Abstract

Plasmodium falciparum contains two genes encoding different isotypes of alpha-tubulin, alpha-tubulin I and alpha-tubulin II. alpha-Tubulin II is highly expressed in male gametocytes and forms part of the microtubules of the axoneme of male gametes. Here we present the characterization of Plasmodium berghei alpha-tubulin I and alpha-tubulin II that encode proteins of 453 and 450 amino acids, respectively. alpha-Tubulin II lacks the well-conserved three amino acid C-terminal extension including a terminal tyrosine residue present in alpha-tubulin I. Investigation of transcription by Northern analysis and RT-PCR and analysis of promoter activity by GFP tagging showed that alpha-tubulin I is expressed in all blood and mosquito stages. As expected, alpha-tubulin II was highly expressed in the male gametocytes, but transcription was also observed in the asexual blood stages, female gametocytes, ookinetes and oocysts. Gene disruption experiments using standard transfection technologies did not produce viable parasites indicating that both alpha-tubulin isotypes are essential for the asexual blood stages. Targeted modification of alpha-tubulin II by the addition of the three C-terminal amino acids of alpha-tubulin I did not affect either blood stage development nor male gamete formation. Attempts to modify the C-terminal region by adding a TAP tag to the endogenous alpha-tubulin II gene were not successful. Introduction of a transgene, expressing TAP-tagged alpha-tubulin II, next to the endogenous alpha-tubulin II gene, had no effect on the asexual blood stages but strongly impaired formation of male gametes. These results show that alpha-tubulin II not only plays an important role in the male gamete but is also expressed in and essential for asexual blood stage development.