SummaryRMgm-4591
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Successful modification | The parasite was generated by the genetic modification |
The mutant contains the following genetic modification(s) | Gene mutation |
Reference (PubMed-PMID number) |
Reference 1 (PMID number) : 30703164 |
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 |
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, Center for Malaria Research |
Name Institute | Pennsylvania State University, University Park, State College |
City | Pennsylvania |
Country | USA |
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Name of the mutant parasite | |
RMgm number | RMgm-4591 |
Principal name | PyCAF1ΔC (AA 1–335) |
Alternative name | |
Standardized name | |
Is the mutant parasite cloned after genetic modification | Yes |
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Phenotype | |
Asexual blood stage | Reduced growth of asexual blood stages |
Gametocyte/Gamete | Reduced male gametocyte production; enhanced production of immature/female gametocytes. |
Fertilization and ookinete | Not tested |
Oocyst | Reduced number of oocysts |
Sporozoite | Not tested |
Liver stage | Not tested |
Additional remarks phenotype | Mutant/mutation Reduced male gametocyte production; enhanced production of immature/female gametocytes. Reduced number of oocysts. The phenotype of gametocytes/oocysts of this mutant is similar to a mutant lacking expression of CCR4-1 (RMgm-4588). 'Here, it was found that PyCCR4-1, a member of the CAF1/CCR4/NOT RNA metabolic complex, acts upon transcripts both directly and indirectly in gametocytes, and results in a reduction of male gametocytemia. In gametocytes lacking PyCCR4-1, as well as those expressing a catalytically dead variant, the initial coordinated wave of male gametocyte activation is lost, and these parasites are ~4-fold less able to productively infect mosquitoes. We find that the deletion of the C-terminal portion of CAF1 in both Plasmodium yoelii and Plasmodium falciparum phenocopies the deletion of pyccr4-1. We also find that the CAF1/CCR4/NOT complex is directly binding some of these transcripts and is likely acting both directly upon mRNAs and indirectly to modulate transcript abundance' Evidence is presented that: 'In eukaryotes, CCR4 and CAF1 function while in association with the other members of the CAF1/CCR4/NOT complex and is found in nuclear and cytosolic granular structures. Because PyCCR4-1 lacks an obvious LRR domain by which it can associate with the rest of the complex, we used immunofluorescence and live fluorescence assays to further validate these interactions. First, using transgenic PyCCR4-1::GFP parasites, we observed that PyCCR4-1 localized to cytoplasmic puncta in asexual blood stage parasites, and is similarly localized in both male and female gametocytes. Moreover, this expression profile extends to oocysts, oocyst sporozoites, and salivary gland sporozoites, where PyCCR4-1 was seen both in cytosolic puncta and located diffusely throughout the parasite. However, PyCCR4-1 was not detected above background in liver stage parasites. Thus, the near constitutive expression and localization of PyCCR4-1 in cytoplasmic foci in Plasmodium resembles that of its orthologues in model eukaryotes. Next, using either full length PyCAF1::GFP or PyCAF1ΔC::GFP transgenic parasites, we observed a similar expression and localization pattern to that of PyCCR4-1::GFP. Moreover, colocalization of PyCAF1ΔC::GFP and PyNOT1 signals were observed. Together, these data further indicate that the truncated PyCAF1ΔC::GFP variant can remain associated with the rest of its complex and yet phenocopies these PyCCR4-1-associated effects.' |
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Details of the target gene | |||||||||||||||||||||||||||
Gene Model of Rodent Parasite | PY17X_1428300 | ||||||||||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_0811300 | ||||||||||||||||||||||||||
Gene product | CCR4-associated factor 1 | ||||||||||||||||||||||||||
Gene product: Alternative name | CAF1 | ||||||||||||||||||||||||||
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Details of the genetic modification | |||||||||||||||||||||||||||
Short description of the mutation | C-terminal truncation of caf1 | ||||||||||||||||||||||||||
Inducable system used | No | ||||||||||||||||||||||||||
Short description of the conditional mutagenesis | Not available | ||||||||||||||||||||||||||
Additional remarks inducable system | |||||||||||||||||||||||||||
Type of plasmid/construct | (Linear) plasmid double cross-over | ||||||||||||||||||||||||||
PlasmoGEM (Sanger) construct/vector used | No | ||||||||||||||||||||||||||
Modified PlasmoGEM construct/vector used | No | ||||||||||||||||||||||||||
Plasmid/construct map | |||||||||||||||||||||||||||
Plasmid/construct sequence | |||||||||||||||||||||||||||
Restriction sites to linearize plasmid | |||||||||||||||||||||||||||
Selectable marker used to select the mutant parasite | hdhfr | ||||||||||||||||||||||||||
Promoter of the selectable marker | unknown | ||||||||||||||||||||||||||
Selection (positive) procedure | pyrimethamine | ||||||||||||||||||||||||||
Selection (negative) procedure | No | ||||||||||||||||||||||||||
Additional remarks genetic modification | |||||||||||||||||||||||||||
Additional remarks selection procedure | |||||||||||||||||||||||||||
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