Summary

RMgm-653
Malaria parasiteP. berghei
Genotype
MutatedGene model (rodent): PBANKA_1232200; Gene model (P.falciparum): PF3D7_0517400; Gene product: FACT complex subunit SPT16, putative (FACT-L; FACT)
Details mutation: 'promoter-swap' mutant; the promoter of fact-l replaced by the promoter of PBANKA_140060
Transgene
Transgene not Plasmodium: GFP (gfp-mu3)
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_0306000; Gene product: 6-cysteine protein (230p, P230p)
Phenotype Gametocyte/Gamete; Fertilization and ookinete; Oocyst;
Last modified: 22 September 2011, 13:22
  *RMgm-653
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) : 21899698
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 507cl1 (RMgm-7)
Other information parent lineP.berghei ANKA 507cl1 (RMgm-7) is a reference ANKA mutant line which expresses GFP under control of a constitutive promoter. This reference line does not contain a drug-selectable marker (PubMed: PMID: 16242190).
The mutant parasite was generated by
Name PI/ResearcherE. Laurentino, C.J. Janse, A.P. Waters
Name Group/DepartmentLeiden Malaria Research Group
Name InstituteLeiden University Medical Center
CityLeiden
CountryThe Netherlands
Name of the mutant parasite
RMgm numberRMgm-653
Principal name1076cl1,cl2
Alternative nameΔfactl-gam 2b
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteNormal numbers of female and male gametocytes are produced. Fertility of female gametes is comparable to wild type female gametes as shown by crossing with wild type male gametes. Male gametocytes showed delayed DNA replication and gamete formation. Male gamete fertility is strongly reduced
Fertilization and ookineteNormal numbers of female and male gametocytes are produced. Fertility of female gametes is comparable to wild type female gametes as shown by crossing with wild type male gametes. Male gametocytes showed delayed DNA replication and gamete formation. Male gamete fertility is strongly reduced, resulting in strongly reduced ookinete formation.
OocystMale gamete fertility is strongly reduced, resulting in strongly reduced ookinete formation. Residual ookinetes generated oocysts that arrested early in development and failed to enter sporogony.
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
In the 'promoter-swap' mutant the promoter of fact-l is replaced by an 'asexual blood stage specific’ promoter that is silent in gametocytes (the promoter of PBANKA_140060). The mutant expresses GFP under the constitutive eef1α promoter.
Transcription of fact-l in  mutant gametocytes was down-regulated compared with wild-type gametocytes.

Protein (function)
FACT is a histone chaperone that has received considerable attention for its function in facilitating RNA transcription and DNA replication by dynamically altering chromatin through modifications of nucleosome structure. FACT is an evolutionarily conserved, abundant nuclear heterodimer/trimer. The large subunit FACT-L, in humans p140h/SPT16, is a homologue of the yeast Spt16/Cdc68 protein (suppressor of Ty). The small subunit of FACT (FACT-S) in human structure specific recognition protein 1 (SSRP1), possesses a C-terminal high-mobility group (HMG) box with an N-terminus homologous to the yeast protein Pob3 (DNA polymerase one binding). In yeast SSRP1 activity is represented by two distinct proteins, Pob3 and Nhp6A (or B) which encodes the missing HMG box. Functionally, both yeast and human FACT act during transcription initiation and displace histones H2A/H2B to effect nucleosome disassembly and facilitate transcription elongation.

BLAST-based homology searches with PBANKA_123220 identified clear homology to the eukaryotic SPT16 protein (e.g. 30% identity, 41% similarity to SPT16 of  Saccharomyces cerevisiae) with an identical arrangement of the Rttp106-like, SPT16 and M24 peptidase domains.

Attempts to disrupt the fact-l gene in P. berghei were unsuccessful (see RMgm-255), indicating an essential role of FACT-L for blood stage development/multiplication

Phenotype
Attempts to disrupt the fact-l gene in P. berghei were unsuccessful (see RMgm-255), indicating an essential role of FACT-L for blood stage development/multiplication.

In the 'promoter-swap' mutant the promoter of fact-l is replaced by an 'asexual blood stage specific’ promoter that is silent in gametocytes (the promoter of PBANKA_140060). Transcription of fact-l in  mutant gametocytes was down-regulated compared with wild-type gametocytes.

Phenotype analyses of the promoter-swap mutant indicate that FACT-L plays an important role in the production of fertile male gametes.

The promoter-swap mutant produced normal numbers of female and male gametocytes. Fertility of female gametes is comparable to wild type female gametes as shown by crossing with wild type male gametes. Male gametocytes showed delayed DNA replication and gamete formation. Male gamete fertility is strongly reduced, resulting in strongly reduced ookinete formation. Residual ookinetes generated oocysts that arrested early in development and failed to enter sporogony.

Additional information
Similar promoter-swap mutants have been generated in the P. berghei reference line cl15cy1 (RMgm-652) and in a reporter reference line of P. berghei that  express GFP in male gametocytes and RFP in female gametocytes/gametes (RMgm-654).

In the same paper a successful 'promoter-swap' mutants are described in which the promoter of fact-l is replaced by the 'asexual blood stage specific’ promoter of PBANKA_091420 that is silent in gametocytes. These mutants are: Δfact-lgam1a, 1044 in cl15cy1; Δfactlgam 1b, 1076cl1, cl3 in 507cl2; Δfact-lgam 1c, 1134cl1,cl2 in 820cl1m1cl1.

Phenotype analyses of a mutant expressing c-myc-tagged FACT-L (RMgm-655) showed that FACT-L is located predominantly in the parasite nucleus with evidence that a small proportion of FACT-L is located in the cytoplasm. Expression was detected in all blood stages with high levels of FACT-L in the nuclei of male gametocytes.

Other mutants
RMgm-255: Unsuccessful attempts to disrupt fact-l
RMgm-652: An independent promoter-swap mutant in which the promoter of fact-l is replaced by an 'asexual blood stage specific’ promoter that is silent in gametocytes (the promoter of PBANKA_140060). This mutant is made in the P. berghei reference line cl15cy1.
RMgm-654: An independent promoter-swap mutant in which the promoter of fact-l is replaced by an 'asexual blood stage specific’ promoter that is silent in gametocytes (the promoter of PBANKA_140060). This mutant expresses GFP in male gametocytes and RFP in female gametocytes/gametes


  Mutated: Mutant parasite with a mutated gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1232200
Gene Model P. falciparum ortholog PF3D7_0517400
Gene productFACT complex subunit SPT16, putative
Gene product: Alternative nameFACT-L; FACT
Details of the genetic modification
Short description of the mutation'promoter-swap' mutant; the promoter of fact-l replaced by the promoter of PBANKA_140060
Inducable system usedNo
Short description of the conditional mutagenesisNot available
Additional remarks inducable system
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 markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationDNA constructs were generated to exchange or ‘swap’ the endogenous fact-l promoter with a promoter of a gene that was expected not to be transcribed in gametocytes (PBANKA_140060). Vector pL0001 (http://www.mr4.org) for integration via double-cross-over homologous recombination, was used as the backbone for four different promoter-swap constructs. This vector contains the dhfr-ts gene of Toxoplasma gondii under the control of the P. berghei dhfr-ts promoter as selectable marker cassette. Three subcloning steps were performed to generate the promoter-swap constructs: first, a 539 bp fragment located 2 kb upstream of fact-l, was PCR-amplified from genomic DNA using primer pair 3190/3191 (ggggtaccTACTATAACATGGTTTTGCC (F); ccaagcttAGGATAAAAACATAGGTGTG (R)). This fragment represents the left flanking target region and was cloned into the KpnI/HindIII restriction sites of pL0001. As the right flanking target region, a 623 bp fragment of the open reading frame of fact-l was PCR amplified using primer pair 3192/3193 (gctctagaTGAACTTTGTAAAAACAAGC (F); tccccgcggGAATTTTCAATGGTAGTTATCA (R)). This fragment was cloned into the SacII/XbaI restriction sites of the vector. Finally, a 2 kb fragment of the 5'UTR promoter region of PBANKA_140060 was amplified from genomic DNA using the primer pair 3196/3197 (CGgaattcATTCGATGTTTACATAATGAC (F); CGggatccCGAATTGACACCATTTTTCC (R) and cloned into the EcoRI/BamHI sites of the vector. The promoter-swap construct (pL1313) was linearized with KpnI/SacII before transfection.
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 (gfp-mu3)
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
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 parasitegfp (FACS)
Promoter of the selectable markereef1a
Selection (positive) procedureFACS (flowsorting)
Selection (negative) procedureNo
Additional remarks genetic modificationThe GFP gene (1 copy) has been inserted into the 230p locus (PBANKA_030600) by double cross-over integration
Additional remarks selection procedureThis reporter mutant expressing GFP does not contain a drug-selectable marker. This mutant has been selected by FACS sorting after transfection based on GFP fluorescence.
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_0306000
Gene product6-cysteine protein
Gene product: Alternative name230p, P230p
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