Figure 4. Identification of the defective gene in mutant 13-20C2 by genetic complementation.

(A) Results of mutant complementation with cosmid libraries and individual cosmids are diagrammed. Mutant 13-20C2 was complemented with cosmid genomic library DNA. Sequence tags (black arrows) were recovered from temperature and drug resistant populations and mapped to a single locus on chromosome III (665,366bp to 711,113bp). To confirm the complementing locus, the original mutant 13-20C2 was complemented with DNA from two cosmid clones, PSBMM94 (no growth at 40°C) that spans the right side of the locus and cosmid PSBLQ51 (100% growth at 40°C compared to growth at 34°C), which spans the center of the locus and includes genes TGGT1_002010 (Gene #2, TgG2a), TGGT1_002020 (Gene #3, TgARP4a), and TGGT1_002030 (Gene #4, hypothetical). To narrow the gene list of cosmid PSBLQ51 further, mutant 13-20C2 was complemented with cosmid DNA bearing either a deletion in Gene #2 (TgG2a) or Gene #3 (TgARP4a). Mutant parasites transfected with individual cosmids were first selected in bleomycin and the drug resistant populations tested for temperature resistant growth by plaque assay. Ability to form plaques is expressed as a percentage of the plaques formed at 40°C versus parallel controls grown at 34°C. To test the ability of genomic fragments to complement, DNA fragments spanning the TgARP4a locus including promoter and 3′-UTR sequences were amplified from mutant 13-20C2 or parental genomic DNA. (B) Sequencing of TgARP4a cDNA from mutant 13-20C2 and parental RHΔhxgprt parasites identified a single transition mutation at 1,862bp (T/C) in the coding sequence of the tsTgAPR4a resulting in a change of isoleucine to threonine at residue 621 of the predicted TgARP4a coding sequence. Mutated codon is shown boxed. (C) Alignment of the helical region that contains the I621T mutation of tsTgARP4a with selected orthologs (Plasmodium falciparum PF14_0218; Saccharomyces cereviseae SacArp4p; Homo sapiens HmALP6B). Hydrophobic residue corresponding to isoleucine 621 (starred column) in TgARP4a is conserved in the analyzed orthologs. Note that the 621-residue in the helix is preserved by conservative substitutions isoleucine and valine. Identical residues as compared to TgARP4a are indicated in the ortholog sequences as a period. (D, E) Predicted folding of the polypeptides I383-L432 (Sc_Arp4) and F614-L665 (TgARP4a). Crystal structure of yeast protein (pdb:3QB0) was used to model a part of the Subdomain 3 of the actin fold of TgARP4a contain the temperature sensitive mutation. The alpha helix is shown in blue and the corresponding residue I621 in TgARP4a to the V385 in yeast Arp4p are shown in red. Bulky side chains of the residues of three α-helixes and one β-sheet that form a hydrophobic pocket are shown and corresponding residues are labeled on the outside. Note the position of I621 in TgARP4a may interact with a hydrophobic pocket that would like be altered by the mutation to threonine in the ts-TgARP4a protein. (F) Ribbon drawing of the helix I617-D626 showing the precise location of the I621T change.