African trypanosome are parasitic protozoa that cause sleeping sickness in humans and nagana among domestic livestocks. We have been pursuing the mechanisms of developmental regulations in one of the species Trypanosoma brucei brucei, and have made some interesting findings in our recent studies. We: (1) identified and analyzed the C-terminal targeting signals in T. brucei glycosomal protein import; (2) identified and characterized an interferon-gamma inducible MAP kinase homolog in bloodstream T. brucei; (3) identified, isolated and characterized the 20S and the activated 20S proteasome but demonstrated the apparent absence of 26 proteasome from T. brucei; (4) demonstrated that T. brucei differentiation from bloodstream into procyclic form may be initiated and completed within any particular phase of cell cycle without crossing any phase boundary; (5) established the feasibility of complementing T. brucei mutants for direct gene clonings. For the future research plan, we propose to: (1) identify genes involved in glycosomal biogenesis by complementing the 7 """"""""glycosomal-import-defective"""""""" T. brucei mutants in our possession; (2) identify the gene encoding the receptor for C- terminal glycosomal import targeting signals in the two hybrid system from yeast; (3) identify the early transcripts induced during T. brucei differentiation by suppression subtractive hybridization. One of the cDNA clones already identified in our preliminary studies turned out to encode the ribosomal L18 protein, known to be over-expressed in procyclic form; (4) identify, by complementation studies, the causative genes in the T. brucei mutants incapable of differentiating or proliferating; (5) identify the protease(s) involved in shedding the variant surface glycoprotein during T. brucei differentiation; (6) analyze the structure of T. brucei proteasome by mass spectrometry; (7) identify the natural protein substrates for T. brucei proteasome; (8) express the T. rhodesiense ornithine decarboxylase gene in transfected T. brucei, and monitor the half-life of the enzyme protein to verify the molecular basis of T. rhodesiense refractoriness to DFMO. It is our hope that the results form these planned studies will advance our understandings on the regulations of T. brucei development.
