyadenylation sites that had been examined were 100-400 nt upstream of the polypyrimidine tract which marked the adjacent trans splice site. We wished to define the sequence requirements for trypanosome mRNA processing more tightly and to generate a predictive algorithm. By scanning all available Trypanosoma brucei cDNAs for splicing and polyadenylation sites, we found that trans splicing generally occurs at the first AG following a polypyrimidine tract of 8-25 nt, giving rise to 5'-UTRs of a median length of 68 nt. We also found that in general, polyadenylation occurs at a position with one or more A residues located between 80 and 140 nt from the downstream polypyrimidine tract. These data were used to calibrate free parameters in a grammar model with distance constraints, enabling prediction of polyadenylation and trans splice sites for most protein-coding genes in the trypanosome genome. The data from the genome analysis and the program are available from: .

Trypanosomatids are widespread parasites that cause three major tropical diseases. In trypanosomatids, as in most other organisms, acetylation is a common protein modification that is important in multiple, diverse processes. This paper describes a new member of the Trypanosoma cruzi acetyltransferase family. The gene is single copy and orthologs are also present in the other two sequenced trypanosomatids, Trypanosoma brucei and Leishmania major. This protein (TcAT-1) has the essential motifs present in members of the GCN5-related acetyltransferase (GNAT) family, as well as an additional motif also found in some enzymes from plant and animal species. The protein is evolutionarily more closely related to this group of enzymes than to histone acetyltransferases. The native protein has a cytosolic cellular location and is present in all three life-cycle stages of the parasite. The recombinant protein was shown to have autoacetylation enzymatic activity.

A major feature of Plasmodium falciparum parasitized red blood cells (pRBC) is their capacity to sequester in the microcirculation. The binding is mediated by PfEMP1 (P. falciparum erythrocyte membrane protein 1), a variable protein encoded by the var gene family. P. falciparum avoids the host antibody response generated against previously used variants by switching the expression of PfEMP1, which may affect the disease outcome. We have here studied var gene transcription over time within the life cycle of the parasite by semi-quantitative PCR and sequencing by employing three sets of degenerate primers to the 5-prime end of the var genes (corresponding to the DBL1alpha-domain). To accurately determine transcript levels, subsequent in-depth analysis was made by amplifying the 10 most frequently expressed var sequences identified in each developmental stage by quantitative PCR (Q-PCR). The maximum peak in var gene transcription seems to vary in time among parasites. In five out of seven parasites, var gene transcription was found to be higher or equal at 22-26h post-invasion compared to 4-10h post-invasion. Our data indicate that the intra-isolate var gene transcription dominance order may change between different developmental stages. The transcription of var genes in field isolates is more complex than in laboratory strains and often changes after in vitro adaption of the parasite. By using semi-quantitative PCR employing degenerate primers combined with quantitative-PCR using specific primers it is possible to monitor var gene transcription in detail during the life cycle of the parasite. The work presented here suggests that trophozoite pRBC is likely to be the optimal source of RNA for predicting the translated var gene species.