RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-4846
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_0824400; Gene model (P.falciparum): PF3D7_0923500; Gene product: cyclin-dependent kinases regulatory subunit, putative (CDKrs)
Phenotype Gametocyte/Gamete;
Last modified: 21 August 2020, 16:48
  *RMgm-4846
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene disruption
Reference (PubMed-PMID number) Reference 1 (PMID number) : 32568069
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone P. berghei ANKA 820cl1m1cl1 (RMgm-164)
Other information parent lineP. berghei ANKA 820cl1m1cl1 (RMgm-164) is a reference ANKA mutant line which expresses GFP under control of a male and RFP under control of a female gametocyte specific promoter. This reference line does not contain a drug-selectable marker (PubMed: PMID: 19438517).
The mutant parasite was generated by
Name PI/ResearcherBalestra AC, Brochet M
Name Group/DepartmentFaculty of Medicine
Name InstituteUniversity of Geneva
CityGeneva
CountrySwitzerland
Name of the mutant parasite
RMgm numberRMgm-4846
Principal nameCDKrs-KO
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteNormal gametocyte production. Only a few microgametocytes formed active exflagellation centres after activation. Severe reduction in exflagellation. The defect was already apparent at 1 min pa, with a >3 fold reduction of cells with more than 2N DNA content and a 3-fold reduction in mitotic spindles.
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of CDKrs and expresses GFP under control of a male and RFP under control of a female gametocyte specific promoter. 

Protein (function)
CDKrs is a protein related to Cks1 and CksHs2, two CDK-associated proteins in Saccharomyces cerevisiae and and human, respectively. In the paper evidence is presented for an interaction between CRK5, SOC2 and CDKrs 

From the Abstract: 'Cell cycle transitions are generally triggered by variation in the activity of cyclindependent kinases (CDKs) bound to cyclins. Malaria-causing parasites have a life cycle with unique cell-division cycles, and a repertoire of divergent CDKs and cyclins of poorly understood function and interdependency. We show that Plasmodium berghei CDK-related kinase 5 (CRK5), is a critical regulator of atypical mitosis in the gametogony and is required for mosquito transmission. It phosphorylates canonical CDK motifs of components in the pre-replicative complex and is essential for DNA replication. During a replicative cycle, CRK5 stably interacts with a single Plasmodium specific cyclin (SOC2), although we obtained no evidence of SOC2 cycling by transcription, translation or degradation. Our results provide evidence that during Plasmodium male gametogony, this divergent cyclin/CDK pair fills the functional space of other eukaryotic cell-cycle kinases controlling DNA replication.'

Phenotype
Normal gametocyte production. Only a few microgametocytes formed active exflagellation centres after activation. Severe reduction in exflagellation. The defect was already apparent at 1 min pa, with a >3 fold reduction of cells with more than 2N DNA content  and a 3-fold reduction in mitotic spindles.

Additional information
Evidence in this study is presented that:
- CRK5 is a key regulator of gametogony and sporogony in the mosquito
- Phosphoproteome kinetics point to direct phosphorylation of the pre-replicative complex by CRK5
- CRK5 is required for both S- and M- phases during P. berghei gametogony
- CRK5 is part of an atypical nuclear cyclin/CDK complex
- CRK5, SOC2 ((PBANKA_1442200)and CDKrs (PBANKA_0824400; (CDK regulatory subunit, CDKrs) have a similar location and complementary functions during gametogony. CDKrs is a protein related to Cks1 and CksHs2, two CDK-associated proteins in Saccharomyces cerevisiae and and human, respectively.
- CRK5, SOC2 and CDKrs have a similar location and complementary functions during gametogony
- SOC2 expression does not follow a temporal cyclin pattern during gametogony and the CSC complex (CRK5/SOC2/CDKrs complex) is stable during the first round of mitosis
- CSC is dynamically phosphorylated during the first round of replication
- Live fluorescence microscopy identified CDKrs-GFP in the nucleus of non-activated microgametocytes and throughout male gametogony, with a spindle-like localisation during mitoses, as found for CRK5
- Ultrastructural analysis by electron microscopy of SOC2-KO and CDKrs-KO activated gametocytes confirms an early arrest during male gametogony as observed in CRK5-KO parasites.

Other mutants


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0824400
Gene Model P. falciparum ortholog PF3D7_0923500
Gene productcyclin-dependent kinases regulatory subunit, putative
Gene product: Alternative nameCDKrs
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 parasitetgdhfr
Promoter of the selectable markerunknown
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modification
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