Figure 1.
Parasite Culturing and Data Characteristics of the P. falciparum IDC Transcriptome Analysis
(A) Giemsa stains of the major morphological stages throughout the IDC are shown with the percent representation of ring-, trophozoite-, or schizont-stage parasites at every timepoint. The 2-h invasion window during the initiation of the bioreactor culture is indicated (gray area).
(B–D) Example expression profiles for three genes, encoding EBA175, DHFR-TS, and ASL, are shown with a loess fit of the data (red line).
(E) MAL6P1.147, the largest predicted ORF in the Plasmodium genome, is represented by 14 unique DNA oligonucleotide elements. The location of each of the oligonucleotide elements within the predicted ORF and the corresponding individual expression profiles are indicated (oligo 1–14). A red/green colorimetric representation of the gene expression ratios for each oligonucleotide is shown below the graph. The pairwise Pearson correlation for these expression profiles is 0.98 ± 0.02.
(F) The percentage of the power in the maximum frequency of the FFT power spectrum was used as an indicator of periodicity. A histogram of these values reveals a strong bias toward single-frequency expression profiles, indicating that the majority of P. falciparum genes are regulated in a simple periodic manner. This bias is eliminated when the percent power was recalculated using random permutations of the same dataset (inset). For reference, the locations of EBA175 (peak B), DHFR-TS (peak C), and ASL (peak D) are shown.
Figure 2.
Overview of the P. falciparum IDC Transcriptome
(A) A phaseogram of the IDC transcriptome was created by ordering the transcriptional profiles for 2,712 genes by phase of expression along the y-axis. The characteristic stages of intraerythrocytic parasite morphology are shown on the left, aligned with the corresponding phase of peak gene expression.
(B–M) The temporal ordering of biochemical processes and functions is shown on the right. Each graph corresponds to the average expression profile for the genes in each set and the mean peak-to-trough amplitude is shown in parentheses.
Figure 3.
Coregulation of Gene Expression along the Chromosomes of P. falciparum Is Rare, While Plastid Gene Expression Is Highly Coordinated
Expression profiles for oligonucleotides are shown as a function of location for Chromosome 2 ([A], Oligo Map). With the exception of the SERA locus (B), coregulated clusters of adjacent ORFs are seldom observed, indicating that expression phase is largely independent of chromosomal position. (C) In contrast to the nuclear chromosomes, the polycistronic expression of the circular plastid genome is reflected in the tight coregulation of gene expression. This is an expanded view of the plastid-encoded genes from Figure 2J. Genomic differences between strain 3D7, from which the complete genome was sequenced, and strain HB3 were measured by CGH. The relative hybridization between the gDNA derived from these two strains is shown as a percent reduction of the signal intensity for 3D7 ([A], CGH Data). Differences between the two strains are predominately located in the subtelomeric regions that contain the highly polymorphic var, rifin, and stevor gene families. Intrachromosomal variations, as observed for the msp2 gene, were rare.
Figure 4.
Temporal Distribution of the Apicoplast-Targeted Proteins and P. falciparum Proteases, Potential Antimalarial Drug Candidates
(A) The expression profiles of all putative plastid-targeted genes represented on our microarray are shown. The yellow box encompasses a highly synchronized group of genes, which are in-phase with plastid genome expression. The average expression profile for this in-phase group of genes is shown and includes most of the known apicoplast-targeted genes as well as many hypothetical genes. For reference, the average expression profile for the plastid genome is shown (dashed gray line).
(B) Proteases represent an attractive target for chemotherapeutic development. The broad range of temporal expression for various classes of proteases and their putative functions are displayed.
Abbreviations: HAP, histo-aspartyl protease (PM III); Clp, caseineolytic protease; sub1, 2, subtilisin-like protease 1 and 2.
Figure 5.
Phaseogram of Putative Vaccine Targets
The similarity of all expression profiles to seven known vaccine candidates (boxed) was calculated. The top 5% of similar profiles correspond to 262 ORFs, 28 of which have been previously associated with plasmodial antigenicity and the process of merozoite invasion.