504 Nucleic Acids Research, 2006, Vol. 34, No. 2 Figure 5. Plasmodium and Toxoplasma selenocysteine tRNAs. (A) Cloverleaf models of P.falciparum (left) and T.gondii (right) tRNASecs. The anticodons are shown in bold. (B) Nucleotide sequence alignments of tRNASecs from P.falciparum, P.yoelii, P.knowlesi, P.vivax, P.berghei and T.gondii. The location of tRNA arms is indicated. The anticodon arm features the Sec anticodon (TCA). the D-stem 6 bp as is commonly observed in other eukaryotic tRNASecs. The anticodon stem in Plasmodium tRNASecs con- tains 6 bp as is found in other tRNASecs with the exception of P.vivax that contains a highly improbable G:A base pairing between the two bases most distal to the anticodon loop within this region. Whether this indicates a sequencing error or is indicative that the anticodon stem in Plasmodium may only require 5 bp must await further investigation. The three extra bases in Plasmodium tRNASec compared with those of animal and Chlamydomonas tRNASecs exist in the long extra arm. The T.gondii tRNASec is shown in Figure 5A, right cloverleaf structure. Its cloverleaf model that includes the CCA terminus is 90 nt long and is very similar to those of other eukaryotic tRNASecs. Evolution of Sec insertion in Plasmodium We constructed phylogenic trees for EFSec/eSelB, SPS and tRNASec (see Supplementary Figures S1–S3). EFSec/eSelB formed a cluster separate from that of EF-Tu, and in all three trees, plasmodial sequences clustered with other eukary- otes, suggesting a common origin of Sec insertion in eukary- otes. In addition, the tRNASec tree suggested that plasmodial tRNASecs formed a cluster that also included T.gondii tRNASec and it was separate from the cluster of animal tRNASecs that also included Chlamydomonas tRNASec. These data suggest that the plasmodial Sec insertion system is perhaps the most divergent among previously characterized eukaryotic Sec insertion systems. Relevance to malaria Growing drug resistance to malaria poses a major problem. For example, chloroquine, a safe and cheap drug, is a medication of choice for the treatment of malaria; however, resistance to this compound in Southeast Asia and many areas of Africa has become a problem. In fact, resistance to all major drugs is a general problem in some areas of Southeast Asia. The development of new drugs and drug targets is an important avenue to combat malaria. In this regard, the following facts support the hypothesis that the selenoproteome of Plasmodium is a promising drug target: (i) the four selenoproteins of P.falciparum are unique to the parasite; (ii) Sec is highly reactive toward electrophilic compounds and potential inhibi- tors; (iii) two of the selenoproteins are likely to be located in the apicoplast, a cell organelle which does not occur in the human host and bears unique metabolic pathways; and (iv) since malarial parasites are particularly susceptible to oxidative stress, interference with redox metabolism repre- sents a promising strategy for antimalarial drug development [reviewed in (35)]. Similarly to functionally characterized selenoproteins in other species, the four Plasmodium seleno- proteins are candidate redox proteins. SUPPLEMENTARY DATA Supplementary Data are available at NAR Online. ACKNOWLEDGEMENTS The authors wish to thank Dr Rimma Iozef for excellent experi- mental support and Drs David Sibley and John B. Dame for providing EST clones. The work is supported by NIH GM061603, and in part by DoE DE-FG07-02ID14380 and Deutsche Forschungsgemeinschaft grants BE 1540/6-3 and GR 2028/1-1. Funding to pay the Open Access publication charges for this article was provided by GM061603. Conflict of interest statement. None declared.