Summary

RMgm-300
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_0804300; Gene model (P.falciparum): PF3D7_0706700; Gene product: DNA mismatch repair protein MSH2, putative (MSH2-2)
PhenotypeNo phenotype has been described
Last modified: 24 January 2010, 15:02
  *RMgm-300
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) : 17583362
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei NK65
Name parent line/clone Not applicable
Other information parent line
The mutant parasite was generated by
Name PI/ResearcherL. Bethke, D. Wirth
Name Group/DepartmentDepartment of Immunology and Infectious Diseases
Name InstituteHarvard School of Public Health
CityBoston
CountryUSA
Name of the mutant parasite
RMgm numberRMgm-300
Principal namePbMSH2-2 mutant (clone A2, B2)
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 stageNot different from wild type
Additional remarks phenotype

Mutant/mutation
In the mutant the gene encoding the mismatch repair protein MSH2-2 has been disrupted using an insertion plasmid designed to integrate via a single cross-over event. This integration event resulted in the presence of two truncated copies of the gene.

Protein (function)
MSH2-2 is a putative mismatch repair (MMR) protein. The most well characterized MMR pathway is the methyl-directed MutHLS system of Escherichia coli. Yeast, humans, and other eukaryotes have multiple homologs of bacterial MutS (MSH) and MutL (MLH), but no MutH. In the genome of P. falciparum two homologs of of MSH2 (bacterial MutS homolog) have been identified PfMSH2-1 (PF14_0254) and PfMSH2-2 (MAL7P1.206).

Phenotype
The phenotype analyses indicate a non-essential role of MSH2-2 during blood stage development, mosquito development and development in the liver under normal growth conditions (see also 'Additional information').

Additional information
The mutant has been analysed in 'drug resistance' and 'microsatelaite instability' assays. These analyses did not provide evidence  that disruption of MSH2-2 results in a strong mutator phenotype which may suggest that PbMSH2-2 is a minor MMR enzyme or that its function overlaps with that of MSH2-1 or other Plasmodium proteins.
In the same study three other genes encoding putative mismatch repair (MMR) proteins have been targeted for disruption in P. berghei, i.e. PB300947.00.0 (PF14_0254; MSH2-1; DNA mismatch repair protein Msh2p, putative),,   PB001297.02.0 (PFE0270c; MSH6; DNA repair protein, putative) and PB000037.02.0 (PF11_0184; MLH1; DNA mismatch repair protein MLH1, putative; mutL homolog). Generation and selection of mutants containing disrupted genes failed for all three genes, suggesting an essential role of these proteins during blood stage development. No information is provided on the (sequence of) DNA constructs used for disruption and on the number of attempts to disrupt these genes.

Other mutants
See RMgm-382, RMgm-383 and RMgm-384 for negative attempts to disrupt putative mismatch repair proteins


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0804300
Gene Model P. falciparum ortholog PF3D7_0706700
Gene productDNA mismatch repair protein MSH2, putative
Gene product: Alternative nameMSH2-2
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct usedPlasmid single cross-over
PlasmoGEM (Sanger) construct/vector usedNo
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Plasmid/construct sequence
Restriction sites to linearize plasmid XcmI
Partial or complete disruption of the genePartial
Additional remarks partial/complete disruption The plasmid designed to disrupt PbMSH2-2 included as a target sequence for homologous recombination, a 1380 bp fragment of PbMSH2-2, which lacked sequence encoding the first 127 amino acids of the protein, including a portion of the putative DNA binding domain, and was also missing the C-terminal 269 amino acids, including the ATP binding domain and the mutS signature motif.
The insertion plasmid was designed to integrate via a single crossover event, resulting in two truncated copies of the gene. No information is provided on the sequence of the primers used to amplify the target sequence for homologous recombination.
Selectable marker used to select the mutant parasitehdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe plasmid designed to disrupt PbMSH2-2 included as a target sequence for homologous recombination, a 1380 bp fragment of PbMSH2-2, which lacked sequence encoding the first 127 amino acids of the protein, including a portion of the putative DNA binding domain, and was also missing the C-terminal 269 amino acids, including the ATP binding domain and the mutS signature motif.
The insertion plasmid was designed to integrate via a single crossover event, resulting in two truncated copies of the gene. No information is provided on the sequence of the primers used to amplify the target sequence for homologous recombination.
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