Summary

RMgm-45
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_1002100; Gene model (P.falciparum): PF3D7_0404400; Gene product: 6-cysteine protein (P36)
Phenotype Liver stage;
Last modified: 13 February 2010, 23:45
  *RMgm-45
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) : 16313615
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone Not applicable
Other information parent line
The mutant parasite was generated by
Name PI/ResearcherT. Ishino, M. Yuda
Name Group/DepartmentSchool of Medicine
Name InstituteMie University
CityMie
CountryJapan
Name of the mutant parasite
RMgm numberRMgm-45
Principal namePbs36(-)1; Pbs36(-)2
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 different from wild type
OocystNot different from wild type
SporozoiteNot different from wild type
Liver stageGliding motility is similar to wild type sporozoites. Hepatocyte traversal activity in vitro (HepG2) was higher than of wild type sporozoites (~7 times higher). Hepatocyte invasion of mutant sporozoites was significantly decreased (~9% of that of wild type). Liver stage development is strongly impaired and parasites do not develop into the schizont stage. Infectivity of sporozoites as measured by infection of rats (Wistar) by intravenous inoculation of sporozoites was reduced (1000 times compared to wildtype).
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of the P36 protein.

Protein (function)
P36 is a member of a small family of proteins, the 6-cysteine (cys) family of (surface) proteins (Thompson J. et al., Mol. Biochem. Parasitol. (2001)118, 147-54). The proteins are characterised by domains of roughly 120 amino acids in size that contain six positionally conserved cysteines (6-cys). Although some species of Plasmodium (may) contain unique members of the 6-cys family, ten members have been identified that are conserved both in structure as well as in genome organisation throughout the genus. Some of the conserved 6-cys proteins are encoded by genes that form paralogous gene-pairs which are closely linked in the genome separated by less then 2 kb of intergenic region. Most members have a GPI anchor and are predicted membrane surface proteins whereas others appear to be secreted and most members are expressed in a discrete stage-specific manner in gametocytes, sporozoites or merozoites.
A paralogue of the p36 gene, p36p (PB000891.00.0; pbs36) is located in the genome next to p36 (in tandem).

Phenotype
The phenotype analysis indicate a role of this protein in infectivity of sporozoites and liver stage development.

Additional information
An independent mutant lacking expression of P36 (RMgm-353) has been analysed. The analyses of this mutant indicate that P36 is not essential during the complete life cycle. Gametocyte production, fertilisation, oocyst and sporozoite production were comparable to wild type parasites. Liver stage development was only analysed by measuring prepatent periods after feeding of infected mosquitoes on mice. No differences were observed in prepatent periods compared to wild type parasites. These observations on liver stage development are in contrast to the phenotype of the mutant describe here.

Other mutants
An independent P. berghei mutant has been generated that lacks expression of P36 (RMgm-353).
A P. berghei mutant (RMgm-42) has been generated that lacks the expression of not only this protein but also its paralogue P36p (PB000891.00.0; p36p).
A P. yoelii mutant (RMgm-43) has been generated that lacks the expression of not only this protein but also its paralogue P36p (PY01340; p36p).


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1002100
Gene Model P. falciparum ortholog PF3D7_0404400
Gene product6-cysteine protein
Gene product: Alternative nameP36
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct usedPlasmid 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 parasitepbdhfr
Promoter of the selectable markerpbdhfr
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 15′-AAAGAGCTCGTGTGACATAAAGAGGAGAG-3′
Additional information primer 1
Sequence Primer 25′-AAAGGATCCTCCAGGCAATGTGTTGTG-3′
Additional information primer 2
Sequence Primer 35′-AAACTCGAGTATATATCCGCCGTTTGTTG-3′
Additional information primer 3
Sequence Primer 45′-AAAGGTACCGAGTTACATAATTAATTTGTGCTTG-3′
Additional information primer 4
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6