Summary

RMgm-182
Malaria parasiteP. yoelii
Genotype
TaggedGene model (rodent): PY17X_0827100; Gene model (P.falciparum): PF3D7_0922900; Gene product: 3-oxoacyl-[acyl-carrier-protein] reductase (FABG)
Name tag: c-myc
Phenotype Sporozoite; Liver stage;
Last modified: 12 March 2009, 21:48
  *RMgm-182
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) : 19068099
MR4 number
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 line17XNL is a non-lethal strain of P. yoelii
The mutant parasite was generated by
Name PI/ResearcherA.M. Vaughan; S.H.I. Kappe
Name Group/DepartmentNot applicable
Name InstituteSeattle Biomedical Research Institute
CitySeattle
CountryUSA
Name of the mutant parasite
RMgm numberRMgm-182
Principal namefabG-myc
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
SporozoiteExpression of myc-tagged FABG in sporozoites (see 'Phenotype').
Liver stageExpression of myc-tagged FABG in liver stages (see 'Phenotype').
Additional remarks phenotype

Mutant/mutation
The mutant expresses a cmyc-tagged (C-terminal) form of FABG.

Protein (function)
FABG is an enzyme of the bacterial like type II fatty acid biosynthesis (FAS-II) pathway. In Plasmodium FAS-II enzymes have been localized to the apicoplast, a nonphotosynthetic plastid organelle of cyanobacterial origin.
FAS-II requires acetyl-Coenzyme A (CoA), which can be converted from pyruvate by the pyruvate dehydrogenase complex. Acetyl-CoA carboxylase converts acetyl-CoA to malonyl-CoA, which is tethered to an acyl carrier protein (ACP) by malonyl-CoA:ACP transacylase (FabD). This produces malonyl-ACP, which, in conjunction with acetyl-CoA, is acted upon by β-ketoacyl-ACP synthase III (Fab H) to form β-ketoacyl-ACP. This precursor enters the FAS-II elongation cycle, mediated by FabB/F (β-ketoacyl-acyl-carrier-protein (ACP) synthase), FabG (β-ketoacyl-ACP reductase), FabZ/A (β-hydroxyacyl-ACP dehydratase), and FabI (trans-2-enoyl-ACP reductase). These four FAS-II enzymes iteratively catalyze the addition of two carbon chains to a growing fatty acyl carbon chain via condensation, reduction, dehydration, and reduction steps, respectively.

Phenotype
The phenotype described below is from the analysis of a myc-tagged FABI protein.(RMgm-197). Myc-tagged FABG showed a similar expression pattern.

FabI-myc expression was first detected in salivary gland sporozoites and localized to a spherical structure close to the nucleus. To analyse FabI expression during liver stage development, HepG2:CD81 hepatoma cells were infected with sporozoites . At 7 h post infection when intracellular sporozoites initiate transformation to trophozoites, apicoplast morphology as determined by FabI-myc staining,  was similar to that of salivary gland sporozoites. At 14 h, parasite nuclear division commenced and the apicoplast initiated its division as indicated by the dumbbell shape. By 24 h, the apicoplast had formed a branched lariat-shaped structure, which became more elaborate with advanced liver stage development at 30 h. By 40 h the apicoplast had differentiated into hundreds of intertwining structures that appeared to be segregating. These results show that FabI expression is initiated in salivary gland sporozoites and that there is robust apicoplast-specific FabI expression throughout liver stage development. At 48 h a time point when the P. yoelii liver stage schizont undergoes merozoite formation, FabI-myc expression was greatly reduced when compared with 40 h. This strongly suggests that FabI expression is downregulated shortly before or during exo-erythrocytic merozoite formation. FabI expression could not be detected in blood stages. 

Additional information
The phenotype analyses of  mutant that lack expression of  other enzymes (FABB/F, FABI; RMgm-183; RMgm-197) of the FASII pathway indicate that these enzymes are not essential for blood stage development and mosquito stage development. These analyses indicate an essential role of these enzymes in the formation of infective liver stage merozoites demonstrating the importance of FASII pathway for synthesis of fatty acids during late liver stage development.

Other mutants
RMgm-180: A mutant expressing myc tagged FABI
RMgm-181: A mutant expressing myc tagged FABZ
RMgm-183: A mutant lacking expression of FABB/F
RMgm-197: A mutant lacking expression of FABI
RMgm-184: A mutant lacking expression of FABZ


  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PY17X_0827100
Gene Model P. falciparum ortholog PF3D7_0922900
Gene product3-oxoacyl-[acyl-carrier-protein] reductase
Gene product: Alternative nameFABG
Details of the genetic modification
Name of the tagc-myc
Details of taggingC-terminal
Additional remarks: taggingQuadruple myc tag
Commercial source of tag-antibodiesPrimary: rabbit polyclonal anti-myc antibody (Santa Cruz Biotechnology, Santa Cruz, CA)
Type of plasmid/constructPlasmid single cross-over
PlasmoGEM (Sanger) construct/vector usedNo
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Plasmid/construct sequence
Restriction sites to linearize plasmid BsaI
Selectable marker used to select the mutant parasitetgdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationA transgenic P. yoelii line was generated expressing a myc epitope-tagged FabG under the control of the endogenous FabG promoter. The quadruple myc tag was fused to the carboxyl (C)-terminus of FabG and was followed by the 3' UTR from P. berghei dihydrofolate reductase/thymidylate synthase (DHFR/TS)
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 1TAccgcggATCATAAAGCGATAAAATTAACCC
Additional information primer 1fabG, coding (no stop) + 1 kb upstream (KspI) forward
Sequence Primer 2TAtctagaACTTGATAATCCACCATCAATTAT
Additional information primer 2fabG, coding (no stop) + 1 kb upstream (XbaI) reverse
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