Mutant/mutation
The mutant lacks expression of Plasmepsin 4 (PM4; plasmepsin IV) and expresses the GFP-Luciferase fusion protein under control of the (schizont specific) ama-1 promoter.

Protein (function)
Plasmepsin4 is an aspartic protease of the digestive vacuole and is involved in hemoglobin degradation. P. falciparum has four genes encoding digestive vacuole plasmepsins. Most other Plasmodium species, including P. berghei, have only a single gene encoding a digestive vacuole plasmepsin, plasmepsin4.

Phenotype
Asexual blood stages show a (slightly) reduced growth rate and reduced hemozoin formation.

Additional information

Figure: Generation of the P. berghei mutants ∆pm4 (RMgm-808), ∆bp1 (RMgm-816) and ∆bp2 (RMgm-809).
(primer sequences can either be found in Lin et al. (2015). J. Exp. Med. or obtained from the Leiden malaria Research Group).
(A) Schematic representation of gene-deletion constructs targeting the open reading frame (ORF) of genes expressing plasmepsin 4 (pm4), berghepain 2 (bp2) or berghepain 1 (bp1) by double cross-over homologous recombination, and wild-type (wt) gene loci before and after disruption. The constructs contain a drug-selectable marker cassette (SM; black box) and gene target regions (hatched boxes). Primer positions (arrows) for diagnostic PCRs are shown (see Table S4 for primer sequences and expected product sizes). (B) Diagnostic PCR (left) and Southern analysis of pulsed field gel-separated chromosomes (center) confirm correct disruption of pm4 in mutant ∆pm4-b. Northern analysis of blood-stage mRNA (right) confirms the absence of pm4 transcripts in the ∆pm4-b mutant. The following primers were used for diagnostic PCRs: 5' integration (5’ in): L5516/L4096; 3' integration: (3’ in) L1662/L5517; SM (hdfhr::yfcu): L4698/L4699; pm4 ORF: L5518/L5519. Separated chromosomes were hybridized using an hdhfr probe that recognizes the DNA-construct integrated into the pm4 locus on chromosome 10. Northern blot was hybridized using a PCR probe recognizing the pm4 ORF (primers L5518/L5519) and with an oligonucleotide probe L644R recognizing the large subunit rRNA (as loading control). (C) Diagnostic PCR (left) confirms the correct deletion of the bp2 gene in mutant ∆bp2-a. RT-PCR analysis of blood stage mRNA (right) shows the absence of bp2 transcription in ∆bp2-a blood-stages. The following primer pairs were used for diagnostic PCR analyses: 5’ in, RS835/RS32; 3’ in, RS110/RS836; SM (tgdhfr/ts), RS404/RS405; bp2 ORF, RS514/RS515. For RT-PCR the following primers were used: tub (tubulin), RS782/RS783 and bp2, RS515/RS516. (D) Diagnostic PCR (left) and Southern analysis of pulsed field gel-separated chromosomes (center) confirm the correct disruption of the bp2 gene in mutant ∆bp2-b. Northern blot analysis of blood stage mRNA (right) confirms the absence of bp2 transcripts in ∆bp2-b. The following primers were used for diagnostic PCRs: 5’ in, L5024/L3211; 3’ in, L5025/L1662; SM (hdhfr::yfcu), L4698/L4699; bp2 ORF, L5026/L5027. Separated chromosomes were hybridized using an hdhfr probe that recognizes the DNA-construct integrated into the bp2 locus on chromosome 9. Northern blot was hybridized using a PCR probe recognizing the bp2 ORF (primers L5026/L5027) and with an oligonucleotide probe L644R recognizing the large subunit rRNA (as loading control). (E) Southern analysis of pulsed field gel-separated chromosomes (left) confirms the correct disruption of bp1 in mutant ∆bp1. Northern analysis of blood-stage mRNA (right) confirms the absence of bp1 transcripts in mutant ∆bp1. Separated chromosomes were hybridized using an 3’UTR pbdhfr probe that recognizes the DNA-construct integrated into the bp1 locus on chromosome 13, the endogenous dhfr/ts on chromosome 7 and the GFP-luciferase reporter cassette in the 230p locus on chromosome 3. Northern blot was hybridized using a PCR probe recognizing the bp1 ORF (primers L7422/L7423) and with an oligonucleotide probe L644R recognizing the large subunit rRNA (as loading control). See Table S4 for primers used for generation of probes.

Other mutants
See the link for other 'plasmepsin 4' mutants