Serial analysis of gene expression (SAGE) in Plasmodium falciparum: application of the technique to A-T rich genomes

The advent of high-throughput methods for the analysis of global gene expre ssion, together with the Malaria Genome project open up new opportunities f or furthering our understanding of the fundamental biology and virulence of the malaria parasite. Serial analysis of gene expression (SAGE) is particu larly well suited for malarial systems, as the genomes of Plasmodium specie s remain to be fully annotated. By simultaneously and quantitatively analyz ing mRNA transcript profiles from a given cell population, SAGE allows for the discovery of new genes. In this study, one reports the successful appli cation of SAGE in Plasmodium falciparum, 3D7 strain parasites. from which a preliminary library of 6880 tags corresponding to 4146 different genes was generated. It was demonstrated that P. falciparum is amenable to this tech nique, despite the remarkably high A-T content of its genome. SAGE tags as short as 10 nucleotides were sufficient to uniquely identify parasite trans cripts from both nuclear and mitochondrial genomes. Moreover. the skewed A- T content of parasite sequence did not preclude the use of enzymes that are crucial for generating representative SAGE libraries. Finally, a few modif ications to DNA extraction and cloning steps of the SAGE protocol proved us eful for circumventing specific problems presented by A-T rich genomes. (C) 2001 Elsevier Science B.V. All rights reserved.