PFF1540w is absent from the HB3 isolate genome sequence database, perhaps due to the incompleteness of the sequence coverage, but the predicted PFF1540w paralog within the Chr2 locus (containing PFB0895c; Figure 6A), itself a pseu- dogene, identiﬁes a virtually identical predicted ortholog and pseudogene in the HB3 isolate. Thus, additional support is provided for the theme that sub-telomeric pseudogenes are excluded from mechanisms generating divergence, or selec- tion for antigenic diversity, despite their inclusion in highly variant gene families. As a result, pseudogenes are found conserved across isolate boundaries. Generation of diversity within the hypervariable loop of 2TM proteins The stark discrepancy of the sequence diversity within the Pfmc-2TM loop, versus the highly conserved ﬂanking regions, suggests that the parasite possesses a mechanism for creating hypervariability within a discrete region, perhaps in concert with immune pressure that selects for antigenic variants. Extrapolating this logic to the riﬁn and stevor genes, the parasite may also have a mechanism for generating diversity within the larger loops contained within these genes. The sub-telomeric location of variable gene families, such as var, riﬁn and stevor, likely confers an enhanced capacity for gene diversiﬁcation and ampliﬁcation through mechanisms of recombination, including that between non-homologous chromosomal ends (35). The clustering of telomeres at the nuclear periphery is thought to play a role in facilitating recombination (35,36). The relative roles of single crossover recombination events versus gene conversion mechanisms involving replacements of short fragments of sequence infor- mation remain unclear. It is possible that the DNA segments encoding the loop regions are prone to strand breaks, and the repair mechanism requires extensive base-pair alignments mediated by the nucleotide conservation within the extended loci and a gene conversion event involving donor sequences. In addition, single nucleotide mutations might accumulate within the loop regions during this process, if introduced by a mutagenic DNA polymerase that functions in translesion DNA repair, such as Plasmodium orthologs of Rev1p and Rev3p (37). The P.falciparum var family has been shown to undergo recombination-mediated variation during meiosis (36,38), which contributes to great sequence diversity between paralogs and across isolates for the erythrocyte sur- face membrane protein 1 (PfEMP1). Analysis of progeny of a genetic cross between HB3 · 3D7 and HB3 · Dd2 demon- strated the generation of non-parental var genotypes (36); and these genotypes appeared more frequently than expected from the calculated meiotic crossover rate of P.falciparum (39). In addition, an HB3 · HB3 self-cross led to the appear- ance of a new var form as detected by an additional restric- tion fragment. The high conservation of the extended Pfmc-2TM and stevor loci in P.falciparum could contribute to gene conver- sion events by allowing for the alignment of chromosome ends as a basis for ectopic recombination events between paralogous genes. To seek examples of recombination or gene conversion, we studied a cloned progeny of a 3D7 · 3D7 self- cross and three cloned progeny from a 3D7 · HB3 cross. Sequencing of the hypervariable loop for all 13 Pfmc-2TM Nucleic Acids Research, 2006, Vol. 34, No. 22 6705 and 39 stevor genes in the progeny of the 3D7 · 3D7 self- cross did not reveal any new sequences (data not shown). Although a lack of new Pfmc-2TM or stevor forms implies that there were no ectopic recombination events between paralogous genes, allelic recombination events cannot be ruled out. PCR ampliﬁcation of the 3D7 Pfmc-2TM genes of all three progeny cloned from the 3D7 · HB3 cross and sequencing of genomic DNA from two of these progeny also revealed a complete conservation of the inherited mem- bers of the Pfmc-2TM family (the inherited genes in the progeny were traced through the mapping of chromosomal recombination events). Thus, no new Pfmc-2TM genes were seen following a non-self cross. Moreover, a partial survey of stevor genes isolated using ‘universal’ PCR primers from a single 3D7 · HB3 progeny also failed to show novel sequences. These results indicate that diversity within the hypervariable loop of the Pfmc-2tm and stevor gene families is not mediated through meiotic recombination events to the extent seen in vars; although it is possible that the much larger size of var genes increases the chances of intragenic recombination events. Because cultured 3D7 parasites do not develop novel Pfmc-2TM or stevor genes, it is possible that mitotic recombination events are not responsible for the sequence diversity that was observed in the proposed loop. However, proper determination of mitotic recombination events would require cloning of individual parasites, since single mutation events would be difﬁcult to detect from a bulk culture unless those parasites represented a signiﬁcant portion of the population. It is possible that the large diversity seen in speciﬁc regions of the 2TM gene families may be acquired through a slower mechanism such as genetic drift. It is also plausible that in the absence of immune pressure, recombination events within these gene families do not occur more frequently than the natural recombination rate determined for P.falciparum. In addition, a possible pathway for the accumulation of single mutations within the loop regions should be considered. Nevertheless, the wide varia- tion seen within the loop of the 2TM proteins implies a parasite-mediated mechanism for generation of antigenic diversity, and perhaps a mechanism of antigenic variation. CONCLUSIONS The shared architecture of a 2TM topology and hypervariable loop unites the Pfmc-2TM, Riﬁn and Stevor proteins and indicates similar functions and likely erythrocyte membrane localization as their ﬁnal trafﬁcking and functional destina- tion. The ampliﬁcation of the 2TM protein-encoding genes into large families, and the profound divergence within each family and across isolates within a region conﬁned to a proposed membrane surface loop, combine to suggest that antibody-mediated immune pressure has expanded their repertoire and shaped their topology. The Pfmc-2TM genes are contained within a locus that is ampliﬁed, in varying lengths of conservation, 13 times in the P.falciparum genome. The striking nucleotide conservation across the length of this locus, when contrasted with the high nucleotide diversity con- ﬁned to the hypervariable loop within the Pfmc-2TM genes, suggests that the parasite possesses a mechanism of creating divergence within a discrete, bounded region. The investment