6702 Nucleic Acids Research, 2006, Vol. 34, No. 22 Figure 4. Alignments of the divergent loop and flanking sequences for Pfmc-2TM family members from isolates 3D7, GB4, Liberia, Dd2, 7G8, HB3 and Ghana. 3D7 isolate genes are denoted by their protein names; genes from other isolates are numbered in the order of their discovery. The TM domains are indicated by blue boxes and a green box indicates the hypervariable loop. Translated sequences corresponding to the sense and antisense PCR primers are indicated in lowercase font. The bottom line reflects 85% consensus. Cysteine residues are highlighted in yellow. To rule out the trivial possibility that the ampliﬁcation of the highly conserved extended Pfmc-2TM locus is the result of genome sequence assembly or sequencing errors within the 3D7 genome sequence project database [accessed at www.plasmodb.org; (21)], the presence of 13 Pfmc-2TM loci was conﬁrmed by sequencing of PCR products ampliﬁed using gene-speciﬁc, rather than universal Pfmc-2TM primers (data not shown). In addition, real-time genomic DNA PCR estimations of gene copy number indicated at least 10 copies, by comparing ampliﬁcation rates using universal versus gene- speciﬁc Pfmc-2TM primers and single copy gene-speciﬁc control primers (data not shown). Moreover, the genome sequences for the Ghanaian and HB3 isolates each contain at least 11 Pfmc-2TM genes (Figure 4), as well as extended regions of nucleotide conservation in the Ghanaian and HB3 isolates with untranslated region (UTR) ﬂanking regions