THE GENE FAMILY OF EF-HAND CALCIUM-BINDING PROTEINS FROM THE FLAGELLUM OF TRYPANOSOMA-BRUCEI

The flagellum of Trypanosoma brucei contains calmodulin, and a separat e family of antigenically related EF-hand calcium-binding proteins whi ch we call calflagins. The following study evaluates the structure and genomic organization of the calflagin family. Genomic Southern blots indicated that multiple copies of calflagin genes occurred in T. bruce i, and that all of these copies were contained in a single 23 kb XhoI- XhoI fragment on chromosomes 15 and 16. mRNAs of 1.2 and 1.6 kb were i dentified in bloodstream and procyclic life-cycle stages. Genomic frag ments of 2.5 and 1.7 kb were cloned that encoded calflagin sequences. The calflagin genes were arranged tandemly along the genomic fragments . Three new members of the calflagin family were sequenced from a cDNA clone and the two genomic clones. Two unrelated families of 3' flanki ng sequences were downstream from the calflagin genes. An open reading frame that was unrelated to any calflagin sequence was at the 5' end of the.2.5 kb genomic fragment. The deduced amino acid sequences of th e genomic clones (called Tb-24 and Tb-1.7g) were similar to the previo usly described Tb-17. Each encoded an approximately 24 kDa protein whi ch contained three EF-hand calcium-binding motifs and one degenerate E F-hand motif. The cDNA encoded a protein (called Tb-44A) which was app roximately twice as large as the other calflagins. The large size resu lted from a nearly direct repeat of 186 amino acids. In general, varia bility among the T. brucei calflagins was greater than observed for re lated proteins from Trypanosoma cruzi. We demonstrate that this variab ility resulted from amino acid substitutions at the N-terminus, C-term inal extensions, and duplication of internal segments.