RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-4465
Malaria parasiteP. berghei
Genotype
TaggedGene model (rodent): PBANKA_0208100; Gene model (P.falciparum): PF3D7_0105200; Gene product: RAP protein, putative (putative heptatricopeptide repeat protein (HPR))
Name tag: mCherry-3xMyc
Transgene
Transgene not Plasmodium: GFP tagged to the promoter and aminoterminus of the mitochondrial HSP70-3
Promoter: Gene model: PBANKA_0711900; Gene model (P.falciparum): PF3D7_0818900; Gene product: heat shock protein 70 (HSP70)
3'UTR: Gene model: PBANKA_1340000; Gene product: dihydrofolate synthase/folylpolyglutamate synthase, putative (PbDHFS-FPGS)
Replacement locus: Gene model: PBANKA_0208100; Gene product: RAP protein, putative (putative heptatricopeptide repeat protein (HPR))
Phenotype Asexual bloodstage; Gametocyte/Gamete;
Last modified: 15 August 2018, 12:45
  *RMgm-4465
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene tagging, Introduction of a transgene
Reference (PubMed-PMID number) Reference 1 (PMID number) : 30102371
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone Not applicable
Other information parent line
The mutant parasite was generated by
Name PI/ResearcherHillebrand A, Matuschewski K, Schmitz-Linneweber C
Name Group/DepartmentMolecular Genetics
Name InstituteHumboldt University
CityBerlin
CountryGermany
Name of the mutant parasite
RMgm numberRMgm-4465
Principal namePBANKA_0208100::mCherry
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stagePBANKA_0208100::mCherry expression. Evidence for mitochondrial localisation
Gametocyte/GametePBANKA_0208100::mCherry expression. Evidence for mitochondrial localisation in gametocytes
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant expresses a C-terminal mCherry-tagged version of a putative heptatricopeptide repeat protein (HPR) and expresses GFP under control of the hsp70 promoter

Protein (function)
The protein belongs to an organellar helical-hairpin-repeat protein family that was termed heptatricopeptide repeat (HPR) proteins

Phenotype
PBANKA_0208100::mCherry expression. Evidence for mitochondrial localisation in asexual blood stages and in gametocytes

Additional information
Transcription of the mitochondrial genome has been shown to lead to polycistronic transcripts, with mRNAs and rRNA fragments transcribed together as one precursor molecule. Even transcripts representing the entire genome have been detected. This expression organization suggests that post-transcriptional regulatory events, such as RNA processing, RNA stabilization, and RNA degradation, play an important role in mitochondria, similar to post-transcriptional gene regulation in the apicoplast. These processes are expected to require a large number of RNA-binding proteins (RBPs), but most of the components and mechanisms of the RNA-processing machinery remain unknown even in human mitochondria.All organellar RBPs must originate from the nuclear genome and be transferred to the organelles post-translationally. The largest class of organellar RBPs is helical-hairpin-repeat proteins called pentatricopeptide repeat proteins (PPRs). These proteins contain a tandem-repeat motif with up to 35 repetitions. Structurally, each repeat forms two alpha-helical elements that fold back onto each other. Alpha helices from consecutive repeats are stacked to form an extended RNA-interacting surface. Within this surface, each repeat is responsible for binding one base in a singlestranded RNA molecule. Two dedicated amino acids are key for specific RNA base recognition. Of the ∼450 predicted PPR proteins in Arabidopsis, approximately two thirds are localized to the mitochondria, with the remainder are found in the plastid. PPR proteins play an important role in RNA processing and transcript stabilization. Binding of a number of PPR proteins to mRNAs acts as a roadblock against exonucleolytic decay. Eventually, only small sequences that are protected by the PPR proteins remain; these can be detected by sequencing smallRNAs, providing a method for identifying PPR protein binding sites. A protein family with a set of functions and structure similar to PPR proteins, called the octatricopeptide repeat (OPR) family, is more prevalent in single-celled photosynthetic algae, e.g. Chlamydomonas reinhardtii. In Plasmodium, there are two annotated PPR proteins; one (PF3D7 1406400; PBANKA 1035800) is predicted to localize to the apicoplast and the other (PF3D7 1233300; PBANKA 1448000) to the mitochondrion. To our knowledge, no OPR proteins have been described in Apicomplexa to date. We discovered a novel helical-hairpin-repeat protein family distinct from PPR and OPR proteins, which we termed heptatricopeptide repeat (HPR) proteins, and demonstrated that individual HPRs are targeted to Plasmodium mitochondria. Although these proteins seem particularly abundant in apicomplexan parasites, they were also found in other species within the  Alveolata. In fact, while they were not dected in bacteria, HPR proteins are found in most eukaryotic groups analyzed, including humans.

Other mutants


  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0208100
Gene Model P. falciparum ortholog PF3D7_0105200
Gene productRAP protein, putative
Gene product: Alternative nameputative heptatricopeptide repeat protein (HPR)
Details of the genetic modification
Name of the tagmCherry-3xMyc
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/yfcu
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationTo create fluorescently tagged proteins, we amplified the sequence directly upstream of the stop codon (ranging in size from 1326 to 2460 bp) and cloned the amplicon into the pBAT-SIL6 vector system in frame with the mCherry 3xMyc tag sequence.
The construct contains a GFP-expression cassette with the gfp gene under control of the hsp70 promoter.
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

  Transgene: Mutant parasite expressing a transgene
Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nameGFP tagged to the promoter and aminoterminus of the mitochondrial HSP70-3
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
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/yfcu
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationTo create fluorescently tagged proteins, we amplified the sequence directly upstream of the stop codon (ranging in size from 1326 to 2460 bp) and cloned the amplicon into the pBAT-SIL6 vector system in frame with the mCherry 3xMyc tag sequence.
The construct contains a GFP-expression cassette with the gfp gene under control of the hsp70 promoter.
Additional remarks selection procedure
Other details transgene
Promoter
Gene Model of Parasite PBANKA_0711900
Gene Model P. falciparum ortholog PF3D7_0818900
Gene productheat shock protein 70
Gene product: Alternative nameHSP70
Primer information details of the primers used for amplification of the promoter sequence  Click to view information
Primer information details of the primers used for amplification of the promoter sequence  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
3'-UTR
Gene Model of Parasite PBANKA_1340000
Gene productdihydrofolate synthase/folylpolyglutamate synthase, putative
Gene product: Alternative namePbDHFS-FPGS
Primer information details of the primers used for amplification the 3'-UTR sequences  Click to view information
Primer information details of the primers used for amplification the 3'-UTR sequences  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
Insertion/Replacement locus
Replacement / InsertionReplacement locus
Gene Model of Parasite PBANKA_0208100
Gene productRAP protein, putative
Gene product: Alternative nameputative heptatricopeptide repeat protein (HPR)
Primer information details of the primers used for amplification of the target sequences  Click to view information
Primer information details of the 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