FORS-D Analysis in P. falciparum can Differentiate Classes of Genes Under Selection

FORS-D is a measure of the contribution of base order to the stem loop potential of a nucleic acid sequence and can also give information on evolutionary pressures on sequences to move away from secondary structure. Negative FORS-D values in a gene are associated with exons and nucleotide substitutions such as SNPs. An analysis of P. falciparum genes under selection pressure shows a correlation between negative FORS-D values and SNP density for genes that drug targets but not for drug transporters or antigenic variation genes. Analysis of the dhfr gene shows that a majority of rare mutations that associate with drug resistance also fall into regions with negative FORS-D values. These data suggest that FORS-D values might be predictors for drug target genes and drug resistance mutations in these genes

In vivo analysis of translation initiation sites in Plasmodium falciparum

Regulation of gene expression in the malaria parasite Plasmodium falciparum is tightly controlled and little is known about the many steps involved. One step i.e. translation initiation is also poorly understood and in P. falciparum, choice of the translation initiation site (TIS) is a critical decision largely due to the high frequency of AUGs in the relatively long 50 untranslated regions of parasite mRNAs. The sequences surrounding the TIS have a major role to play in translation initiation and this report evaluates these sequences by mutational analysis of the heat shock protein 86 gene, transient transfection and reporter assays in the parasite. We find that purines at the -3 and +4 positions are essential for efficient translation in P. falciparum, similar to other eukaryotes. Interestingly, a U at the -1 position results in 2.5-fold higher reporter activity compared to wild type. Certain classes of protein biosynthetic genes show higher frequencies of U at the -1 position, suggesting that these genes may exhibit higher levels of translation. This work defines the optimal sequences for TIS choice and has implications for the design of efficient expression vectors in an important human pathogen