African trypanosomes are not passively transmitted, but they undergo several rounds of differentiation and proliferation within their intermediate host, the tsetse fly. At each stage, the survival and successful replication of the parasites improve their chances of continuing the life cycle, but little is known about specific molecules that contribute to these processes. Procyclins are the major surface glycoproteins of the insect forms of Trypanosoma brucei. Six genes encode proteins with extensive glutamic acid–proline dipeptide repeats (EP in the single-letter amino acid code), and two genes encode proteins with an internal pentapeptide repeat (GPEET). To study the function of procyclins, we have generated mutants that have no EP genes and only one copy of GPEET. This last gene could not be replaced by EP procyclins, and could only be deleted once a second GPEET copy was introduced into another locus. The EP knockouts are morphologically indistinguishable from the parental strain, but their ability to establish a heavy infection in the insect midgut is severely compromised; this phenotype can be reversed by the reintroduction of a single, highly expressed EP gene. These results suggest that the two types of procyclin have different roles, and that the EP form, while not required in culture, is important for survival in the fly.

Procyclins are the major surface glycoproteins of insect forms of Trypanosoma brucei. We have previously shown that a conserved 16-mer in the 3 untranslated region (UTR) of procyclin transcripts functions as a positive element in procyclic-form trypanosomes. A systematic analysis of the entire 297-base 3 UTR has now revealed additional elements which are involved in posttranscriptional regulation: a positive element which requires the first 40 bases of the 3 UTR and at least one negative element between nucleotides 101 and 173 (the LII domain). Deletion of either positive element resulted in a >8-fold reduction in the amount of protein but only an ϳ2-fold decrease in the steady-state level of mRNA, suggesting that regulation also occurred at the level of translation. In contrast, deletion of LII caused a threefold increase in the steady-state levels of both the mRNA and protein. LII-16-mer double deletions also gave high levels of expression, suggesting that the 16-mer functions as an antirepressor of the negative element rather than as an independent activator. All three elements have an effect on RNA turnover. When either positive element was deleted, the half-life (t 1/2 ) of the mRNA was reduced from ϳ50 min (the t 1/2 of the wild-type 3 UTR) to <15 min, whereas removal of the LII element resulted in an increased t 1/2 of ϳ100 min. We present a model of posttranscriptional regulation in which the negative domain is counteracted by two positive elements which shield it from nucleases and/or translational repressors.The differentiation of bloodstream forms of Trypanosoma brucei into procyclic forms which replicate in the tsetse fly midgut is marked by the synthesis of a new surface coat composed of procyclins (otherwise known as procyclic acidic repetitive proteins [PARPs]) and the shedding of the variant surface glycoprotein (VSG) coat, which covers the parasites in the mammalian host (31,42,54). It has been estimated that each cell is covered by approximately six million procyclin molecules (9) which are attached to the surface membrane by glycosylphosphatidylinositol (GPI) anchors (15, 16). The parasites divide by binary fission, with a population doubling time of ϳ9 to 10 h in culture, so there is a constant requirement for high levels of procyclin synthesis in order to maintain the density of the coat.Like the majority of genes in trypanosomatids, the procyclin genes form part of polycistronic transcription units (reviewed in reference 50). The trypanosome strain T. brucei 427 contains four procyclin expression sites which are located on separate chromosomes (43). Each expression site consists of tandemly linked procyclin genes (␣ and ␤), followed by a locus-specific procyclin-associated gene (PAG) (6, 28, 51). Two expression sites also contain an additional gene, GRESAG 2 (gene related to ESAG 2), which is very similar to a gene in the VSG expression site (4).When bloodstream form trypanosomes are triggered to differentiate into procyclic forms, there is a 5-to 10-fold increase in transcription initiatio...

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