Summary

RMgm-679
Malaria parasiteP. berghei
Genotype
TaggedGene model (rodent): PBANKA_0524800; Gene model (P.falciparum): Not available; Gene product: early transcribed membrane protein small exported protein (SEP1; ETRAMP; Pbsep1)
Name tag: FLAG
Phenotype Asexual bloodstage;
Last modified: 26 December 2011, 22:21
  *RMgm-679
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) : 22106924
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone 8417HP
Other information parent lineA reference wild type clone from the ANKA strain of P. berghei (Janse et al., Exp. Parasitol. 68, 274-282).
The mutant parasite was generated by
Name PI/ResearcherCurà, C; Pace, T; Ponzi, M.
Name Group/DepartmentDipartimento di Malattie Infettive, Parassitarie ed Immunomediate
Name InstituteIstituto Superiore di Sanità
CityRome
CountryItaly
Name of the mutant parasite
RMgm numberRMgm-679
Principal nameF-sep1
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationNo
Phenotype
Asexual blood stageSee 'Additional remarks phenotype'.
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant expresses a (C-terminal) FLAG-tagged version of SEP1. The endogenous sep1 gene is not tagged. The FLAG-tagged copy of sep1 is introduced into the genome into the (silent) c/d-ssu-rRNA locus. The FLAG-tagged copy of sep1 is under the control of the upstream and downstream regulatory regions of the endogenous sep1.

Protein (function)
Early transcribed membrane protein (ETRAMP) family member. Plasmodium conserved family with greater than ten members in P. falciparum. ETRAMPs are abundantly expressed early in the intraerythrocytic cycle and are small (frequently less than 200 aa) integral membrane proteins that are localized within the parasitophorous vacuolar membrane (PVM). All members have signal peptides plus a transmembrane domain. The ETRAMP/SEP proteins of P. yoelii and P. berghei (8-11 genes) show homology to members of the ETRAMP family of proteins of P. falciparum (14 genes) but the orthologous relationship of the different members is not completely resolved.

P. berghei sep genes, Pbsep1, Pbsep2 and Pbsep3 (PBANKA_052480, PBANKA_052420 and PBANKA_050110, respectively) are 3 ETRAMP members which reside in the subtelomeric regions of chromosome 5. The three genes share the upstream regulatory region and differ in their 3’UTRs. The encoded proteins (13-16 KDa) are nearly identical in the first 81 amino acids, which include a predicted signal peptide, a lysine-rich domain and a TM region, while differ in their C-terminal portion.
Mutants lacking expression of SEP1 have been generated (RMgm-16, RMgm-18) which show a normal phenotype during the complete life cycle, comparable to wild type parasites.
Multiple attempts to disrupt sep2 en sep3 (RMgm-64, RMgm-65) indicate that these proteins are essential for blood stage proliferation.
SEP1 is an integral membrane of the parasitophorous vacuole membrane of blood stages.

Phenotype
The phenotype has not been analysed in detail. Antibodies against SEP1 recognized the endogenous SEP1 in Western analysis. However, these antibodies failed to recognise the FLAG-tagged version of SEP1 (SEP1-F). This might be be explained by the fact that the immune serum was raised against the C-terminal peptide (18 amino acids) of SEP1 and the fusion with FLAG may have affected its conformation. As expected, α-FLAG monoclonal antibody detected SEP1-F.

See RMgm-680 and RMgm-681 for more detailed analyses of mutants expressing FLAG-tagged SEP2 and SEP3, showing transport of SEP2 and SEP3 into the cytoplasm of the host erythrocyte. In the paper additional analyses are shown for determination of motifs for export of these proteins.

Additional information
SEP2 and SEP3 are components of the parasitophorous vacuole membrane (PVM). During blood stage development vesicle-like structures containing these proteins detach from the PVM en route to the host cytosol. These SEP-containing vesicles remain associated with the infected erythrocyte ghosts most probably anchored to the membrane skeleton.

Other mutants
See RMgm-680 and RMgm-681 for more detailed analyses of mutants expressing FLAG-tagged SEP2 and SEP3


  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0524800
Gene Model P. falciparum ortholog Not available
Gene productearly transcribed membrane protein small exported protein
Gene product: Alternative nameSEP1; ETRAMP; Pbsep1
Details of the genetic modification
Name of the tagFLAG
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 parasitepbdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationSee the paper for details of the constructs, (sequence of) primers and methods used to tag sep1 with FLAG
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