Genome Sequences, Homology Search, and Comparative Sequence Analysis
A total of 5865 nonredundant query sequences of characterized and predicted proteases from 1066 organisms were obtained from the Merops database (http://www.merops.ac.uk, release 5.8 of March 19, 2002), which has a catalog and a structure-based classification of proteases. The BLASTP searches with default setting were targeted to the predicted and annotated Plasmodium falciparum genome that was published in the PlasmoDB (http://plasmodb.org/; Kissinger et al. 2002). A cut-off criteria of E-value was adopted to define protease homologs. Partial sequences ( Conserved domains/motifs in P. falciparum sequences were identified by searching InterPro release 5.1, which integrates Pfam 7.3, PRINTS 33.0, PROSITE 17.5, ProDom 2001.3, SMART 3.1, TIGRFAMs 1.2, and the current SWISS-PROT + TrEMBL data.
Multiple alignments were obtained by the program T-coffee (Notredame et al. 2000), followed by manual editing according to the structure information. Graphic presentation of the alignment and consensus sequences were deduced by the program BOXSHADE 3.21 (http://www.ch.embnet.org/software/BOX_form.html). Phylogenetic trees were inferred by the neighbor-joining method (Saitou and Nei 1987) using MEGA2.0 (http://www.megasoftware.net/). Unweighted Maximum Parsimony (as implemented in PAUP 4.0) and Maximum Likelihood (as implemented in PHYLIP) were used to examine whether the inferred phylogeny is sensitive to any tree-making method. The bootstrap resampling with 1000 pseudoreplicates was carried out to assess support for each individual branch. Bootstrap values of polytomies.
Microarray Expression Analysis Using Asynchronous Erythrocytic P. falciparum Culture
An en masse gene expression profile was obtained using microarray chips arrayed with 6239 Malaria Genome Array Oligomers (Operon Technologies), designed by Dr. Joe DeRisi of the University of California at San Francisco (http://derisilab.ucsf.edu/). These 6239 70-mers mapped to 4407 predicted open reading frames which covered >90% of the available P. falciparum genome sequences. In order to obtain all genes transcribed throughout the erythrocyte stage of the parasite, we extracted and pooled mRNAs from P. falciparum 3D7 culture samples (Trager and Jensen 1976) collected at four 12-h intervals to achieve an asynchrony (shown in Fig. 3). Probes were labeled with fluorescent dyes using mRNAs purified from the asynchronous culture as a template. Messager RNAs were purified using oligo T cellulose, and reverse transcription was conducted to incorporate aminoallyl dUTP into the cDNAs. The Cy3 and Cy5 NHS esters were then coupled to amine groups of the cDNA, and dye-labeled probes were hybridized with the microarray slides under standard conditions (3xSSC, 50% formamide, 0.1% SDS, 10 mg/mL salmon sperm DNA, 68°C). The slide was scanned with a GenePix 4000B (Axon Instrument) at default PMT settings, 100% power. The array data were analyzed initially with GenePixPro software (Axon Instrument), then with global normalization. The expression level is indicated by the mean signal intensity of all corresponding oligomers in triplicates on the microarray slides (MRA-452) obtained from Malaria Research and Reference Resource Center (http://www.malaria.mr4.org/). Two sets of negative controls were included in the DeRisi design: (1) 20 oligomers from yeast intergenic region with the mean intensity 529, (2) 33 P. falciparum genes cloned into a plasmid, including 16 ribosomal proteins, 17 tRNA genes, LSU, Clp, and tufA. Their mean intensity was 598.
Reverse Transcription Polymerase Chain Reaction
RT-PCR was performed using the same mRNA described above as template. Reverse transcription was conducted using SuperScript II (Invitrogen). The PCR cycle: 95°C 1 min; (95°C 1 min, 54°C 30 sec, 52°C 30 sec, 65°C 1 min) x 35, 65°C 10 min, hold at 4°C. The primer sequences used to amplify 10 target genes without corresponding oligomers in the array set, and putative calpain, metacaspase, and signal peptidase I are included in the Supplemental Table 1.