RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-1230
Malaria parasiteP. berghei
Genotype
TaggedGene model (rodent): PBANKA_1103300; Gene model (P.falciparum): PF3D7_0503600; Gene product: myosin B (MyoB; PfM-B)
Name tag: GFP
Phenotype Asexual bloodstage; Fertilization and ookinete; Sporozoite; Liver stage;
Last modified: 27 March 2015, 18:23
  *RMgm-1230
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) : 25802338
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 2.34
Other information parent lineP. berghei ANKA 2.34 is a cloned, gametocyte producer line of the ANKA strain (PubMed: PMID: 15137943)
The mutant parasite was generated by
Name PI/ResearcherYusuf NA; Tewari R; Holder AA
Name Group/DepartmentDivision of Parasitology
Name InstituteMRC National Institute for Medical Research
CityLondon
CountryUK
Name of the mutant parasite
RMgm numberRMgm-1230
Principal namePbMyoBGFP
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageExpression of MyoBGFP in schizonts. MyoB-GFP was detected as a distinct single dot in developing merozoites within schizonts with the fluorescence concentrated at the extreme apical end of the cell.
Gametocyte/GameteNot different from wild type
Fertilization and ookineteExpression of MyoBGFP in ookinets. MyoB-GFP was detected as a distinct single dot in mature ookinetes with the fluorescence concentrated at the extreme apical end of the cell.
OocystNot different from wild type
SporozoiteExpression of MyoBGFP in sporozoites. MyoB-GFP was detected as a distinct single dot in sporozoites with the fluorescence concentrated at the extreme apical end of the cell. In salivary gland sporozoites, in addition to the bright apical spot of fluorescence, a more diffuse fluorescence within the body of the parasite was also observed. This was not observed in any other stage.
Liver stageIn mature liver-stage schizonts 55 h after invasion by sporozoites, individual merozoites displayed an apical dot localisation for PbMyoBGFP.
Additional remarks phenotype

Mutant/mutation
The mutant expresses a C-terminal GFP-tagged version of myosin B

Protein (function)
Myosin B (MyoB) is one of the two short class XIV myosins encoded in the Plasmodium genome. Class XIV myosins are characterised by a catalytic ‘head’, a modified ‘neck’ region and the absence of a ‘tail’ region. Myosin A (MyoA), the other class XIV myosin in Plasmodium, has been established as a component of the glideosome complex important in motility and cell invasion but MyoB is not well characterised.
The malaria parasite genome contains six genes that encode myosins. Two of these genes code for short class XIV myosins, which are restricted to Apicomplexan parasites and other members of the Alveolata. These myosins differ from most others in that, while they have the highly conserved 'head' region that binds actin and hydrolyses ATP, and the 'neck' region that acts as the lever arm and contains IQ-motifs to which myosin light chains bind, they have no 'tail' region. The tail normally allows oligomerisation, cargo binding and determines the subcellular location of the myosin.

Phenotype
Expression of MyoBGFP in the invasivie stages (merozoites, ookinetes sporozoites). MyoB-GFP was detected as a distinct single dot  with the fluorescence concentrated at the extreme apical end of the cell. In salivary gland sporozoites, in addition to the bright apical spot of fluorescence,  a more diffuse fluorescence within the body of the parasite was also observed. This was not observed in any other stage.


Additional information
Through analysis of MyoB in P. falciparum, P. knowlesi and P. berghei evidence is presented that MyoB is expressed in all invasive stages (merozoites, ookinetes and sporozoites) of the life cycle and the protein is found in a discrete apical location in these polarised cells. In P. falciparum, MyoB is synthesised very late in schizogony/merogony and its location in merozoites is distinct from, and anterior to, that of a range of known proteins present in the rhoptries, rhoptry neck or micronemes. Unlike MyoA, MyoB is not associated with glideosome complex proteins, including the MyoA light chain, Myosin A tail domain interacting protein (MTIP). A unique MyoB light chain (MLC-B) was identified that contains a calmodulin-like domain at the C-terminus and an extended Nterminal region. MLC-B localises to the same extreme apical pole in the cell as MyoB and the two proteins form a complex.

Other mutants


  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1103300
Gene Model P. falciparum ortholog PF3D7_0503600
Gene productmyosin B
Gene product: Alternative nameMyoB; PfM-B
Details of the genetic modification
Name of the tagGFP
Details of taggingC-terminal
Additional remarks: tagging
Commercial source of tag-antibodies
Type of plasmid/construct(Linear) plasmid single 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 parasitehdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationMyoB was modified by single homologous recombination to insert a sequence coding for a C-terminal GFP tag, using the p277 vector that contains a human dhfr selection cassette. A 978 bp sequence of myoB starting 2.4 kb downstream of the ATG initiator codon and omitting the stop codon was amplified using primers 11 and 12 and inserted into the vector using KpnI and ApaI restriction sites. The construct was linearised using HincII and then used to transfect the P. berghei ANKA 2.34 line by electroporation.

primer 11: ccccggtaccATACCGTAAGTGATGTAACATATACTATTAC
primer 12: ccccgggcccTTCGAGCTCTTTCATATGCTTATATTTTCG
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