1170 Nucleic Acids Research, 2006, Vol. 34, No. 4 Variability in gene expression One of the few differentially expressed genes that we observed was MAL13P1.344. Its expression proﬁle was virtually ident- ical in 3D7 and Dd2, but was nearly opposite in the HB3 strain (Figure 2B). This gene is a member of the superfamily of ATP- binding cassette transporters from the OABP subfamily and is an example of a gene which is expressed completely out of phase between strains. The time of maximal transcript abund- ance is shifted by 22 h, with a Pearson correlation value of 0.92. The DNA microarray results for MAL13P1.344 have been conﬁrmed by northern blot analysis using RNA from ring-stage and schizont-stage parasites (Figure 2C). Such anti-correlation is very rare in the datasets. Large changes in gene expression were also detected for PFC0110w—the cytoadherence linked asexual protein (CLAG 3.1), PF11_0512—ring-infected erythrocyte surface antigen 2 (RESA-2) and a pair of erythrocyte membrane protein 1 (PfEMP1)—PF08_0103 and PFB0010w. These genes all encode surface proteins likely to be involved in antigen pre- sentation. In fact, we have examined the Gene Ontology (GO) parameters (19) for genes with the greatest difference in Pearson correlation between HB3 and 3D7 using the LACK tool (20), and we ﬁnd a signiﬁcant enrichment (P-value < 0.000005) for extracellular and plasma membrane-associated genes involved in cell adhesion, defense and immunity, antigenic variation and host-pathogen interac- tion all of which are properties of the P.falciparum cell surface (data not shown). We also considered the cases where a gene expression pro- ﬁle from a particular oligo is detectable in at least one, but not all strains. Leaving basic technical microarray issues aside, this situation could only arise if there are true transcriptional differences or if deletions and signiﬁcant polymorphisms are present between the strains. For every oligo, we summed the number of missing data points across the time courses and selected those whose difference in missing values was greater than 50% in a given strain pair, also requiring that the oligo passed our quality control criteria in at least one of the two strains. This analysis identiﬁed 556 such cases between HB3 and Dd2, 347 between Dd2 and 3D7 and 278 between HB3 and 3D7 (Supplementary Table S10). Over half of these cases occur in hypothetical genes. The remaining cases are almost solely comprised of surface antigens including vars, var pseudogenes, riﬁns and stevors. Lack of observed expression in HB3 and Dd2 when com- pared to 3D7 can be explained in several instances by the presence of highly polymorphic loci within the genome. In some cases, such as merozoite surface protein 2 (MSP2, PFB0300c) and S-antigen (PF10_0343), the representative 70mer oligonucleotide lies within regions that are known to contain high levels of sequence diversity among strains (21,22). Other genes, such as knob associated histidine rich protein (KAHRP, PFB0100c) and erythrocyte membrane pro- tein 3 (PfEMP3, PFB0095c), may be completely absent, as has been previously shown for Dd2 (23). However, while HB3 appears to contain PfEMP3 and KAHRP, these genes are likely to be highly polymorphic since previous CGH work failed to detect signal using a 70mer oligonucleotide (10). Although the differential performance of some probes may be due to DNA polymorphisms as discussed above, 70mer oligos may tolerate slight sequence variability without com- promising overall signal (24). Therefore, it is relevant to con- sider to what extent the inter-strain differences in expression may be explained by a high degree of polymorphism. CGH analysis of chromosomal variability between strains suggests that at most, 20% of these changes are due to deletions or polymorphisms in the genome [(10), E.D.W. and J.L.D. per- sonal communication]. Over 1000 genes in the 3D7 genome are represented by more than one oligo on our P.falciparum DNA microarray. These probes are very useful for intragene probe validation. Furthermore, variability between these probes, when observed between strains, may provide evidence for deletions and/or insertions, or alternative splicing. Within a given strain, par- ticularly for the reference strain 3D7, these differences may also simply illustrate the difﬁculty in annotating intron–exon boundaries. We determined the Pearson correlation for all pairs of oligos in genes represented by multiple oligos. For most genes, there is excellent intra-strain correlation (above 0.9 for over 70% of the data) in the expression proﬁles meas- ured by any pair of oligos (Supplementary Table S11). We also calculated the maximal difference in Pearson correlation between all pairs of oligos for each ORF and remarkably, the most dissimilar pair for any given strain was 80% likely to be dissimilar in another strain. For oligo pairs that do not behave commonly across strains, these differences suggest either sequence variations or potential alternative splicing events. Transcript abundance While the DNA microarrays used here do not quantify RNA copy number within a cell directly, we can qualitatively estim- ate the relative abundance of each transcript throughout the IDC by examining the intensity signals for individual microar- ray probes. We calculated the average sum of the median intensity (AVG SOM) for each individual oligo throughout the IDC for all three strains and note a remarkable correlation (Supplementary Table S12). The 150 oligos with the highest AVG SOM from 3D7 are in the top 5% of AVG SOMs (372 oligos) of both HB3 and Dd2, which strongly suggests that the most highly abundant transcripts are common to all three strains. These results were independent of hybridization effects due to oligo probe properties as demonstrated by the lack of signiﬁcant correlation between the AVG SOM values and the free energy, complexity score, G-C content or self- binding energy of the microarray probes (data not shown). Unlike the majority of abundant transcripts that are in com- mon across the three strains, a small subset of these high intensity genes is unique to one strain. For 3D7 these include: KAHRP, S-antigen, PfEMP3, a sexual stage speciﬁc antigen PFD0310w, MSP 2 and CLAG3.2. As described above, many of these genes are either deleted or highly polymorphic in the Dd2 and HB3 strains. There are also highly abundant tran- scripts unique to the HB3 strain including PFI1750c, PF11_0038 and PF14_0733 which encode predicted hypothet- ical proteins. In addition, a large 20 kb region on chromo- some 4 (134 000–153 500 bp) near PFD0100c encodes several over-represented transcripts in the HB3 strain, including a putative reticulocyte binding protein, PFD0110w. The pres- ence of an established reticulocyte binding protein PFD0110w