Summary

RMgm-483
Malaria parasiteP. berghei
Genotype
Genetic modification not successful
DisruptedGene model (rodent): PBANKA_1126900; Gene model (P.falciparum): PF3D7_0628200; Gene product: protein kinase PK4 | eukaryotic translation initiation factor 2-alpha kinase
PhenotypeNo phenotype has been described
Last modified: 21 December 2011, 16:26
  *RMgm-483
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 Unknown
Reference (PubMed-PMID number) Reference 1 (PMID number) : 20584882
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone Not applicable
Other information parent line
Attempts to generate the mutant parasite were performed by
Name PI/ResearcherM. Zhang; V. Nussenzweig
Name Group/DepartmentMichael Heidelberger Division, Department of Pathology
Name InstituteNew York University School of Medicine
CityNew York
CountryUSA

  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1126900
Gene Model P. falciparum ortholog PF3D7_0628200
Gene productprotein kinase PK4 | eukaryotic translation initiation factor 2-alpha kinase
Gene product: Alternative name
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct usedUnknown
PlasmoGEM (Sanger) construct/vector usedNo
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Plasmid/construct sequence
Restriction sites to linearize plasmid
Partial or complete disruption of the geneUnknown
Additional remarks partial/complete disruption
Selectable marker used to select the mutant parasiteUnknown
Promoter of the selectable markerUnknown
Selection (positive) procedureUnknown
Selection (negative) procedureUnknown
Additional remarks genetic modificationIn the paper it is reported that multiple attempts to disrupt protein kinase PK4 in P. berghei were unsuccessful, indicating an essential role of PK4 during asexual blood stage development.

No information/details is provided on the number of attempts to disrupt the gene, the construct used to disrupt the gene and the primers to amplify the target regions of pk4.

One control mechanism of protein synthesis in eukaryotic cells involves the phosphorylation of the α-subunit of eukaryotic translation-initiation factor-2 (eIF-2α). During initiation of protein synthesis, a ternary complex of Met-tRNA, GTP, and the eukaryotic initiation factor 2 (eIF2) is delivered to the translation initiation machinery. During this translation process, eIF2-GTP is hydrolyzed to eIF2-GDP and released from the machinery. A guanine nucleotide exchange factor (eIF2B) facilitates the exchange of eIF2-GDP to eIF2-GTP, which is a requisite for a new round of translation. The phosphorylation of eIF2α blocks the recycling of eIF2-GTP and downregulates protein synthesis. The phosphorylation of eIF2α is associated with the appearance of stress granules in the cytoplasm of stressed cells. Three eIF2α kinases, IK1 (PF14_0423; eukaryotic initiation factor 2alpha kinase 1), IK2 (PfA0380w; PBANKA_020580; serine/threonine protein kinase, putative), and PK4 (PFF1370w; PBANKA_112690; protein kinase PK4), have been identified in Plasmodium.

See RMgm-482 for a P. berghei mutant lacking expression of IK2 (PfA0380w; PBANKA_020580).

Evidence has been presented that P. falciparum IK1 (PF14_0423; eukaryotic initiation factor 2alpha kinase 1) is not required for P. falciparum asexual and sexual blood stage development and sporozoite formation (Fenell C et al. 2009. Malar J. 8, 99)

See also RMgm-566 for unsuccessful attempts to disrupt PBANKA_112690

Disruption of the P. falciparum ortholog has been attempted (Solyakov et al., 2011, Nat Commun, 2:565).
The gene is likely essential for asexual proliferation as integration of the KO vector was not achieved. Accesibility of the locus to recombination was verified by C-terminal tagging.

Disruption of the P. falciparum ortholog has been attempted (Solyakov et al., 2011, Nat Commun, 2:565). After transfection with a KO vector a strong PCR signal diagnostic for gene disruption was observed in transfected populations indicating that this gene is not essential for asexual proliferation. Cloning will however be required to validate this interpretation for this
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 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
Sequence Primer 3
Additional information primer 3
Sequence Primer 4
Additional information primer 4
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6