Summary

RMgm-619
Malaria parasiteP. berghei
Genotype
Genetic modification not successful
DisruptedGene model (rodent): PBANKA_0715500; Gene model (P.falciparum): PF3D7_0413500; Gene product: phosphoglucomutase-2 (PGM2)
PhenotypeNo phenotype has been described
Last modified: 24 July 2011, 12:10
  *RMgm-619
Successful modificationThe gene/parasite could not be changed/generated by the genetic modification.
The following genetic modifications were attempted Gene disruption
Number of attempts to introduce the genetic modification 3
Reference (PubMed-PMID number) Reference 1 (PMID number) : 21689687
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone P. berghei ANKA cl15cy1
Other information parent lineA reference wild type clone from the ANKA strain of P. berghei (PubMed: PMID: 17406255)
Attempts to generate the mutant parasite were performed by
Name PI/ResearcherT. Hills; A. Srivastava; AP Waters; J.C. Pizarro
Name Group/DepartmentThe SGC (Structural Genomics Consortium)
Name InstituteUniversity of Toronto
CityToronto
CountryCanada

  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0715500
Gene Model P. falciparum ortholog PF3D7_0413500
Gene productphosphoglucomutase-2
Gene product: Alternative namePGM2
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct usedPlasmid double cross-over
PlasmoGEM (Sanger) construct/vector usedNo
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Plasmid/construct sequence
Restriction sites to linearize plasmid HindIII, EcoRI
Partial or complete disruption of the geneComplete
Additional remarks partial/complete disruption
Selectable marker used to select the mutant parasitetgdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationPbPGM2 appears to be essential to P. berghei asexual blood stage development by the repeated failure to disrupt the gene. On all three occasions, the mutants failed to grow under selection pressure and integration of the selection cassette at the desired locus was not observed suggesting that PGM2 is essential for asexual stages of the parasite. However, it is possible to tag the protein with GFP at its C-terminus via a single cross over mechanism (see RMgm-620).

Two P. falciparum genes code for putative phosphoglycerate mutases (PGMases; PfPGM1 - PF11_0208 and PfPGM2 - PFD0660w), a widespread protein group characterized by the involvement of histidine residues in their catalytic mechanism. Phosphoglycerate mutases (EC 5.4.2.1; PGMase) catalyze the reversible conversion of 2-phosphoglycerate (2-PG) to 3-phosphoglycerate (3-PG).This is an essential component of the glycolysis pathway providing 2-PG to the enzyme enolase and also of the gluconeogenesis pathway where it supplies 3-PG to the phosphoglycerate kinase. Members of the PGM family function as phosphotransferases or phosphohydrolases acting upon a variety of substrates. Aside from metabolic functions, a proportion of PGM family members are involved in cell signaling and/or regulation.

In this study the P. berghei homologue of PGM2 (PBANKA_071550)was studied. The P. berghei homolog of PGM1 is PBANKA_092810
Additional remarks selection procedure
Primer information: Primers used for amplification of the target sequences  Click to view information
Primer information: Primers used for amplification of the target sequences  Click to hide information
Sequence Primer 1CCaagcttGTATATGTATTTTACAAATAGTTATTAATGTAC
Additional information primer 1GU0602 (HindIII); 5'targeting forward
Sequence Primer 2CCccgcggTTTTTATGACACACTGAAAAATATCATATAC
Additional information primer 2GU0603 (SacII); 5'targeting reverse
Sequence Primer 3CCctcgagTCATTTATATGAATTTTTTGCAACTAAAATATG
Additional information primer 3GU0604 (XhoI); 3'targeting forward
Sequence Primer 4CCgaattcAAACGAAACGCCGCATAATACC
Additional information primer 4GU0605 (EcoRI); 3'targeting reverse
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6