The VSG expression site of T. brucei contains several Expression Site Associated Genes which are expressed at high levels in a stage-specific manner. We have previously cloned and sequenced an ESAG which we named T- LR (for Trypanosome Leucine-rich Repeat) and others have called ESAG 8. The protein sequence predicted from T-LR contains two interesting domains. One has homology to the RING finger motif in regulatory proteins which bind DNA in a sequence-specific fashion, while the other is a leucine-rich repeat (LRR) sequence found in wide variety of prokaryotic and eukaryotic proteins involved in protein-protein interactions. Thus, it is likely that T-LR has an important regulatory role in parasite physiology. The overall aim of this project is to elucidate the role of T-LR and to identify the molecular mechanisms underlying its function. We also plan to identify and characterize other regulatory molecules with which T-LR may interact. We will express recombinant T-LR proteins in bacterial, yeast, insect or mammalian expression systems and produce specific antibodies against the potential nucleic acid- and protein-binding domains. The antibodies will be used to characterize the T-LR protein in terms of size, post- translational modification, and stage-specific expression and to determine its subcellular location. We will then identify molecules which interact with T-LR within the trypanosome cell. These nucleic acids and proteins will be characterized and their genes cloned. In addition, we will modulate the normal expression of the T-LR gene by knockout of the expressed T-LR gene in bloodstream forms and over-expression of T-LR in bloodstream and procyclic forms. Changes in trypanosome growth, morphology and expression of other specific genes will identify cellular processes involving T-LR. These studies are designed to identify new molecular processes that provide the basis for development of novel parasite control strategies. In addition, like the study of several other phenomena in T. brucei, they should also provide new insights into the fundamental molecular biology of all eukaryotic organisms.
