RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-4060
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_1303400; Gene model (P.falciparum): PF3D7_1439500; Gene product: oocyst rupture protein 2, putative (ORP2; NFYC)
Transgene
Transgene not Plasmodium: GFP
Promoter: Gene model: PBANKA_0711900; Gene model (P.falciparum): PF3D7_0818900; Gene product: heat shock protein 70 (HSP70)
3'UTR: Gene model: PBANKA_1340000; Gene product: dihydrofolate synthase/folylpolyglutamate synthase, putative (PbDHFS-FPGS)
Replacement locus: Gene model: PBANKA_1303400; Gene product: oocyst rupture protein 2, putative (ORP2; NFYC)
Phenotype Oocyst; Sporozoite; Liver stage;
Last modified: 24 December 2016, 17:28
  *RMgm-4060
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene disruption, Introduction of a transgene
Reference (PubMed-PMID number) Reference 1 (PMID number) : 27982038
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone 8417HP
Other information parent line
The mutant parasite was generated by
Name PI/ResearcherCurrà C, Siden-Kiamos I
Name Group/DepartmentFoundation for Research and Technology-Hellas
Name InstituteInstitute of Molecular Biology and Biotechnology
CityHeraklion
CountryGreece
Name of the mutant parasite
RMgm numberRMgm-4060
Principal nameorp2(-)
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteNot different from wild type
Fertilization and ookineteNot tested
OocystSlightly higher oocyst production compared to wild type. Sporozoites are formed inside oocysts. Oocysts do not rupture and oocysts do not release sporozoites.
SporozoiteSlightly higher oocyst production compared to wild type. Sporozoites are formed inside oocysts. Oocysts do not rupture and oocysts do not release sporozoites.
Liver stageNo infection of mice after mosquito bite. No infection of mice after infection with sporozoites that are artificially released from oocysts (motility of these sporozoites was not tested/reported)
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of ORP2 end expresses GFP under control of the constitutive hsp70 promoter.

Protein (function)
In the paper it is shown that each of two HFD (histone-fold domain)-containing proteins of P. berghei gene (IDs PBANKA_0902500 and PBANKA_1303400), which are named oocyst rupture protein 1 (ORP1) and ORP2, have essential and similar functions in the rupture of the oocyst capsule. Evidence is presented that protein interaction via the HFD is critical for function. ORP1 is located in the oocyst capsule, whereas ORP2 is re-localized from the oocyst cytoplasm to the capsule at the time when mature sporozoites have formed.
The histone-fold domain (HFD) is found in histones and in proteins with a role in transcriptional regulation such as the TATA-box-binding protein-associated factor TAFII and in the CCAAT-binding transcription factor subunits NF-YB and NF-YC. The only example of a protein with a HFD acting outside the nucleus is son-of-sevenless, a protein with multiple domains containing two HFDs, which are involved in binding to lipids. The HFD is B70 amino acids in length and forms three helices separated by small linker sequences. In a heterodimer the proteins are organized in head-to-tail orientation, resulting in a compact ‘handshake’ interaction. In the well-studied NF-Y complex, the heterodimer NF-YB and NF-YC interacts with a third subunit, NF-YA. Although the heterodimer binds DNA in a nonspecific manner, the NF-YA subunit confers binding specificity to the CCAAT motif.
ORP1 (PBANKA_0902500, 950 amino acids) contains a carboxyterminal HFD. ORP2 has an amino-terminal HFD, which is most similar to NF-YC of plants and animals, and HAP5 of yeast. Both HFDs comprises the three a-helices characteristic of the HFD and the short aC helix found in NF-YB/NF-YC proteins. Neither of the two proteins contains any other recognizable motifs and outside the HFD there is only a low degree of similarity comparing the two. The two proteins are considerably longer than NF-YB and NF-YC of animals. BLAST searches of the Plasmodium genome failed to reveal any protein with similarity to NF-YA, which suggested that these two proteins may not be part of a classical NF-Y DNA-binding complex.

Phenotype
Slightly higher oocyst production compared to wild type. Sporozoites are formed inside oocysts. Oocysts do not rupture and oocysts do not release sporozoites.
No infection of mice after mosquito bite. No infection of mice after infection with sporozoites that are artificially released from oocysts (motility of these sporozoites was not tested/reported).

Additional information
See RMgm-4059 for a mutant lacking ORP1 (PBANKA_0902500).
See RMgm-4061 for a mutant with a mutated histone-fold domain (HFD) of ORP1. Phenotype analyses of this mutant indicates that HFD is essential for ORF function
See RMgm-4062  and RMgm-4063 for mutants expresing GFP-tagged ORP1 version and a mCherry-tagged version of ORP2. Analyses of these mutants show  that ORP1 is located in the oocyst capsule, whereas ORP2 is re-localized from the oocyst cytoplasm to the capsule at the time when mature sporozoites have formed.

Other mutants
See above


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1303400
Gene Model P. falciparum ortholog PF3D7_1439500
Gene productoocyst rupture protein 2, putative
Gene product: Alternative nameORP2; NFYC
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/yfcu
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

  Transgene: Mutant parasite expressing a transgene
Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nameGFP
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
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/yfcu
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modification
Additional remarks selection procedure
Other details transgene
Promoter
Gene Model of Parasite PBANKA_0711900
Gene Model P. falciparum ortholog PF3D7_0818900
Gene productheat shock protein 70
Gene product: Alternative nameHSP70
Primer information details of the primers used for amplification of the promoter sequence  Click to view information
Primer information details of the primers used for amplification of the promoter sequence  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
3'-UTR
Gene Model of Parasite PBANKA_1340000
Gene productdihydrofolate synthase/folylpolyglutamate synthase, putative
Gene product: Alternative namePbDHFS-FPGS
Primer information details of the primers used for amplification the 3'-UTR sequences  Click to view information
Primer information details of the primers used for amplification the 3'-UTR sequences  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
Insertion/Replacement locus
Replacement / InsertionReplacement locus
Gene Model of Parasite PBANKA_1303400
Gene productoocyst rupture protein 2, putative
Gene product: Alternative nameORP2; NFYC
Primer information details of the primers used for amplification of the target sequences  Click to view information
Primer information details of the 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