498 Nucleic Acids Research, 2006, Vol. 34, No. 2 TAAAAGG-30; as: 50-AAAAGAGTTATTTTGTATATGTT- CC-30. Selenoprotein #4, N: 50-ATGGACACAAATGAAAA- TATGAAA-30; as: 50-CCTATTTTTTAATGACGGCC-30; C: 50-TTATCGTCAGGATTTGCGATTATTG-30; nC: 50-TTTT- TTTGTACTTTTAATTCATCC-30; CD: 50-CTAATTTTTT- CCTTTTATTATTTTA-30; gf: 50-ACTTCACGTGTACATT- CTTC-30. Selenocysteine insertion into proteins supported by Plasmodium SECIS elements pEGFP-C-3 Vector (Clontech) was used for preparing green ﬂuorescent protein (GFP) fusion constructs. A GFP-SelH expression construct containing a mouse SelH SECIS element was described previously (22). A GFP-Sel1 construct contain- ing a P.falciparum Sel1 SECIS element was obtained by cloning Sel1 coding and 30-untranslated region (30-UTR) sequences, which were ampliﬁed with primers 50- GAGAACTCGAGAGATTATGAAATATTTTTAAGAG-30 and 50-CTTTACCTGCAGCAAAAGCATATAAATGAA- TATAACATCATTTC-30 into an XhoI/PstI restriction site immediately downstream of the GFP coding region. A GFP-Sel4 fusion construct was obtained by cloning a PCR fragment, ampliﬁed with primers 50-GTATCCTGCAGCTAA- CATCCCTTTAAAGCTATG-30 and 50-CGATGGATCCGT- CATGAAGATATTTTCATTTATATAACATTGTGTAGC- 30, into a PstI/BamHI site. NIH3T3 cells were transfected with these constructs as well as GFP-SelH (positive control) and GFP alone (negative control) constructs using Lipofectamin (Invitrogen) in the presence of a Plus Reagent (Invitrogen). After transfection, the cells were metabolically labeled with 75Se and harvested after 24 h, and proteins were elec- trophoresed on an SDS–PAGE gel and transferred onto a PVDF membrane. A 75Se radioactivity proﬁle was detected with a PhosphorImager system (Amersham). RESULTS AND DISCUSSION Sec insertion in Apicomplexa To determine if apicomplexan protozoa encode selenopro- teins, we initially analyzed P.falciparum, T.gondii and Cryp- tosporidium genomes for the presence of homologs of known selenoproteins. The full set of human selenoproteins (17), as well as Chlamydomonas MsrA (19,20), Gallus gallus SelU (27) and protein disulﬁde isomerase from Emiliania huxleyi (28) were used as query sequences. BLAST searches revealed no selenoprotein homologs in P.falciparum and Cryptosporid- ium, but detected a distant homolog of SelW, in T.gondii (data not shown). We further searched Plasmodium and Table 1. Computational search for Plasmodium selenoproteins Species name and strain Database P.falciparum 3D7 strain P.yoelii 17XNL strain P.berghei ANKA strain P.chabaudi AS strain P.knowlesi H strain P.vivax (multiple strains) Whole genome Contigs Whole genome shotgun Contigs GSS GSS Size (bp) 23 181 054 26 029 223 29 163 218 17 078 234 20 348 340 6 917 471 Cryptosporidium genomes for the presence of EFSec/eSelB, SBP2 and SPS homologs. These proteins were identiﬁed in P.falciparum, but were absent in Cryptosporidium. Thus, these results suggested that P.falciparum and T.gondii utilize the Sec insertion, whereas Cryptosporidium does not, and veriﬁed the recent prediction that Plasmodium utilizes the Sec insertion system (21). Identification of SECIS elements in Plasmodium The presence of the Sec insertion system and the lack of known selenoproteins in P.falciparum suggested that this organism might have evolved selenoproteins that are unique to this organism. Initially, we used the default pattern of SECISearch to quickly screen for possible SECIS elements and selenoprotein genes in P.falciparum. Two strong candi- dates were identiﬁed (see below), suggesting that indeed Plasmodia evolved a unique selenoproteome. To increase speciﬁcity of the selenoprotein searches in Plasmodia, entire genomes of P.falciparum and P.yoelii and partial genomic sequences of P.vivax, P.chabaudi, P.knowlesi and P.berghei were then analyzed with a loose pattern of SECISearch (Table 1). A total of 38 010 P.falciparum hits satisﬁed primary and secondary structure criteria of the SECIS consensus, and application of thermodynamic criteria reduced the set to 212 SECIS candidates. Other Plasmodium genomes produced variable numbers of candi- dates (Table 1). In the next step, all SECIS candidates were compared with each other using BLASTN and candidates with a homology score below e ¼ 0.001 were extracted. This step reduced the number of P.falciparum SECIS candi- dates to 88. Further manual analysis of these structures and analyses of predicted ORFs ﬁltered out all candidates except 4 (Table 1). The four P.falciparum SECIS candidates generated by SECISearch are shown in Figure 1. One of the matches (SECIS #3) was also predicted by Mourier et al. (21). This recent study also reported a bioinformatics search for SECIS elements, but identiﬁed only a single candidate structure. It was suggested that if additional SECIS elements are present in P.falciparum, they may be non-canonical (21). However, the four SECIS candidates identiﬁed in our work are in full compliance with the current consensus SECIS model. The four structures satisﬁed all criteria of eukaryotic SECIS elements, including the presence of two major helices, unpaired AA in the apical bulge and unpaired A as the nucleotide preceding the Quartet (9). In addition, the Quartet (SECIS core) had a UGAN. . .NGAN sequence, which forms four non-Watson–Crick base pairs (29) and is the most characteristic feature of eukaryotic SECIS elements. Evolutionary criteria 88 41 28 21 51 5 Selenoproteins found #1, #2, #3 and #4 #1, #2, #3 and #4 #1 and #3 #1, #2, #3 and #4 #1, #2, #3 and #4 #1, #2 and #3 Number of SECIS candidates after Primary/secondary Free energy structure analyses analysis 38 010 45 141 49 944 33 877 28 261 6850 212 1255 274 1958 1907 767