RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5095
Malaria parasiteP. yoelii
Genotype
TaggedGene model (rodent): PY17X_1027900; Gene model (P.falciparum): PF3D7_1417200; Gene product: NOT family protein, putative (NOT1)
Name tag: EGFP
Phenotype Asexual bloodstage; Gametocyte/Gamete; Oocyst; Sporozoite; Liver stage;
Last modified: 2 November 2021, 12:40
  *RMgm-5095
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene tagging
Reference (PubMed-PMID number) Reference 1 (PMID number) : 34673764
MR4 number
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
The mutant parasite was generated 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
CityPennsylvania
CountryUSA
Name of the mutant parasite
RMgm numberRMgm-5095
Principal namePyNOT1::GFP
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageBoth NOT1 paralogues were expressed and localized at the same times in the life cycle, and the expression pattern for PyNOT1 and PyNOT1-G is cytosolic and at least partially punctate. Of note, the NOT1 paralogues were expressed in both asexual and sexual blood-stage parasites with a nonuniform, cytosolic distribution with some puncta visible.
Gametocyte/GameteBoth NOT1 paralogues were expressed and localized at the same times in the life cycle, and the expression pattern for PyNOT1 and PyNOT1-G is cytosolic and at least partially punctate. Of note, the NOT1 paralogues were expressed in both asexual and sexual blood-stage parasites with a nonuniform, cytosolic distribution with some puncta visible.
Fertilization and ookineteNot tested
OocystBoth paralogous proteins were expressed in mosquito stage parasites, with localization shifting from a cytosolic diffuse pattern in oocysts, to a more nucleus-proximal pattern in oocyst sporozoites, and, finally, to a more apical pattern in salivary gland sporozoites.
SporozoiteBoth paralogous proteins were expressed in mosquito stage parasites, with localization shifting from a cytosolic diffuse pattern in oocysts, to a more nucleus-proximal pattern in oocyst sporozoites, and, finally, to a more apical pattern in salivary gland sporozoites.
Liver stageNo expression of either protein was observed in mid- or late-liver stage parasites.
Additional remarks phenotype

Mutant/mutation
The mutant expresses a C-terminal GFP-tagged version of NOT1.

Protein (function)
'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.'

Phenotype
Plasmodium’s NOT1 paralogues localize to cytosolic puncta: 'To determine if the 2 paralogues of NOT1 (NOT1 and NOT1-G) are used at different points in the Plasmodium life cycle, we used conventional reverse genetics approaches to append a C-terminal GFP tag to each NOT1 paralogue in P. yoelii  and assessed their expression and localization. Both NOT1 paralogues were expressed and localized at the same times in the life cycle, and the expression pattern for PyNOT1 and PyNOT1-G is cytosolic and at least partially punctate. This is consistent with the localization of other members of the CAF1/CCR4/NOT complex in Plasmodium, model eukaryotes, and human cells. Of note, the NOT1 paralogues were expressed in both asexual and sexual blood stage parasites with a nonuniform, cytosolic distribution with some puncta visible. Both paralogous proteins were expressed in mosquito stage parasites, with localization shifting from a cytosolic diffuse pattern in oocysts, to a more nucleus-proximal pattern in oocyst sporozoites, and, finally, to a more apical pattern in salivary gland sporozoites. No expression of either protein was observed in mid- or late-liver stage parasites. Together, these expression and staining patterns match what is commonly seen for members of the CAF1/CCR4/NOT complex in other eukaryotes, as well as what we observed previously in P. yoelii with 2 other members of this complex: CAF1 and CCR4-1 [21]. Coupled with proteomic data showing their association with CCR4-1, this strongly indicates that these NOT1 paralogues are resident members of the CAF1/CCR4/NOT complex in P. yoelii.

Unsuccessful attempts to disrupt the gene encoding NOT1 indicate an essential function during asexual blood-stage growth (see RMgm-5092).


Additional information

Other mutants


  Tagged: Mutant parasite with a tagged 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
Name of the tagEGFP
Details of taggingC-terminal
Additional remarks: tagging
Commercial source of tag-antibodies
Type of plasmid/construct(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
Selectable marker used to select the mutant parasitehdhfr
Promoter of the selectable markerunknown
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationPlasmids used for appending a C-terminal GFPmut2 tag to a protein were based upon pSL0442, which inserts a the coding sequence for GFPmut2 with the P. berghei DHFR 3′ UTR, a HsDHFR expression cassette, and the plasmid backbone into that genomic locus.
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