6698 Nucleic Acids Research, 2006, Vol. 34, No. 22 Percoll-sorbitol gradients and the cell pellets were resus- pended in 2% paraformaldehyde, 0.1% glutaraldehyde (EM grade) in PBS. The samples were dehydrated through a cold ethanol series, inﬁltrated ﬁrst with ethanol-LRWhite (London Resin Company, UK), overnight at 4 C, then in pure LRW at 4 C for up to 24 h. The blocks were hardened at 50 C over- night. Sections were cut using a Diatome diamond knife on a Leica Ultracut S ultramicrotome and contrasted with aqueous uranyl acetate for 15 min. Thin sections were incubated with an anti-c-myc mAb (1:25) followed by 6 nM gold-labeled goat anti-mouse IgG (1:100, Aurion). Samples were exam- ined in a JEOL 100 CX-II EM operating at 80 Kv and imaged on Kodak 4489 EM ﬁlm. Real-time PCR assays of gene transcription Gene-speciﬁc primers (Supplementary Table 1) were empiri- cally designed for the 13 Pfmc-2TM genes and two control genes, namely, arginyl-tRNA synthetase (PFL0900c) and glutamyl-tRNA synthetase (PF13_0170). The control genes were identiﬁed in the P.falciparum 3D7 genome sequence database [(21); http://www.plasmodb.org] as fulﬁlling tran- script expression criteria for ‘housekeeping’ control genes which display consistent transcript expression patterns throughout the parasite life cycle. Real-time PCR was per- formed using an ABI Prism 7900HT sequence detector (Applied Biosystems). Reactions were prepared in 25 ml volumes using SYBR Green PCR master mix (Applied Biosystems) and 1 mM primers. Speciﬁcity of ampliﬁcation was conﬁrmed by melting-curve analysis for each PCR prod- uct. Duplicate PCR were analyzed for each sample. The number of Pfmc-2TM genes were estimated using the 2ddct method (User Bulletin 2, ABI), as well as an adjacent paralo- gous 2TM gene family, Hyp5 (16), that is present in the genomes of the P.falciparum 3D7 and Dd2 isolates. Real- time PCR experiments were performed on several dilutions of genomic DNA from different isolates using universal primers for Pfmc-2TM and Hyp5 genes, with gene-speciﬁc primers for PFL0900c and PF13_0170 used as single copy gene references. Sequence analyses The non-redundant (NR) database of protein sequences (National Center for Biotechnology Information, NIH, Bethesda, MD) and the P.falciparum genome nucleotide sequence database [(21); www.plasmodb.org] were searched using the BLASTP or TBLASTN programs (22). Iterative database searches were conducted using the PSI-BLAST program with either a single sequence or an alignment used as the query, with the PSSM inclusion expectation E-value threshold of 0.01 (unless speciﬁed otherwise); the searches were iterated until convergence. Multiple alignments were constructed using the T_Coffee and MUSCLE programs, fol- lowed by manual correction based on the PSI-BLAST results (23,24). Potential transmembrane regions were predicted using TopPred (25) and TMHMM-2 (26). The Ghanaian P.falciparum isolate sequence information was used by permission and was accessed via the BLAST server at The Wellcome Trust Sanger Institute (http://www.sanger.ac.uk/ cgi-bin/blast/submitblast/p_falciparum). The P.falciparum HB3 isolate raw sequence reads (The Broad Institute) were obtained from the NCBI Trace Archive (http://www.ncbi. nlm.nih.gov/Traces/trace.cgi) and formatted for database searches using the TBLASTN program. Nucleotide sequence alignments of extended loci containing Pfmc-2TM genes were performed using ClustalW (27). RESULTS AND DISCUSSION Classification of Pexel/VTS-containing proteins To determine a representative catalog of Pexel/VTS- containing proteins we have combined the catalogs compiled by Marti et al. (12) and Hiller et al. (10); performed exhaus- tive BLAST screening of Plasmodium sp. genome sequence databases using all identiﬁed Pexel/VTS-containing proteins and position-speciﬁc score matrix (PSSM) proﬁles as queries; and conducted extensive genome walking to identify ORFs within sub-telomeric regions of the P.falciparum genome sequence. A similar analysis was recently described (9,16) and appears to correlate well with our resulting catalog, which contains over 300 Pexel/VTS-containing proteins that are predicted to be trafﬁcked to the erythrocyte. These pro- teins can be grouped into several families of proteins acc- ording to their paralogous relationships, protein domain architectures and transmembrane topologies (Figure 1). These groups include two superfamilies, the 2TM proteins and the PHIST domain-containing proteins, which together represent >75% of all the Pexel/VTS-containing proteins. The remaining proteins include the knob-associated proteins KAHRP and MESA; a family of 20 serine/threonine protein kinases of the R45 family as well as a family of 15 uncharac- terized predicted 1TM proteins. The 1TM proteins are grouped together based upon their similarity in architectures; namely, overall size, C-terminal TM domain, the presence of signal peptide sequences plus Pexel/VTS motifs at their N-termini, two exon gene structure and, in some instances, paralogous relationships. The 2TM superfamily is encoded by seven or more paralogous gene families, that have a shared architecture of 300 amino acid lengths and a 2TM topology. Both the Riﬁn proteins, encoded by over 130 genes, and the related Stevor proteins, with >35 genes, are placed within this topological superfamily as well as the recently described Pfmc-2TM family (28). Protein domain architecture of the 2TM superfamily members Alignment of 13 members of the Pfmc-2TM family reveals a high degree of amino acid conservation throughout the pro- tein, including the TM and signal peptide domains [described in Ref. (28); see also Supplementary Figure 1]. Notably, the overall conservation within the Pfmc-2TM family highlights a marked divergence within the 17 amino acid long hyper- variable region between the two TM domains (boxed regions in Supplementary Figure 1). Three members of the family (PF11_0014, PFC1080c and MAL7P1.58) differ within the C-terminal-most 30 amino acids due to a shared single nucle- otide deletion and resulting frameshift. It is not known if this mutation results in non-functional or non-expressed proteins, or if it indicates alternate protein functions. Align- ments of full-length amino acid sequences from 23 Stevor