Mutant/mutation
The mutant lacks expression of ITP-ase (PBANKA_0618300) and NDH.

'We proceeded to generating an itpase(-);ndh(-) double mutant in order to determine whether P. berghei NDH and ITPase could compensate each other’s function in the single knockout lines. To this end we first removed the yFCU-hDHFR marker by negative 5-fluorocytosine selection from the itpase(-) clone (RMgm-4580) and then transfected the ndh deletion plasmid into the selection marker-free itpase(-) mutant; limiting dilution cloning was used to generate an isogenic itpase(-);ndh(-) line.

Protein (function)
Cellular metabolism generates reactive oxygen species. The oxidation and deamination of the deoxynucleoside triphosphate (dNTP) pool results in the formation of non-canonical, toxic dNTPs that can cause mutations, genome instability and cell death. House-cleaning or sanitation enzymes that break down and detoxify non-canonical nucleotides play major protective roles in nucleotide metabolism. In this paper 4 putative sanitation enzymes were identified using bioinformatic approaches ((dUTPase: PBANKA_0921300, ITPase: PBANKA_0618300, Ap4AH: PBANKA_1235300, NDH: PBANKA_1361900).

Based on structural features, sanitation enzymes belong to four superfamilies:

ITPases (inosine triphosphate pyrophosphatase) are defined by the Ham1 (6-n-HydroxylAMinopurine sensitive) domain consisting of a long central beta-sheet forming the floor of the active site;

dUTPases (deoxy-uridine triphosphatase) which present a trimeric fold, each with an eight-stranded jelly-roll beta barrel;

the NuDiX (nucleotide diphosphate linked to some other moiety, X) superfamily is defined by the NuDiX box domain G-x(5)-E-x(5)-[UA]-x-R-E-x(2)-E-E-x-G-U, where U is a hydrophobic residue and x any residue;

The fourth group contains all-α-helical NTPases which are active against dNTPs as well as dNDPs with the generation of dNMPs as an end product.

From the Abstract:
'While all four proteins are expressed constitutively across the intraerythrocytic developmental cycle, neither ITPase nor NDH are required for parasite viability. dutpase and ap4ah null mutants on the other hand are not viable suggesting an essential function for these proteins for the malaria parasite.

Phenotype
Although the two genes are not essential for blood-stage development, mutant parasites were less virulent with prolonged host survival rates.
Normal oocyst and sporozoite production. Reduced motility of sporozoites. Normal sporozoite infectivity to mice. Normal liver stage development (prepatent period in mice).

Additional information
In order to address expression timing and localization we generated transgenic berghei parasite lines expressing each protein fused to green fluorescent protein (GFP) at its C-terminus. ap4ah and ndh were tagged at the endogenous loci, while we expressed dUTPase-GFP and ITPase-GFP episomally under the control of the native promoter using 986 and 865 basepairs of genomic sequence preceding each open reading frame, respectively. Protein expression in transfected parasites was followed by live microscopy and revealed a clear cytoplasmic GFP signal for all four proteins in asexual blood stages (ring, trophozoite and schizont) as well as gametocytes. Western blot analyses showed GFP-tagged dUTPase and ITPase to run higher than their expected molecular weights, while Ap4AH::GFP and NDH::GFP were detected at their predicted positions; NDH with a clear doublet and a smaller 25 kDa band correlating with the size of the cleaved GFP tag.

Other mutants