The EP and GPEET procyclins of Trypanosoma brucei are the surface glycoproteins of parasites in the midgut of tsetse flies (Glossina sp.). The procyclin genes are at the beginning of polycistronic transcription units and are followed by at least one procyclin-associated gene (PAG). Whereas the procyclin genes are highly expressed at all levels of gene expression, PAG mRNA levels are about two orders of magnitude downregulated by unknown mechanisms. The PAGs have no known function and encode putative proteins, however, there is no evidence that the PAG mRNAs are translated. In this study, three additional PAGs were identified. We show that the mRNA levels of PAG1, PAG2 and PAG3 are transiently increased during differentiation of bloodstream forms to procyclic forms. Sequentially knocking out all eight PAGs resulted in a cell line without obvious phenotype, indicating that the PAGs are not essential for cyclical transmission through tsetse flies. Deletion of the first PAG in each procyclin locus led to increased mRNA levels of downstream positioned PAGs. Nascent RNA analysis suggests that most of the effects were due to increased transcription elongation in the knockouts.

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