TRYPANOSOMA-BRUCEI - MOLECULAR-CLONING OF HOMOLOGS OF SMALL GTP-BINDING PROTEINS INVOLVED IN VESICLE TRAFFICKING

Members of the rab subfamily of GTPases have been implicated as import ant components in vesicle trafficking in the eukaryotes. Individual ra b proteins have a remarkable degree of specific subcellular localizati on. As a first approach to the study of these proteins in the protozoa n parasite Trypanosoma brucei we have undertaken a survey of the rab s ubfamily using a strategy based on degenerate polymerase chain reactio n utilizing the miniexon and the highly conserved WDTAGQE box which co mprises part of the GTP-binding site. A number of T. brucei partial cD NAs were obtained from procyclic stage cDNA, and seven of these clones (designated rtb1, 3-7, 9) were clearly members of the rab family. Nor thern analysis of poly(A)-selected RNA indicates that rtb, 3, 4, and 7 are constitutively expressed at low levels in both life-stages of T. brucei. By Southern analysis of trypanosome genomic DNA and specific P CR from an isolated genomic clone, we show that two of the genes, RTB1 and RTB7, are adjacent or at least closely linked in the T. brucei ge nome, while the other five are dispersed. These data provide important molecular reagents for dissecting the unusual secretory pathway in th is organism. (C) 1995 Academic Press Inc.