RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-4061
Malaria parasiteP. berghei
Genotype
MutatedGene model (rodent): PBANKA_0902500; Gene model (P.falciparum): PF3D7_1146600; Gene product: oocyst rupture protein 1, putative | CCAAT-box DNA binding protein subunit B (ORP1; NFYB)
Details mutation: Deletion of part of the α2 to αC domains of the Histone Fold Domain (amino 52 acids 797 to 860).
Phenotype Oocyst; Sporozoite; Liver stage;
Last modified: 24 December 2016, 17:25
  *RMgm-4061
Successful modificationThe 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) : 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-4061
Principal nameorp1-hfd(-)
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
OocystNormal oocyst production compared to wild type. Sporozoites are formed inside oocysts. Oocysts do not rupture and oocysts do not release sporozoites.
Sporozoiteormal 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.
Additional remarks phenotype

Mutant/mutation
The mutant expresses a mutated form of ORP1: part of the α2 to αC domains of the Histone Fold Domain (HFD) is deleted (amino 52 acids 797 to 860).

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
Normal 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.
This phenotype is similar to the phenotype of a mutant lacking expression of ORP1 (RMgm-4059).
Phenotype analyses of this mutant indicates that HFD is essential for ORP1 function

Additional information
The mutant protein still maintained a peripheral localization in oocysts consistent with the localization of the GFP fusion of the C-terminally truncated ORP1 to the oocyst capsule (see mutant RMgm-4062), which tindicates that localization is due to motifs in the N-terminal part of the protein and is independent of the dimerization domain

See RMgm-4059 for a mutant lacking ORP1(PBANKA_0902500).
See RMgm-4060 for a mutant lacking ORP2 (PBANKA_1303400).
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


  Mutated: Mutant parasite with a mutated gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0902500
Gene Model P. falciparum ortholog PF3D7_1146600
Gene productoocyst rupture protein 1, putative | CCAAT-box DNA binding protein subunit B
Gene product: Alternative nameORP1; NFYB
Details of the genetic modification
Short description of the mutationDeletion of part of the α2 to αC domains of the Histone Fold Domain (amino 52 acids 797 to 860).
Inducable system usedNo
Short description of the conditional mutagenesisNot available
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 markerunknown
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe construct comprised of the last 890 bp of the first exon of the orp1 gene, a 25 bp region encoding c-myc in frame with the last 270 bp of the coding region followed by a stop codon and 595 bp of the 3’-FR. A 597 bp fragment downstream of the 3’-FR of orp1 was used as a 3’ targeting sequence. The construct contains the drug selectable cassette encoding hDHFR fused to yeast Fcu (of vector pBATSIL6). Integration of the construct by double cross-over resulted in 189 bp deletion of the orp1 ORF encoding part of the α2 to αC domains of the HFD (amino 52 acids 797 to 860).
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