CONTRIBUTIONS OF THE PROCYCLIN 3'-UNTRANSLATED REGION AND CODING REGION TO THE REGULATION OF EXPRESSION IN BLOOD-STREAM FORMS OF TRYPANOSOMA-BRUCEI

When bloodstream forms of Trypanosoma brucei differentiate into procyc lic forms they rapidly synthesise a new surface coat composed of procy clins. Procyclin genes are transcribed in bloodstream forms at approxi mately one-tenth of the rate in procyclic forms, but little, if any, m RNA can be detected, indicating that further down-regulation must occu r post-transcriptionally. We have examined the role of the 297 bp proc yclin 3' untranslated region (UTR) in regulating expression in bloodst ream forms and have identified three discrete elements: a dominant. ne gative element between positions 101 and 173, and two positive element s. When chloramphenicol acetyl transferase (CAT) was used as the repor ter gene, deletion of the negative element caused a similar to 6-fold increase in the level of steady slate mRNA and > 30-fold increase in C AT activity, suggesting that both RNA stability and translation were a ffected. Similar results were obtained with glutamic acid/alanine-rich protein (GARP), the T. congolense analogue of procyclin, indicating t hat the 3' UTR acts independently of the coding region. In contrast, w hen trypanosomes were stably transformed with a construct in which the procyclin coding region was linked to a truncated form of the 3' UTR which lacked the negative element, they expressed high levels of mRNA, but no protein could be detected in cell lysates or culture supernata nts. These results imply that the procyclin coding region exerts yet a nother layer of control which prevents inappropriate expression of the protein in the mammalian host. (C) 1997 Elsevier Science B.V.