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Biology Articles » Protistology » Data-Mining Approaches Reveal Hidden Families of Proteases in the Genome of Malaria Parasite » Figures

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- Data-Mining Approaches Reveal Hidden Families of Proteases in the Genome of Malaria Parasite

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Figure 1. Multiple alignment of the catalytic domains of the putative P. falciparum calpain (MAL13P1.310) and the representative human calpains using T-coffee program followed by manual correction. The catalytic domain region is predicted to be from amino acid residue 985 to 1453 by pfam HMM algorithm. The three conserved amino acids, C(1035), H(1371), N(1391), that are part of the active sites are highlighted with arrowheads. Graphic presentation of the alignment and the consensus sequence were obtained by the program BOXSHADE 3.21. Conserved residues are shaded with black and gray. The accession numbers of calpain protein sequences used for alignment refer to Figure 2.

figure 1

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Figure 2. The phylogenetic tree of the calpains, inferred by the neighbor-joining method based on the amino acid sequences with Poisson corrected distance. The option of complete deletion of gaps was used for tree construction. 1000 bootstrap replicates were used to infer the reliability of branching points. Bootstrap values of >50% are presented. The scale bar indicates the number of amino acid substitutions per site. The parasite sequences are underlined. The putative P. falciparum calpain and P. yoelii yoelii ortholog are highlighted in red and blue, respectively. The accession number for each sequence is included in the parentheses after the species name.

figure 2

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Figure 3. The phylogenetic tree of the caspases, inferred by the neighbor-joining method based on the amino acid sequences with Poisson corrected distance. The option of complete deletion of gaps was used for tree construction. 1000 bootstrap replicates were used to infer the reliability of branching points. Bootstrap values of >50% are presented. The scale bar indicates the number of amino acid substitutions per site. The protozoan sequences are underlined.

figure 3

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Figure 4. Multiple alignment of the catalytic regions of the putative P. falciparum metacaspase (PF13_0289) and the representative proteolytically active caspases using Clustal X1.8 followed by manual correction. The catalytic dyad H (404) and C (460), which are part of the active sites, is highlighted with arrowheads. Graphic presentation of the alignment and the consensus sequence were obtained by the program BOXSHADE 3.21. Conserved residues are shaded with black and gray. The accession numbers of caspase protein sequences used for alignment refer to Figure 3.

figure 4

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Figure 5. The phylogenetic tree of the Signal peptidases I (SP1), inferred by the neighbor-joining method based on the amino acid sequences with Poisson corrected distance. The option of complete deletion of gaps was used for tree construction. 1000 bootstrap replicates were used to infer the reliability of branching points. Bootstrap values of >50% are presented. The scale bar indicates the number of amino acid substitutions per site. The putative P. falciparum calpain and P. yoelii yoelii ortholog are highlighted in red and blue, respectively. The SP1 homolog in Arabidopsis is termed Chloroplast thylakoidal processing peptidase. Imp is the abbreviation for mitochondrial inner membrane peptidase.

figure 5

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Figure 6. Multiple alignment of the catalytic regions of the putative P. falciparum SP1 (PF13_0118) and the representative proteolytically active signal peptidase I using T-coffee program followed by manual correction. The catalytic dyad Ser (S175) and Lys (K274), which are part of the active sites, is highlighted with arrowheads. Graphic presentation of the alignment and the consensus sequence were obtained by the program BOXSHADE 3.21. Conserved residues are shaded with black and gray. The accession numbers of SP1 protein sequences used for alignment refer to Figure 5.

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Figure 7. Four P. falciparum 3D7 culture samples were collected at 12-h intervals, and pooled to achieve a total asynchrony.

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Figure 8. (A) Expression of ten putative proteases without corresponding oligomers in the microarray set. RT-PCR was conducted to examine the transcriptional expression of the ten putative proteases using specific primers based on the prediction. Lane a in each sample represents the negative control in which RT-PCR was conducted without reverse transcriptase. Lane 1b: PF14_0281; Lane 2b: PFL1635w; Lane 3b: MAL6P1.153; Lane 4b: PFL1925w; Lane 5b: PFC0950C; Lane 6b: MAL8P1.16; Lane 7b: MAL6P1.88; Lane 8b: MAL8P1.128; Lane 9b: PFA0400c; Lane 10b: PFI1545c.M indicates 1 kb DNA ladder. (B) Expression of putative calpain, caspase, and SP1 genes using two pairs of primers. RT-PCR was conducted to confirm the transcriptional expression of putative calpain, metacaspase, and SP-1 genes, using 2 pairs of specific primers for each gene. Lanes "a" represent negative controls in which RT-PCR was conducted without reverse transcriptase.M indicates 1 kb DNA ladder. Lanes 1b and 2b: MAL13P1.310; Lanes 3b and 4b: PF13_0289; Lanes 5b and 6b: PF13_0118. See Suppl. Table 1 for the predicted size of RT-PCR products.

figure 8

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