Molecular Microbiology (1989) 3(11), 1511-1519 Cloning of a (3-tubulin gene from Plasmodium falciparum C. J. Delves,^ R. G. Ridley,^ M. Goman,^ S. P. Holloway,=2 J. E. Hyde^ and J. G. Scaife^* ^Department of Moiecuiar Biology, University of Edinburgh, King's Buiidings, Mayfield Road, Edinburgh EH9 3JR, UK. ^Department of Biochemistry and Applied Molecular Bioiogy, University of Manchester institute of Science and Technology, PO Box 88, Manchester M60 10D, UK. Summary We describe the isolation and characterization of a gene for p-tubulin from the malaria parasite, Plasmo- dium falciparum. This organism appears to contain a single gene encoding f^-tubulin. A single transcript from this gene can be detected in the total RNA of the parasite's asexual blood stages. The complete sequence for the gene has been elucidated. It has two introns, one of which has a position identical to that of a related parasite, Toxoplasma gondii. The gene shows the usual preference for codons with A or T in the third position. The predicted amino acid sequence is compared with that of T. gondii and the human host. Further comparisons between these and fungal sequences of p-tubulins resistant to benomyl, a drug binding this protein, highlight differences that could be exploited in the development of parasite-specific anti- tubulin drugs. Introduction Microtubules are filamentous proteins comprising the principal structural components of mitotic spindles, cilia, flagella and neuronal processes in eukaryotic cells (Bershadsky and Vasiliev, 1989). They are formed by the polymerization of dimers of non-identical a- and p-tubulln subunits, which have regions of homology (Valenzuela ef al., 1981). In some species {Toxoplasma gondii, Nagel and Boothroyd, 1988; Saccharomyces cerevisiae, Neff et al., 1983; Hiraoka etal., 1984), one or both of the subunits are encoded by single-copy genes. Others have multigene families whose members are either tandemly repeated {Trypanosoma, Seebeck et ai., 1983; Leishmania, Land- fear et ai., 1983) or dispersed throughout the genome Received 17 May, 1989. 'For correspondence. Tel. (031)667 1081. {Drosophila, Sanchez etal., 1980; chicken, Lopata etal., 1983; human. Cowan etal., 1981; Lee etai., 1983). Tubulins show a high degree of evolutionary conser- vation. For instance, hybrid microtubules can be created easily in vitro by co-polymerization of tubulins from different species (Snyder and Mclntosh, 1976); further- more, a chimaeric p-tubulin containing both mammalian and yeast sequences can be incorporated into the micro- tubules of mouse celis without impairing their growth rate or morphology (Bond ef al., 1986). Nevertheless, significant functional differences be- tween tubulins of higher and lower eukaryotes have been documented. Drug-binding studies have been parti- cularly informative. Colchicine is toxic to mammalian cells at concentrations that have no effect on yeast tubulins (Kilmartin, 1981), whereas benzimidazoles bind selectively to nematode tubulin and inhibit microtubule formation (Dawson ef al., 1984). Among the fungi, susceptibility to these drugs varies widely and can be changed by mutation within the p-tubulin gene (Thomas ef ai., 1985; Orbach ef ai., 1986; Jung ef ai., 1989). Thus the tubulins attract considerable interest as potential targets for drug development. The malaria parasite, Piasmodium faiciparum, remains an important pathogen for which there is a need for new drugs. We therefore decided to study the tubulins of this organism, seeking differences from their analogues in the host which might be exploited in drug design. We were encouraged in this approach by the finding that anti- tubulin drugs do arrest the growth of blood-stage para- sites in culture (Usanga ef al., 1986). In this paper we report the cloning and characterization of a gene for p-tubulin from P. falciparum. The gene and its adjacent DNA have been completely sequenced, allowing us to compare its amino acid sequence with those of humans, a related parasite, Toxopiasma gondii, and some fungi. These comparisons indicate which regions of p-tubulin may bind the benzimidazoles and suggest a part of the P. faiciparum protein which might be exploited as a site differentially susceptible to this class of drugs. Results Isolation of the p-tubulin gene in order to clone the p-tubulin gene(s) of P. falciparum we