ABSTRACT In eukaryotes, RNA polymerase (pol) I exclusively transcribes the large rRNA gene unit (rDNA) and mRNA is synthesized by RNA pol II. The African trypanosome, Trypanosoma brucei, represents an exception to this rule. In this organism, transcription of genes encoding the variant surface glycoprotein (VSG) and the procyclins is resistant to α-amanitin, indicating that it is mediated by RNA pol I, while other protein-coding genes are transcribed by RNA pol II. To obtain firm proof for this concept, we generated a T. brucei cell line which exclusively expresses protein C epitope-tagged RNA pol I. Using an anti-protein C immunoaffinity matrix, we specifically depleted RNA pol I from transcriptionally active cell extracts. The depletion of RNA pol I impaired in vitro transcription initiated at the rDNA promoter, the GPEET procyclin gene promoter, and a VSG gene expression site promoter but did not affect transcription from the spliced leader (SL) RNA gene promoter. Fittingly, induction of RNA interference against the RNA pol I largest subunit in insect-form trypanosomes significantly reduced the relative transcriptional efficiency of rDNA, procyclin genes, and VSG expression sites in vivo whereas that of SL RNA, αβ-tubulin, and heat shock protein 70 genes was not affected. Our studies unequivocally show that T. brucei harbors a multifunctional RNA pol I which, in addition to transcribing rDNA, transcribes procyclin genes and VSG gene expression sites.

Due to trans-splicing and polycistronic transcription, the 5′ end structure of precursor RNAs of protein coding genes in Trypanosoma brucei has not yet been characterized. In eukaryotes, in general, the 5′ ends of transcripts generated by RNA polymerase (pol) I and pol II are different. Pol I derived precursor RNAs contain an unmodified tri- or diphosphate group at their 5′ ends. In contrast, pol II primary transcripts, the 5′ triphosphate (initially also part of the pre-mRNA) is rapidly modified by the addition of methylated guanosine triphosphate, immediately after transcription initiation. We determined the 5′ end structure of precursor RNAs of the rRNA gene and the RNA pol I transcribed protein coding gene by the differential display of RNA ligase mediated amplification of cDNA ends (DDRLACE) method. Comparing the ability of the 5′ end of RNA transcripts to ligate with an RNA primer following different pre-treatments, the structure of the 5′ end of RNA transcripts was characterized. We found that: (1) the 5′ end of putative precursor RNAs from a pol I transcribed protein coding gene and the rRNA gene was uncapped; (2) ∼20% of the putative rRNA precursor contained a 5′ tri- or diphosphate group, representing the primary transcript and ∼80% of the putative rRNA precursor were dephosphorylated and contained a 5′ hydroxyl group; (3) the majority of putative neomycin resistance gene precursor RNAs, driven by the procyclin gene promoter (a pol I promoter), contained a 5′ hydroxyl group. The procyclin-neo primary transcript, as being those containing a 5′ tri- or diphosphate, was below a detectable level in the steady state RNA; and (4) we did not detect pol I transcribed precursor RNAs that contained a 5′ monophosphate group. The observation that the putative pre-RNAs derived from the procyclin gene promoter, similar to those of rRNA do not have a 5′ capped structure, is consistent with the notion that transcription of pol I transcribed protein coding genes is crucially dependent on trans-splicing for the cap addition.

ABSTRACT The recent advent of gene-targeting techniques in malaria (Plasmodium) parasites provides the means for introducing subtle mutations into their genome. Here, we used the TRAPgene of Plasmodium berghei as a target to test whether an ends-in strategy, i.e., targeting plasmids of the insertion type, may be suitable for subtle mutagenesis. We analyzed the recombinant loci generated by insertion of linear plasmids containing either base-pair substitutions, insertions, or deletions in their targeting sequence. We show that plasmid integration occurs via a double-strand gap repair mechanism. Although sequence heterologies located close (less than 450 bp) to the initial double-strand break (DSB) were often lost during plasmid integration, mutations located 600 bp and farther from the DSB were frequently maintained in the recombinant loci. The short lengths of gene conversion tracts associated with plasmid integration intoTRAP suggests that an ends-in strategy may be widely applicable to modify plasmodial genes and perform structure-function analyses of their important products.

Most Apicomplexan parasites, including the human pathogens Plasmodium, Toxoplasma, and Cryptosporidium, actively invade host cells and display gliding motility, both actions powered by parasite microfilaments. In Plasmodium sporozoites, thrombospondin-related anonymous protein (TRAP), a member of a group of Apicomplexan transmembrane proteins that have common adhesion domains, is necessary for gliding motility and infection of the vertebrate host. Here, we provide genetic evidence that TRAP is directly involved in a capping process that drives both sporozoite gliding and cell invasion. We also demonstrate that TRAP-related proteins in other Apicomplexa fulfill the same function and that their cytoplasmic tails interact with homologous partners in the respective parasite. Therefore, a mechanism of surface redistribution of TRAP-related proteins driving gliding locomotion and cell invasion is conserved among Apicomplexan parasites.

gel electrophoresis following surface biotinylation andradioiodination. Metabolic labeling with(35S)cysteine and electrophoretic analysis also revealed foreachcloned isolate apredominant protein that corresponded insize tothemajor surface protein demonstrated bysurface labeling techniques. Immunoprecipitation studies witha polyclonal antiserum specifically directed against the90-kDa majorcysteine-rich protein purified froma subclone ofthesheep isolate (02-4A1) showed that thecysteine-rich protein andthemajor surface protein are identical. Thesurface location oftheantigen wasfurther corroborated bythereaction offluorescence-labeled antibodies raised against the90-kDa 02-4A1 cysteine-rich protein withtheentire surface oflive trophozoites ofthehomologous clone. Theability ofthecloned Giardia isolates toundergo variations oftheir cysteine-rich surface protein (CRISP) wasdemonstrated bythespontaneous appearance ofnewCRISPsinclonally derived populations during prolonged invitro culturing andincultures ofthe02-4A1clone thathadsurvived treatment withthecytotoxic anti-90-kDa CRISPantiserum specific forthesurface antigen ofthis clone. The surviving progeny weredevoid oftheoriginal CRISP,asjudgedbyresistance totheimmuneserum. Subsequent cysteine metabolic labeling oftherecloned surviving trophozoites demonstrated alarge numberof newvariants, eachexpressing asingle CRISPthat varied significantly inmolecular weight fromthose inthe different cloned lines. These studies suggest that thepresence ofCRISPsandtheir variations arenotrestricted toGiardia isolates obtained fromhumansbutareuniversal phenomena amongtheGiardia duodenalis types of organisms.