RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-4747
Malaria parasiteP. berghei
Genotype
TaggedGene model (rodent): PBANKA_1416900; Gene model (P.falciparum): PF3D7_1318400; Gene product: structural maintenance of chromosomes protein 2, putative (SMC2)
Name tag: GFP
Phenotype Asexual bloodstage; Gametocyte/Gamete; Fertilization and ookinete; Oocyst; Sporozoite; Liver stage;
Last modified: 6 April 2020, 17:16
  *RMgm-4747
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) : 32049018
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 2.34
Other information parent lineP. berghei ANKA 2.34 is a cloned, gametocyte producer line of the ANKA strain (PubMed: PMID: 15137943).
The mutant parasite was generated by
Name PI/ResearcherPandey R, Tewari R
Name Group/DepartmentSchool of Life Sciences, Queens Medical Centre
Name InstituteUniversity of Nottingham
CityNottingham
CountryUK
Name of the mutant parasite
RMgm numberRMgm-4747
Principal nameSMC2GFP
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageWe found that SMC2 and SMC4 are expressed during both schizogony and male gametogenesis. In early schizonts within host red blood cells, we observed discrete foci in the parasite cell adjacent to the nuclear DNA for both SMC2 and SMC4, whereas in mature schizonts, the signal was dispersed throughout the nucleus.
Gametocyte/GameteWe found that SMC2 and SMC4 are expressed during both schizogony and male gametogenesis. During male gametogenesis, the proteins were also dispersed throughout the nucleus. To validate the SMC4 subcellular location, fractionation of cytoplasmic and nuclear extracts derived from purified gametocytes revealed the presence of SMC4 in the nucleus.
Fertilization and ookineteIn addition, we observed SMC4GFP distributed either as dispersed in the nucleus or at a discrete focus adjacent to the DNA throughout the parasite life cycle, including in female gametocytes, in ookinetes, during oocyst development, and in the liver stages
OocystIn addition, we observed SMC4GFP distributed either as dispersed in the nucleus or at a discrete focus adjacent to the DNA throughout the parasite life cycle, including in female gametocytes, in ookinetes, during oocyst development, and in the liver stages
SporozoiteIn addition, we observed SMC4GFP distributed either as dispersed in the nucleus or at a discrete focus adjacent to the DNA throughout the parasite life cycle, including in female gametocytes, in ookinetes, during oocyst development, and in the liver stages
Liver stageIn addition, we observed SMC4GFP distributed either as dispersed in the nucleus or at a discrete focus adjacent to the DNA throughout the parasite life cycle, including in female gametocytes, in ookinetes, during oocyst development, and in the liver stages
Additional remarks phenotype

Mutant/mutation
The mutant expresses a C-terminal GFP-tagged form of SMC2

Protein (function)
An essential component of chromosome dynamics is a family of structural maintenance of chromosomes proteins, originally described in budding yeast as stability of minichromosomes (SMC) proteins, which are implicated in chromosome segregation  and condensation. Most eukaryotes have at least six genes encoding SMC proteins (each 110–170 kDa, with a central hinge region and N- and C-terminal globular domains with Walker A and Walker B motifs forming the ATPase head domain). The six SMCs can be classified as subunits of condensin (SMC2 and SMC4, required for chromosomal condensation), cohesin (SMC1 and SMC3, required for chromosomal segregation), and the SMC5-SMC6 complex (involved in DNA repair and homologous recombination. Higher eukaryotic organisms have two condensin complexes, condensin I and condensin II, whereas many single-celled organisms such as yeast have only one condensin complex.
SMC2 and SMC4 form the core structure for both condensin I and condensin II in higher eukaryotes and interact with three additional non-SMC components: one kleisin and two Heat protein subunits. Kleisin Ig (CAP-H), Heat IA (CAP-D2), and Heat IB (CAP-G) form the condensin I complex, whereas Kleisin IIb (CAP-H2), Heat IIA (CAP-D3), and Heat IIB (CAP-G2) form the condensin II complex. 
Domain analysis of Plasmodium SMC2 and SMC4 revealed a conserved domain architecture for both SMC2 and SMC4 . A comparative sequence analysis revealed low 
sequence similarity and identity (29%–34%), except for the SMC4 homolog in Arabidopsis thaliana (65%), although there was similarity in size and overall domain structure when compared with the proteins in the other studied organisms. 
We found the P. berghei SMC4 N-terminal ATPase domain divided in two by a 44 amino acid insertion; a similar pattern has been observed in other Plasmodium species.
P. berghei SMC2 (PBANKA_1416900), SMC4 (PBANKA_1108700)

Phenotype
We found that SMC2 and SMC4 are expressed during both schizogony and male gametogenesis. In early schizonts within host red blood cells, we observed discrete foci in the parasite cell adjacent to the nuclear DNA for both SMC2 and SMC4, whereas in mature schizonts, the signal was dispersed throughout the nucleus. During male gametogenesis, the proteins were also dispersed throughout the nucleus. To validate the SMC4 subcellular location, fractionation of cytoplasmic and nuclear extracts derived from purified gametocytes revealed the presence of SMC4 in the nucleus. 
In addition, we observed SMC4GFP distributed either as dispersed in the nucleus or at a discrete focus adjacent to the DNA throughout the parasite life cycle, including in female gametocytes, in ookinetes, during oocyst development, and in the liver stages.

Additional information
To examine whether these foci are centromeric or centrosomal, we used two approaches: we performed a colocalization experiment using parasites expressing SMC4GFP crossed with those expressing NDC80mCherry, a kinetochore/centromeric 
marker protein, and we performed immunofluorescence assays using anti-centrin and anti-a-tubulin, together with anti-GFP antibodies. Live imaging using the SMC4GFP and NDC80mCherry genetic cross showed colocalization of SMC4 and NDC80 in early schizonts and discrete foci of NDC80mCherry alone in gametocytes. A similar pattern was observed in ookinetes nd oocysts, with centromeric colocalization of SMC4 and NDC80. In early schizonts, immunofluorescence assays with anti-centrin antibodies revealed that SMC4 is located between centrin and DAPI-stained nuclear DNA, confirming the non-centrosomal localization of SMC4. However, partial colocalization was observed with anti-a-tubulin antibodies in schizonts and during male gametogenesis.

To examine the colocalization of SMC2 and SMC4 proteins, we generated transgenic parasite lines expressing either SMC2mCherry or SMC4GFP and crossed them genetically. The progeny, expressing both SMC2mCherry and SMC4GFP, showed colocalization of the two proteins during schizogony and gametogenesis consistent with SMC2 and SMC4 heterodimer complex formation at both stages.

From the Abstract
'During early schizogony, SMC2/SMC4 localize to a distinct focus, identified as the centromeres by NDC80 fluorescence and chromatin immunoprecipitation sequencing (ChIP-seq) analyses, but do not form condensin I or II complexes. In mature schizonts  and during male gametogenesis, there is a diffuse SMC2/SMC4 distribution on chromosomes and in the nucleus, and both condensin I and condensin II complexes form at these stages. Knockdown of smc2 and smc4 gene expression reveals essential 
roles in parasite proliferation and transmission. The condensin core subunits (SMC2/SMC4) form different complexes and may have distinct functions at various stages of the parasite life cycle.

Other mutants


  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1416900
Gene Model P. falciparum ortholog PF3D7_1318400
Gene productstructural maintenance of chromosomes protein 2, putative
Gene product: Alternative nameSMC2
Details of the genetic modification
Name of the tagGFP
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
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationGFP-tagged vectors were designed using the p277 plasmid vector
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