RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-978
Malaria parasiteP. yoelii
Genotype
TaggedGene model (rodent): PY17X_1418400; Gene model (P.falciparum): PF3D7_1318200; Gene product: glycerol-3-phosphate 1-O-acyltransferase (G3PAT; apiG3PAT)
Name tag: 4xMyc
Phenotype Liver stage;
Last modified: 27 December 2013, 22:10
  *RMgm-978
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene tagging
Reference (PubMed-PMID number) Reference 1 (PMID number) : 24330260
MR4 number
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Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. yoelii
Parent strain/lineP. y. yoelii 17XNL
Name parent line/clone Not applicable
Other information parent line
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The mutant parasite was generated by
Name PI/ResearcherS.E. Lindner; S.H.I. Kappe; A.M. Vaughan
Name Group/DepartmentSeattle Biomedical Research Institute
Name InstituteSeattle Biomedical Research Institute
CitySeattle
CountryUSA
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Name of the mutant parasite
RMgm numberRMgm-978
Principal namePy apiG3PATmyc
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
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Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot different from wild type
Liver stageapiG3PATmyc expression in apicoplast of liver stages
Additional remarks phenotype

Mutant/mutation
The mutant expresses a C-terminal 4xMyc-tagged version of apicoplast glycerol-3-phosphate acyltransferase, putative (apiG3PAT).

Protein (function)
Fatty acids are a major component of the phospholipids that make up all cellular membranes and one hypothesis would be that the fatty acids are required for the enormous amount of membrane biosynthesis necessary late in liver stage development for the formation of tens of thousands of exoerythrocytic merozoites. The precursor of phospholipid biosynthesis is phosphatidic acid. Phosphatidic acid is formed in a three-step enzymatic reaction, the first step of which is the conversion of dihydroxyacetone phosphate (DHAP) to glycerol 3-phosphate by glycerol 3-phosphate dehydrogenase (G3PDH). Two fatty acid side chains are then transferred to glycerol 3-phosphate, firstly by glycerol 3-phosphate acyltransferase (G3PAT) to form lysophosphatidic acid and then lysophosphatidic acid acyltranferase (LPAAT) to form phosphatidic acid. Analysis of the Plasmodium genome has also uncovered two sets of genes for phosphatidic acid biosynthesis and one set is predicted to target to the apicoplast. In the rodent malaria parasite P. yoelii  G3PDH and G3PAT are indeed ocalized to the apicoplast only during liver stage development, where they are essential for production of viable merozoites. Unexpectedly, there appears to be no specific apicoplast-targeted LPAAT.

Analysis of a mutant lacking expression of apiG3PAT (RMgm-977) provides evidence for an essential role of  G3PDH for the maturation of liver stage schizonts and formation of viable liver-stage merozoites

Phenotype
Analysis of a mutant lacking expression of apiG3PAT (RMgm-977) provides evidence for an essential role of  G3PDH for the maturation of liver stage schizonts and formation of viable liver-stage merozoites

Analysis of the mutant expressing the tagged version of apiG3PAT showed apiG3PDHmyc expression in apicoplast of liver stages. No expression was detected in blood stages and sporozoites

Additional information

Other mutants
RMgm-977: A mutant lacking expression of apicoplast glycerol-3-phosphate acyltransferase, putative (apiG3PAT).

  Tagged: Mutant parasite with a tagged gene
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Details of the target gene
Gene Model of Rodent Parasite PY17X_1418400
Gene Model P. falciparum ortholog PF3D7_1318200
Gene productglycerol-3-phosphate 1-O-acyltransferase
Gene product: Alternative nameG3PAT; apiG3PAT
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Details of the genetic modification
Name of the tag4xMyc
Details of taggingC-terminal
Additional remarks: tagging
Commercial source of tag-antibodies
Type of plasmid/constructPlasmid 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 parasitetgdhfr
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modification
Additional remarks selection procedureThe transgenic myc-epitope expressing parasites were created by gene replacement and thus contained a single epitope-tagged copy of the gene under its endogenous promoter.
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
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Conceptual design of the database: Dr. C.J. Janse (Leiden Malaria Research Group, Leiden, The Netherlands).
Technical design and realization: StudioDrie StudioDrie; S. Mededovic and A. van Wigcheren