6700 Nucleic Acids Research, 2006, Vol. 34, No. 22 Figure 2. Immunolocalization of epitope-tagged proteins. (A) Schematic representation of the SFM (Stevor-FLAG-myc) and 2TMFM (Pfmc-2TM-FLAG-myc) recombinant proteins. For the respective genes, two FLAG epitopes (F) were placed between the 2TM domains and three myc epitopes (M) were inserted at the carboxy terminus. The lengths, in amino acids, of the chimeric proteins are indicated at the bottom. The restriction sites specific for the insertion of tags are indicated. (B) Immunofluorescence assays (IFA) showing the localization of SFM and 2TMFM in the transformed lines, pHL-SFM and pHL-2TMFM. IFA studies were performed on air-dried P.falciparum transformed parasites. Infected erythrocytes were stained with FITC-conjugated anti-c-myc mAb and with anti-FLAG polyclonal antibodies followed by goat anti-rabbit Alexa 594-conjugated IgG. As a negative control, air-dried pHL-dhfr-luc P.falciparum parasites gave no signal (data not shown). sequenced for a region that spans the hypervariable loop of Pfmc-2TM genes from the P.falciparum African isolates, Liberia and GB4; the Southeast Asian isolate, Dd2; and the South American isolate, 7G8. The PCR products were ampli- ﬁed using universal PCR primers speciﬁc to the Pfmc-2TM gene family and ﬂanking the loop region (see primer loca- tions in Supplementary Figure 1). It was not attempted to exhaustively clone the entire repertoire of Pfmc-2TM genes from each isolate; rather, representative numbers of genes were obtained. In addition, 11 Pfmc-2TM gene sequences were obtained from the P.falciparum Ghanaian isolate gen- ome sequence project database (The Wellcome Trust Sanger Institute), as well as 11 genes identiﬁed by TBLASTN screening of raw sequences from the genome sequence pro- ject for the P.falciparum HB3 Honduran isolate (The Broad Institute). The translated amino acid sequences within the loop region are highly divergent in all Pfmc-2TM versions (Figure 4), supporting the hypothesis that the divergence is selected for by immune pressure. Two exceptions to this rule were observed: the gene MAL7P1.58 in the 3D7 isolate, which is highly conserved across isolates and is located within the Chr7 internal cluster of var genes in the 3D7 genome sequence; and the gene fragment PFB0960c in the 3D7 isolate, which is predicted to be a pseudogene because the 3D7, Ghanaian and Dd2 versions possess frameshift mutations and the 3D7 and Ghanaian versions additionally lack a start methionine. Isolate-speciﬁc differences were also examined for the hypervariable loop within the Stevor protein family. Using ‘universal’ primers within the highly conserved regions ﬂanking the 2TM domains, 11 stevor gene sequences were cloned from the Dd2 isolate, supplemented with 20 genes and 14 genes, respectively from the HB3 and Ghanaian isolate sequence databases (Figure 5). The overwhelming majority of the sequences, both across paralogs and between isolates, showed extensive diversity that is localized to the loop region. As observed with the Pfmc-2TM genes, the most conserved stevor gene is a pseudogene fragment that is present in the 3D7 (PFA0105w), HB3, and Ghana isolates, and four additional predicted orthologs were identiﬁed. In summary, the hypervariable loop examined for Pfmc- 2TM and Stevor sequences from multiple isolates, as well as within 3D7 paralogs for the Riﬁn family (Supplementary Figure 3), is highly divergent within a precise region encod- ing the erythrocyte surface-exposed loop of the 2TM super- family genes, and this diversity is likely in response to host immune pressure that is encountered due to exposure at the erythrocyte surface. Following, a general theme is described of variable versus invariant genes, and this distinction is correlated with gene location within the chromosomes; status as intact or pseudogenes; and the ability of genes to undergo positive selection for antigenic variants via antibody-mediated immune pressure. Amplification and nucleotide conservation of extended Pfmc-2TM loci containing Pexel/VTS-encoding genes In the course of determining if the high degree of nucleotide conservation within Pfmc-2TM paralogs extends to adjacent non-coding regions, it was noticed that the genes reside