RMgmDB - Rodent Malaria genetically modified Parasites


Malaria parasiteP. yoelii
Genetic modification not successful
DisruptedGene model (rodent): PY17X_1027900; Gene model (P.falciparum): PF3D7_1417200; Gene product: NOT family protein, putative (NOT1)
PhenotypeNo phenotype has been described
Last modified: 1 November 2021, 16:49
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 ≥ 5
Reference (PubMed-PMID number) Reference 1 (PMID number) : 34673764
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. yoelii
Parent strain/lineP. y. yoelii 17XNL
Name parent line/clone Not applicable
Other information parent line
Attempts to generate the mutant parasite were performed by
Name PI/ResearcherHart KJ, Lindner SE
Name Group/DepartmentDepartment of Biochemistry and Molecular Biology, The Huck Center for Malaria Research
Name InstitutePennsylvania State University

  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PY17X_1027900
Gene Model P. falciparum ortholog PF3D7_1417200
Gene productNOT family protein, putative
Gene product: Alternative nameNOT1
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct used(Linear) plasmid double cross-over
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 geneComplete
Additional remarks partial/complete disruption
Selectable marker used to select the mutant parasitehdhfr
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe unsuccessful attempts to disrupt this gene indicate an essential function during asexual blood-stage growth.

Additional information:
'In agreement with the critical/essential role of NOT1 to eukaryotic RNA metabolism, our attempts to delete py17x_1027900 were largely unsuccessful. However, in 1 of 2 technical duplicates of 6 independent transfection attempts, we were able to obtain a 100% transgenic population with an extremely slow growth phenotype. It was only possible to produce this parasite line because the population was entirely transgenic, as the presence of even a small number of wild-type parasites with de novo resistance to pyrimethamine would likely have rapidly outgrown these transgenic parasites in the mouse. These data align with results from both PlasmoGEM (P. berghei) and piggyBac (P. falciparum) genetic screens that noted the importance of this gene to asexual blood-stage growth. Due to the severe asexual blood-stage defect, which closely aligned with observations of NOT1-related phenotypes in other eukaryotes, we propose that this “NOT Family Protein” is truly the NOT1 protein of Plasmodium parasites. Finally, it is notable that in contrast to other eukaryotes, PyNOT1 can be deleted and indicates that it is not strictly essential.'

'We identified a protein annotated only as “NOT Family Protein” (PY17X_1027900) that was found to be associated with CCR4-1. Bioinformatic analyses using PlasmoDB and BLASTp identified that the protein encoded by PY17X_1027900 mostly closely matched eukaryotic NOT1 proteins but lacked a bioinformatically predictable TTP-binding domain that is present in the gene currently annotated as NOT1 (PY17X_0945600). We concluded that these 2 not1 genes were paralogues due to the high degree of sequence conservation in specific domains at both the DNA and protein levels (DNA: CAF1-binding domain 72% identity, NOT4-binding domain 66% identity; Protein: CAF1-binding domain 41% identical/63% positive, CAF40-binding domain 33%/57%, NOT4-binding domain 48%/62%, NOT1 domain 50%/73%). Moreover, these genes are highly conserved, syntenic, and only present across Plasmodium species and 2 other species of the Aconoidasida class (Genera: Theileria, Babesia) but not in other apicomplexans, model eukaryotes, or humans.'
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