RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-4317
Malaria parasiteP. berghei
Genotype
MutatedGene model (rodent): PBANKA_0607700; Gene model (P.falciparum): PF3D7_1209300; Gene product: C2H2 zinc telomere repeat-binding zinc finger protein; finger protein Zfp, putative (TRZ, Zfp)
Details mutation: a truncated form of Zfp (lacking the C-terminal C2H2 ZnFs)
Transgene
Transgene not Plasmodium: mCherry
Promoter: Gene model: PBANKA_1133300; Gene model (P.falciparum): PF3D7_1357100; Gene product: elongation factor 1-alpha (eef1a)
3'UTR: Gene model: PBANKA_0719300; Gene product: bifunctional dihydrofolate reductase-thymidylate synthase, putative (dhfr/ts)
Replacement locus: Gene model: PBANKA_0607700; Gene product: C2H2 zinc telomere repeat-binding zinc finger protein; finger protein Zfp, putative (TRZ, Zfp)
Phenotype Asexual bloodstage; Oocyst;
Last modified: 7 September 2017, 16:08
  *RMgm-4317
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene mutation, Introduction of a transgene
Reference (PubMed-PMID number) Reference 1 (PMID number) : 28851851
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/ResearcherGopalakrishnan AM; Kumar N
Name Group/DepartmentDepartment of Tropical Medicine, School of Public Health and Tropical Medicine, Vector-Borne Infecti
Name InstituteTulane University
CityNew Orleans
CountryUSA
Name of the mutant parasite
RMgm numberRMgm-4317
Principal nameZfp-Trunc
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNormal growth/multiplication characteristics of blood stages. Mice infected with Zfp-Trunc parasites survived longer than mice infected with wild type parasites.
Gametocyte/GameteNot different from wild type
Fertilization and ookineteNot different from wild type
OocystOocyst numbers in mosquitoes infected with Zfp-Trunc parasites were 75% to 80% lower than those seen with mosquitoes infected with wild type parasites.
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant expresses a mutated form of Zfp (lacking the C-terminal C2H2 ZnFs) and expresses mCherry under control of the constitutive eef1a promoter.

Protein (function)
Studies on identification and genome-wide mapping of recombination have revealed a correlation between hotspot activity and enrichment of trimethylated histone 3 at lysine 4 (H3K4me3). Further studies in mammals have identified PRDM9, a meiosis-specific chromatin-modifying protein, as the key determinant of recombination hotspots and a master transcription regulator of entry into meiosis. The PRDM9 protein contains a C-terminal module comprised of multiple C2H2-type zinc fingers (ZnF) which binds the degenerate 13-bp motif that was previously found to be associated with ~40% of human hotspots, formation of doublestrand breaks (DSB), and recruitment of the recombination machinery leading to exchange of chromosomal material and DNA repair. Additionally, the ZnF domain (Zfd) in PRDM9 is fused to a SET domain which catalyzes the trimethylation of histone 3 lysine 4 (H3K4) and histone 3 lysine 36 (H3K36) associated with the hotspots.
The SET domain initially characterized in Drosophila is an evolutionarily well-conserved domain associated with many chromosomal proteins and with proteins necessary for histone lysine trimethylation in mammalian cells.
The C2H2-type Zfd, represented by two cysteine and two histidine residues separated by ~12 amino acids acting as ligands to a zinc atom, are widespread in most DNA-binding proteins. In animals, proteins with multiple C2H2 ZnFs are transcription factors regulating gene expression and acting during cell differentiation and development. Binding of C2H2 ZnF proteins to DNA often helps recruit other proteins which mediate epigenetic changes, including posttranslational modifications of histones. These include histone methylases as well as enzymes catalyzing demethylation of H3K4me3 and other histone methyl transferases transferring methyl groups to H3K9 and H3K27.
While proteins with runs of multiple (>3) instances of C2H2 ZnF are common in animals, the P. berghei Zfp (PbZfp) protein is the only such example conserved across Plasmodium species. The protein is comprised of a poorly conserved N-terminal low-complexity region and a C-terminal region with 11 C2H2 ZnFs, which is where the sequence similarity is most pronounced. The C2H2 ZnFs are arranged in an initial run of 3 ZnFs followed by a disordered poorly conserved linker connecting them to the remaining 8 ZnFs. Unlike the animal PRDM9 protein, PbZfp lacks the methyltransferase SET domain and does not have KRI domains typical of the members of the KRAB family of animal C2H2 ZnF proteins. Thus, while not directly related to these animal proteins, PbZfp shows an unusual resemblance to animal C2H2 ZnF proteins in having a multiplicity of C2H2 Zfd. The presence of a run of multiple C2H2 ZnFs indicates that the protein might contact an extended binding site as is typical of recombination hotspots. These observations pointed to potential roles in addition to or beyond that of a conventional transcription factor and prompted the authors to investigate if it might play a role in recombination.

Phenotype
See also mutant RMgm-4316 which lacks expression of Zfp (Zfp-KO) which has a comparable phenotype as Zfp-Trunc, which expresses a truncated form of Zfp (lacking the C-terminal C2H2 ZnFs).

Normal growth/multiplication characteristics of blood stages. Mice infected with Zfp-Trunc parasites survived longer than mice infected with wild type parasites.
No differences in gametocyte production and fertilsation.
Oocyst numbers in mosquitoes infected with Zfp-Trunc parasites were 75% to 80% lower than those seen with mosquitoes infected with  wild type parasites.

Additional information
The authors present evidence for: 'KO parasites revealed a total lack of trimethylation of histone 3 at several lysine residues (K4, K27, and K36) without any effect on acetylation patterns (H3K9, H3K14, and H4K16). Reduced DNA damage and reduced expression of topoisomerase-like Spo11 in the KO parasites with normal Rad51 expression further suggest a functional role for PbZfp during genetic recombination that involves DNA double-strand break (DSB) formation followed by DNA repair. These finding raise the possibility of some convergent similarities of PbZfp functions to functions of mammalian PRDM9, also a C2H2 ZnF protein with histone 3 lysine 4 (H3K4) methyltransferase activity. These functions include the major role played by the latter in binding recombination hotspots in the genome during meiosis and trimethylation of the associated histones and subsequent chromatin recruitment of topoisomerase-like Spo11 to catalyze DNA DSB formation and DMC1/Rad51-mediated DNA repair and homologous recombination' .

Other mutants
RMgm-4316- A mutant lacking expression of Zfp


  Mutated: Mutant parasite with a mutated gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0607700
Gene Model P. falciparum ortholog PF3D7_1209300
Gene productC2H2 zinc telomere repeat-binding zinc finger protein; finger protein Zfp, putative
Gene product: Alternative nameTRZ, Zfp
Details of the genetic modification
Short description of the mutationa truncated form of Zfp (lacking the C-terminal C2H2 ZnFs)
Inducable system usedNo
Short description of the conditional mutagenesisNot available
Additional remarks inducable system
Type of plasmid/construct(Linear) plasmid 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 markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationWe generated parasites expressing truncated (Trunc) PbZfp, lacking the C-terminal C2H2 ZnFs. A truncation plasmid was designed to introduce a stop codon within the coding sequence at the start of the Zfd.
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 namemCherry
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct(Linear) plasmid 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 markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modification
Additional remarks selection procedure
Other details transgene
Promoter
Gene Model of Parasite PBANKA_1133300
Gene Model P. falciparum ortholog PF3D7_1357100
Gene productelongation factor 1-alpha
Gene product: Alternative nameeef1a
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_0719300
Gene productbifunctional dihydrofolate reductase-thymidylate synthase, putative
Gene product: Alternative namedhfr/ts
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_0607700
Gene productC2H2 zinc telomere repeat-binding zinc finger protein; finger protein Zfp, putative
Gene product: Alternative nameTRZ, Zfp
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