Characterization and disruption of a new Trypanosoma brucei repetitive flagellum protein, using double-stranded RNA inhibition

In Trypanosoma brucei, we have cloned a gene similar to 5 kb downstream of the glucose transporter gene cluster, containing a variable number of 102 b p repeats. This gene encodes a protein with no homologues in the data bases . Antibodies raised against the 34 amino acids repeated motif recognized pr oteins ranging from 145 to 270 kDa, depending on strains, in both bloodstre am and procyclic forms of T. brucei. A correlation was established between the apparent molecular mass of the detected proteins and the number of 34 a mino acid repeats which varies from 3 to 40. We have called this protein th e flagellum transition zone component (FTZC) due to its localization to the proximal region of the axoneme, within the transition zone. FTZC is the on ly reported example of a trypanosomal protein present in the transition zon e. To determine the role of FTZC we developed a new strategy of gene inacti vation based on conditional expression of double-stranded RNA. In the prese nce of tetracycline, expression of the double-stranded RNA, we observed a c omplete disappearance of FTZC in the EATRO 1125 and EATRO 427 strains of T. brucei. Molecular ablation of FTZC does not generate any obvious phenotype such as, lethality, modification of growth rate or cellular shape, in the growth conditions used. (C) 2000 Elsevier Science B.V. All rights reserved.