Summary

RMgm-23
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_0622921; Gene model (P.falciparum): Not available; Gene product: 18S ribosomal RNA (small subunit (ssu) of the d-type ribosomal rna gene unit)
Phenotype Oocyst; Sporozoite;
Last modified: 28 August 2015, 16:35
  *RMgm-23
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) : 11292830
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 cl15cy1
Other information parent lineA reference wild type clone from the ANKA strain of P. berghei (PubMed: PMID: 17406255).
The mutant parasite was generated by
Name PI/ResearcherR.M.L. van Spaendonk, C.J. Janse, A.P. Waters
Name Group/DepartmentLeiden Malaria Research Group
Name InstituteLeiden University Medical Center
CityLeiden
CountryThe Netherlands
Name of the mutant parasite
RMgm numberRMgm-23
Principal name59cl1; 63cl1
Alternative nameDdis1; Ddis2
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
OocystSmall growth delay (retardation) of oocysts
SporozoitePossibly less sporozoites are produced as a result of the growth delay of the oocysts.
Sporozoites are infectious to mice.
Liver stageNot different from wild type
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of  d-type ribosomal rna.

Gene (function)
P. berghei contains four distinct copies of the ribosomal RNA gene units (A-D), each encoding the following rRNA molecules: large subunit (LSU; 28 S) rRNA, small subunit (SSU; 18 S) rRNA and 5.8 S rRNA .
Based on differences in sequence and expression the four gene units are divided into the blood stage A-type (a- and b-unit) and S-type (c- and d-units), which is transcribed mainly in the proliferative stages in the mosquito.  The A-type ribosomes are present in the liver and blood stages of the parasite, and the S-type ribosomes are the predominant types produced during development in the mosquito.

Phenotype
The phenotype analyzes show that the lack of expression of d-type ribosomal RNA does have a relatively minor effect on parasite development, resulting in a delayed maturation of oocysts. The sporozoites formed are infectious to the mammalian host (Swiss mice). See also mutants RMgm-21 and RMgm-22 which lack expression of c-type ribosomal RNA.

Additional information
This mutant contains a disruption of the ssu (small subunit) part of the rRNA locus (disruption by single cross-over integration).

Other mutants
Two other mutants has been generated that lack expression of d-type ribosomal RNA. Mutant RMgm-24 in which the d-type rRNA gene has been disrupted  by double cross-over recombination, resulting in deletion of the 5.8S, ITS2 region and part of the 5'-LSU. In mutant RMgm-25 the d-type rRNA gene unit has been disrupted by double cross-over recombination, resulting in deletion of the SSU, ITS1, 5.8S, ITS2 region, and part of the D-ETS and 5'-LSU.These mutants shows a similar phenotype as described above.
Mutants have been generated lacking expression of the c-type ribosomal RNA. These mutants did not show a distinct phenotype (RMgm-21, RMgm-22).
Mutants have been generated that lack expression of both c- and d-type ribosomal RNA (pers. comm. B. Franke-Fayard; C.J. Janse). These mutants show a strong delayed maturation of the oocysts with reduced numbers of salivary gland sporozoites. Salivary gland sporozoites produced were infective to the mammalian host (Swiss mice).


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0622921
Gene Model P. falciparum ortholog Not available
Gene product18S ribosomal RNA
Gene product: Alternative namesmall subunit (ssu) of the d-type ribosomal rna gene unit
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct used(Linear) plasmid single cross-over
PlasmoGEM (Sanger) construct/vector usedNo
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Plasmid/construct sequence
Click to view information
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not available
Restriction sites to linearize plasmid
Partial or complete disruption of the genePartial
Additional remarks partial/complete disruption Disruption of the ssu (small subunit) part of the rRNA locus
Selectable marker used to select the mutant parasitepbdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationDisruption of the ssu (small subunit) part of the rRNA locus (disruption by single cross-over integration)
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