Summary

RMgm-655
Malaria parasiteP. berghei
Genotype
TaggedGene model (rodent): PBANKA_1232200; Gene model (P.falciparum): PF3D7_0517400; Gene product: FACT complex subunit SPT16, putative (FACT-L; FACT)
Name tag: c-myc
Phenotype Asexual bloodstage; Gametocyte/Gamete;
Last modified: 22 September 2011, 14:10
  *RMgm-655
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) : 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 cl15cy1
Other information parent lineA reference wild type clone from the ANKA strain of P. berghei (PubMed: PMID: 17406255).
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-655
Principal name1054cl1
Alternative namefact-l::c-myc
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageAll blood stages express c-myc::FACT-L as shown by IFA (and Western analysis). Signals are mainly observed in the nuclei of parasites.
Gametocyte/GameteMale and female gametocytes express c-myc::FACT-L as shown by IFA. Signals are mainly observed in the nuclei of parasites with strong levels signals in the nuclei of male gametocytes.
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant expresses a c-myc-tagged (C-terminal) form of FACT-L

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.

Phenotype analyses of the mutant expressing c-myc-tagged FACT-L by IFA and Western analyses indicate that FACT-L is 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.

Additional information
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.

Other mutants
RMgm-255: Unsuccessful attempts to disrupt fact-l
RMgm-652: A 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-653: 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 constitutively expresses GFP.
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.

 


  Tagged: Mutant parasite with a tagged 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
Name of the tagc-myc
Details of taggingC-terminal
Additional remarks: tagging
Commercial source of tag-antibodies
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
Selectable marker used to select the mutant parasitetgdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationFor C-terminal tagging of FACT-L with a c-Myc tag, we used the standard vector pL1080 (http://www.mr4.org) as a backbone. This vector contains a double c-Myc tag. A 1.4 kb fragment of the C-terminus of fact-l was PCR-amplified using the primer pair 2779/3171 (CTGATgaattcATGCATGATGAAAT (F); TAggatccATTTTTTTTCCTCTTTC (R)), subcloned into TOPO vector (Invitrogen) and subsequently transferred into SacII/BamHI-digested pL1080, resulting in vector pL1289. After linearization with XbaI this construct was transfected into P. berghei (ANKA) parasites of reference line cl15cy1.
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