Successful modification | The parasite was generated by the genetic modification |
The mutant contains the following genetic modification(s) |
Gene tagging
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Reference (PubMed-PMID number) |
Reference 1 (PMID number) : 34673764 |
MR4 number |
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Parent parasite used to introduce the genetic modification |
Rodent Malaria Parasite | P. yoelii |
Parent strain/line | P. y. yoelii 17XNL |
Name parent line/clone |
Not applicable
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Other information parent line | |
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The mutant parasite was generated by |
Name PI/Researcher | Hart KJ, Lindner SE |
Name Group/Department | Department of Biochemistry and Molecular Biology, The Huck Center for Malaria Research |
Name Institute | Pennsylvania State University |
City | Pennsylvania |
Country | USA |
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Name of the mutant parasite |
RMgm number | RMgm-5095 |
Principal name | PyNOT1::GFP |
Alternative name | |
Standardized name | |
Is the mutant parasite cloned after genetic modification | Yes |
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Phenotype |
Asexual blood stage | 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. 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/Gamete | 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. 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 ookinete | Not tested |
Oocyst | 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. |
Sporozoite | 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. |
Liver stage | No 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 |