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

• Published in: Molecular and biochemical parasitology 2000-12, Vol.111 (2), p.283-297
• Main Authors: Bringaud, Frédéric, Robinson, Derrick R., Barradeau, Sébastien, Biteau, Nicolas, Baltz, Dominique, Baltz, Théo
• Format: Article
• Language: English
• Subjects: Amino Acid Motifs; Amino Acid Sequence; Animals; Biochemistry, Molecular Biology; Blotting, Western; Carrier Proteins - genetics; Carrier Proteins - metabolism; Cellular Biology; Double-stranded RNA expression; Flagella - metabolism; Flagella - ultrastructure; Flagellum transition zone; flagellum transition zone component; flagellum transition zone component protein; Fluorescent Antibody Technique; Gene Deletion; Gene inactivation; glucose transporter; glucose transporters; Life Sciences; Mice; Molecular Sequence Data; Protozoan Proteins - genetics; Protozoan Proteins - metabolism; Rabbits; Rats; Repetitive protein; Repetitive Sequences, Amino Acid; RNA, Double-Stranded - genetics; RNA, Double-Stranded - metabolism; Subcellular Fractions - metabolism; Transfection; Trypanosoma brucei; Trypanosoma brucei brucei - genetics; Trypanosoma brucei brucei - growth & development; Trypanosoma brucei brucei - metabolism; Trypanosoma brucei brucei - ultrastructure
• ISSN: 0166-6851; 1872-9428
• DOI: 10.1016/S0166-6851(00)00319-4

In Trypanosoma brucei, we have cloned a gene ∼5 kb downstream of the glucose transporter gene cluster, containing a variable number of 102 bp repeats. This gene encodes a protein with no homologues in the data bases. Antibodies raised against the 34 amino acids repeated motif recognized proteins ranging from 145 to 270 kDa, depending on strains, in both bloodstream and procyclic forms of T. brucei. A correlation was established between the apparent molecular mass of the detected proteins and the number of 34 amino acid repeats which varies from 3 to 40. We have called this protein the flagellum transition zone component (FTZC) due to its localization to the proximal region of the axoneme, within the transition zone. FTZC is the only reported example of a trypanosomal protein present in the transition zone. To determine the role of FTZC we developed a new strategy of gene inactivation based on conditional expression of double-stranded RNA. In the presence of tetracycline, expression of the double-stranded RNA, we observed a complete 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.