family of 20 serine/threonine protein kinases of the R45 family that are predicted to be trafﬁcked to the erythrocyte cytoplasm (8,15). In addition, as much as 25% of the total Pexel/VTS catalog is represented by unique proteins. We have determined such a catalog [see also the recent reviews, (9,16)] by combining the catalogs compiled by Marti et al. (12) and Hiller et al. (10); and additionally performing exhaustive BLAST screening of Plasmodium sp. genome sequence databases and conducting extensive genome walk- ing on the P.falciparum genome sequence. The resulting cata- log contains over 300 genes that, although daunting in its breadth, can be greatly distilled into families of proteins that are grouped according to paralogous relationships, protein domain architectures and transmembrane topologies. Here we describe the sequence diversity and genomic organi- zations of genes and extended loci for the Pexel/VTS- containing 2TM families. The riﬁn, stevor and Pfmc-2TM genes exhibit paralog-speciﬁc and isolate-speciﬁc divergences that are conﬁned to a hypervariable loop region between the 2TM domains. We present evidence that the 2TM protein families are exported to the erythrocyte membrane, thus sup- porting the hypothesis that the hypervariable loop is exposed on the erythrocyte surface and that host immune pressure drives antigenic diversity within the loop. The genes encod- ing 2TM superfamily members are frequently found in sub-telomeric genome neighborhoods that contain Pexel/ VTS-encoding genes that do not exhibit sequence diversity across isolates, such as the PHIST domain-containing protein superfamily. Thus it is proposed that the sub-telomeric regions facilitate molecular mechanisms that promote hyper- variability within genes, and within discrete regions of these genes, which encode proteins that are exposed to the host immune system; whereas invariant erythrocyte cytoplasmic proteins are shielded from this process. MATERIALS AND METHODS Parasite culture and isolate-specific genomic DNA Asexual and gametocyte NF54 isolate P.falciparum parasites were cultivated in vitro as described (17). To synchronize cultures, schizonts were puriﬁed either by magnetic isola- tion using a MACS depletion column (Miltenyi Biotec) in conjunction with a magnetic separator (18), or by a Percoll- sorbitol gradient; and placed back into culture at 0.5% hema- tocrit in RPMI 1640 containing 10% human serum. Genomic DNA from P.falciparum isolates GB4, Liberia, 7G8 and Dd2 were the generous gift of Dr Karen Hayton (NIAID, NIH). A cloned progeny parasite line, 3D7B, from a 3D7 · 3D7 self-cross was kindly provided by Dr Thomas Wellems and was cultured for the puriﬁcation of genomic DNA. Genomic DNA from the parents and progeny of an HB3 · 3D7 cross were the generous gift of Dr Akhil Vaidya. Plasmid construction and parasite transformation Plasmodium falciparum transfection plasmids were con- structed to express stevor (PFF1550w) and Pfmc2TM (PFA0680c) genes which encode tagged proteins having two adjacent FLAG epitopes within the hypervariable loop region plus three adjacent c-myc epitopes at the carboxy termini (Figure 2A). The plasmids were constructed by Nucleic Acids Research, 2006, Vol. 34, No. 22 6697 amplifying the N-terminal portion containing the ﬁrst TM, and the C-terminal portion encoding the second TM, from P.falciparum NF54 isolate genomic DNA using the following primers: Stevor50XhoI (50-CCCTGCAGCTCGAGATGTTA- TTGTTTAACTTTTTAAT); Stevor30FLAGHindIII (50-GG- AAGCTTCTTATCGTCGTCATCCTTGTAATCTGTAGTT- AATGAACCACCATG); Stevor50FLAGHindIII (50-CCAAG- CTTGATTACAAGGATGACGACGATAAGGCTCTTAA- AGCAACCGAAGCG); Stevor30 BamHI (50-GGGGATCCC- TTACATAAATGTTTCTTGC); 2TM50XhoI (50-CCCTGC- AGCTCGAGATGTTTCATTATATTTATAAAAT); 2TM30- FLAGHindIII (50-GGAAGCTTCTTATCGTCGTCATCCTT- GTAATCAATATCAACCGAAAATAAACATAC); 2TM50 FLAGHindIII (50-CCAAGCTTGATTACAAGGATGACGA- CGATAAGACATCATCTTCTGCACTTGGC); 2TM30BglII (50-GGGGATCCTTTTGTTATTTGCTTTTTGTT). The PCR products corresponding to the N-terminal portion and the C-terminal portion of each gene were ligated to reconstitute the full ORF sequences and to introduce two FLAG epitope tags in the middle of the hypervariable loop region (Figure 2A). These full-length products were restriction digested with XhoI and BglII and inserted into the expression vector, pHL-dhfr-3myc, which is a derivative of the pHLH1 vector (19). pHL-dhfr-3myc contains three consecutive c-myc epitopes within an expression cassette that is driven by the P.falciparum 50hrp3 promoter and the 30hrp2 terminator (Figure 2A), to ensure a high expression level of stevor and Pfmc-2TM genes; and an hDHFR expression cassette for selection of transformed parasites with pyrimethamine. Expression vectors were transfected into the NF54 P.falciparum isolate by ‘pre-loading’ erythrocytes as described (20). Stable transformant parasites were selected by culturing in media containing 40 ng/ml pyrimethamine. Transfections were validated by performing plasmid rescue experiments; speciﬁcally, chemically competent Escherichia coli cells were transformed with 100 ng of puriﬁed genomic DNA from selected transformant parasites. Expression of Stevor-FLAG-myc (SFM) in the transformed line, pHL-SFM, and Pfmc2TM-FLAG-myc (2TMFM) in the transformed line, pHL-2TMFM, were conﬁrmed by reverse transcription and real-time PCR using speciﬁc primers for PFF1550w (sense: 50-AAACAAAAGGAAGAGATAAG; antisense: 50-ATCGC- TGCTACATATACAGG) and for PFA0680c (see Supple- mentary Table 1), following the protocol described below. As a control for expression and localization experiments we used the pHL-dhfr-luciferase line (a gift from K. Deitsch), which expresses the luciferase protein and is a derivative of pHLH1 vector (19). Immunofluorescence and immunoelectron microscopy For indirect immunoﬂuorescence microscopy assays, infected erythrocytes were thinly smeared within wells of Teﬂon coated slides and ﬁxed for 20 min in methanol chilled at 80 C. Slides were blocked in 2% BSA in phosphate- buffered saline (PBS) and then incubated with FITC- conjugated anti-c-myc mAb (1:200; Sigma), or with anti-FLAG polyclonal antibodies (1:200; Sigma) followed by goat anti-rabbit Alexa 594-conjugated IgG (1:2000; Mole- cular Probes). For electron microscopy, schizonts and late trophozoites from transformant lines were puriﬁed on