gamma-tubulin in trypanosomes: molecular characterisation and localisation to multiple and diverse microtubule organising centres.

A genomic clone from Trypanosoma brucei, which contains a full length gamma-tubulin gene, was isolated using degenerate oligonucleotide primers. The sequence of this clone predicts a protein of 447 amino acids having a high degree of homology with gamma-tubulins from human and Xenopus laevis (67.2% amino acid identity) and only 57.7% identity with the Plasmodium falciparum gamma-tubulin. Northern blot analysis of poly(A)+ selected RNA from a procyclic culture detects a major transcript of approximately 2.2 kb plus a minor transcript of approximately 3.6 kb. A fusion protein comprising almost the full length gamma-tubulin gene product (amino acids 8-447) plus an amino-terminal histidine tag has been expressed and purified from Escherichia coli and used to raise a polyclonal antibody. Immunofluorescence, using this antibody, shows classical centrosomal localisation in mammalian cells. In T. brucei gamma-tubulin is present in the basal bodies which subtend the flagellum and also at the anterior tip of the cell body where many minus ends of microtubules are located. Furthermore the antibody reveals a small subset of the sub-pellicular microtubules and a discrete dot within the nucleus which alters form with progression through the mitotic cycle. Evidence is also presented for discrete punctate staining within the microtubules of the cell body which may represent the presence of gamma-tubulin on the ends of individual microtubules. Our results indicate that gamma-tubulin is associated with diverse microtubule organising centres and structures in trypanosomes.