1172 Nucleic Acids Research, 2006, Vol. 34, No. 4 of a few genes such as the chloroquine resistance transporter gene pfcrt (3). Currently, there are few macroscopic pheno- types for differentiating between strains of P.falciparum and yet the identiﬁcation of additional diagnostic markers could greatly aid the clinical prediction of malaria severity. This study expands our understanding of P.falciparum transcrip- tional regulation during the IDC by comparing the full tran- scriptomes of two commonly used laboratory strains 3D7 and Dd2 with that of HB3. The major differences between these three strains lie in their original geographical locations and divergent drug sensitivities. Our goal was to detect changes in gene expression patterns between strains and thereby identify pathways and mechanisms responsible for strain-speciﬁc dif- ferences. These changes can then be further exploited to identify regulatory regions contributing to strain adaptation. This study demonstrates that the transcriptomes of the 3D7, Dd2 and HB3 strains of P.falciparum are strikingly similar in the overall gene expression patterns of the majority of genes during the IDC. This conservation implies that many key developmental processes are well preserved among strains. The streamlined nature of the transcriptome indicates that the mechanisms for control of gene expression may be quite simple for all strains. Changes in gene expression between the strains were detec- ted primarily at loci encoding surface antigens involved in immune evasion. These changes could be due to strain- speciﬁc deletions, polymorphisms or gene silencing. Sir2- dependent silencing has recently been demonstrated for var gene regulation in telomeric regions (34,35), and our data suggest that perhaps other regions of the genome may also be similarly silenced in a strain-speciﬁc manner. In addition, we found several differences in transcript abundance between oligos within genes represented by multiple oligos on our microarray, supporting the possibility for changes in gene structure or alternative splicing between strains. We anticipated changes in expression for genes known to be involved in drug resistance. For example, chloroquine res- istance is in part conferred by point mutations in the pfcrt transporter gene which prevents chloroquine from accumulat- ing in the food vacuole (3). Similarly, polymorphisms in the P-glycoprotein (Pgh1) gene, pfmdr1 or overexpression of this gene have both been shown to facilitate meﬂoquine resistance (36). Although up or down regulation of these genes could contribute to drug resistance, we detected no such changes. As previously observed, cells under favorable growth conditions in vitro may not be under any signiﬁcant environmental pres- sure to alter the expression of these genes (37). In cultures containing chloroquine we have detected speciﬁc expression changes for the pfcrt transporter in 3D7 cells (chloroquine sensitive), although this response is clearly not sufﬁcient for survival (M.L., manuscript in preparation). It remains unclear as to the range of environmental signals to which Plasmodium is capable of responding. Since the parasite is sequestered within four membranes inside the red blood cell during most of the infection, it experiences a fairly homeostatic environment throughout development. In fact, a study examining Plasmodium transcripts from infected patients’ blood found few genes that had not been previously detected by in vitro studies (38). The rigidity of the transcrip- tome is strikingly different from what has been seen for dif- ferent S.cerevisiae strains (16). The Plasmodium parasite is highly adapted to its unique environment, outside of which it simply does not survive. The streamlined nature of the tran- scriptome gene expression cascade strongly supports this, with gene expression occurring in a ‘just-in-time’ fashion following a seemingly unchecked commitment to complete the IDC. The strong conservation of the transcriptional program across various strains will be extremely useful in bioinformatic approaches that aim to strengthen the statistical signiﬁcance of highly correlated gene expression proﬁles. The gene expression proﬁle of any given gene during the IDC can now be treated with high conﬁdence due to the reproducibility of the data across strains. In addition, our high resolution 3D7 transcrip- tome data are qualitatively in excellent agreement with the Le Roch et al. (31) 3D7 transcriptome study which used 6 IDC timepoint measurements. By harnessing similarities in gene expression levels, timing and uniqueness within a strain, our three strain transcriptome data will begin to allow for classiﬁcation of the more than half of the P.falciparum genome with no homology to other species. Coupled with emerging sequence data from other Plasmodia species, this will certainly assist in homology searching, gene family recon- struction and regulatory motif ﬁnding. All of our strain data are publicly available at both http://malaria.ucsf.edu/comparison/ and http://PlasmoDB.org (39), the online malaria community resource center. Ultimately, these results will serve to identify desperately needed novel candidates for the development of Plasmodium-speciﬁc drug therapies and vaccines. SUPPLEMENTARY DATA Supplementary Data are available at NAR Online. ACKNOWLEDGEMENTS The authors thank the members of the DeRisi lab for critical reading of this manuscript. This work, J.L.D., M.L. and Z.B. were supported by a grant from the National Institute of Allergy and Infectious Disease (AI53862) and the Burroughs- Wellcome Fund. E.D.W. was supported by a fellowship from the Pharmaceutical Research and Manufacturers of America Foundation. A.T.A. was supported by a grant from the National Science Foundation. Funding to pay the Open Access publication charges for this article was provided by National Institute of Allergy and Infectious Disease (NIAID) (AI53862). Conflict of interest statement. None declared. REFERENCES 1. Sachs,J. and Malaney,P. (2002) The economic and social burden of malaria. Nature, 415, 680–685. 2. Arav-Boger,R. and Shapiro,T.A. (2005) Molecular mechanisms of resistance in antimalarial chemotherapy: the unmet challenge. Annu. Rev. Pharmacol. Toxicol., 45, 565–585. 3. Bray,P.G., Martin,R.E., Tilley,L., Ward,S.A., Kirk,K. and Fidock,D.A. (2005) Defining the role of PfCRT in Plasmodium falciparum chloroquine resistance. Mol. 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