SummaryRMgm-5242
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Successful modification | The 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) : 36040030 |
MR4 number | |
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Parent parasite used to introduce the genetic modification | |
Rodent Malaria Parasite | P. berghei |
Parent strain/line | P. berghei ANKA |
Name parent line/clone | P. berghei ANKA 2.34 |
Other information parent line | P. berghei ANKA 2.34 is a cloned, gametocyte producer line of the ANKA strain (PubMed: PMID: 15137943). |
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The mutant parasite was generated by | |
Name PI/Researcher | Ambekar, SV, Mair GR |
Name Group/Department | Biomedical Sciences, |
Name Institute | Iowa State University |
City | Ames |
Country | USA |
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Name of the mutant parasite | |
RMgm number | RMgm-5242 |
Principal name | Nup221::TurboID |
Alternative name | |
Standardized name | |
Is the mutant parasite cloned after genetic modification | Yes |
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Phenotype | |
Asexual blood stage | Following the successful establishment of TurboID as an efficient in vivo proximity labeling tool in P. berghei, we extended our study to the 4 remaining Plasmodium FG-Nups 205, 221, 313, and 637. Apart from Nup637, each of the FG-Nups has previously been localized by fluorescent protein tagging to the nuclear periphery in P. berghei. Analogous to TurboID-tagging of Nup138, we transfected linearized plasmid constructs promoting integration into the P. berghei genome, thus maintaining expression of each fusion under the control of the endogenous promoter. With similar, coordinated transcription levels of all FG-Nups across the P. berghei intraerythrocytic developmental cycle, Nup205::turboid-HA, Nup221::turboid-HA, Nup313::turboid-HA, and Nup637::turboid-HA could be mapped to the nuclear periphery by anti-HA immunofluorescence assay as well as streptavidin staining. For the first time, we could confirm the localization of Nup637 at the nuclear periphery |
Gametocyte/Gamete | Not tested |
Fertilization and ookinete | Not tested |
Oocyst | Not tested |
Sporozoite | Not tested |
Liver stage | Not tested |
Additional remarks phenotype | Mutant/mutation Protein (function) Phenotype Additional information With an aim to identifying novel Nups, we focused on 8 proteins within our data set that were conserved among Plasmodium spp. but lacked functional annotation: PBANKA_0309200, PBANKA_0309400, PBANKA_0609700, PBANKA_0807900, PBANKA_1211700, PBANKA_1365100, PBANKA_1404700, and PBANKA_1454600. Of those, PBANKA_1211700 and PBANKA_1404700 contained predicted signal peptides and were not pursued any further. Among the remaining candidates, PBANKA_0309200, PBANKA_0309400, and PBANKA_1365100 were similar in spectral abundance (NSAF) to the bait, Nup313. As a first step toward their characterization, we performed C-terminal GFP-tagging with genomic integration to establish their subcellular localization; turboID-HA tagging was used for PBANKA_0309400 as GFP-tagging failed repeatedly (n = 5). Five of the 6 candidate proteins localized to the nuclear periphery typical for FG-repeat nucleoporins in P. berghei. In contrast, PBANKA_0609700 presented a diffuse cytoplasmic localization despite the presence of four predicted transmembrane domains. Apart from the localization to the nuclear periphery, the transcriptome profiles of the new nucleoporins were similar to those of the 5 annotated FG-Nups with the exception of Nup335. We forthwith refer to these newly identified P. berghei nucleoporins according to their predicted molecular weights in kDa as: NUP176 (PBANKA_1365100), NUP269 (PBANKA_1454600), NUP335 (PBANKA_0807900), NUP390 (PBANKA_0309200), NUP434 (PBANKA_0309400). Other mutants
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Details of the target gene | |||||||||||||||||||||||||||
Gene Model of Rodent Parasite | PBANKA_0416300 | ||||||||||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_0905100 | ||||||||||||||||||||||||||
Gene product | nucleoporin NUP221, putative | ||||||||||||||||||||||||||
Gene product: Alternative name | NUP221 | ||||||||||||||||||||||||||
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Details of the genetic modification | |||||||||||||||||||||||||||
Name of the tag | TurboID-3XHA | ||||||||||||||||||||||||||
Details of tagging | C-terminal | ||||||||||||||||||||||||||
Additional remarks: tagging | |||||||||||||||||||||||||||
Commercial source of tag-antibodies | |||||||||||||||||||||||||||
Type of plasmid/construct | (Linear) plasmid single cross-over | ||||||||||||||||||||||||||
PlasmoGEM (Sanger) construct/vector used | No | ||||||||||||||||||||||||||
Modified PlasmoGEM construct/vector used | No | ||||||||||||||||||||||||||
Plasmid/construct map | |||||||||||||||||||||||||||
Plasmid/construct sequence | |||||||||||||||||||||||||||
Restriction sites to linearize plasmid | |||||||||||||||||||||||||||
Selectable marker used to select the mutant parasite | tgdhfr | ||||||||||||||||||||||||||
Promoter of the selectable marker | eef1a | ||||||||||||||||||||||||||
Selection (positive) procedure | pyrimethamine | ||||||||||||||||||||||||||
Selection (negative) procedure | No | ||||||||||||||||||||||||||
Additional remarks genetic modification | For comparison of various biotin ligases, nucleoporin 138 (Nup138) was used as the bait protein. The C-terminal region of the gene was PCR-amplified from P. berghei genomic DNA using Q5 High-Fidelity DNA polymerase. BioID2-3XHA was obtained from Addgene (#74224) and was fused in-frame to the C-terminal end of the Nup138 resulting in plasmid pLIS0574 which also contains the Toxoplasma gondii DHFR selection marker. Each Nup138 tagging construct includes a 1,848 bp fragment encoding the C-terminal region of the 3,714 bp long gene to promote homologous recombination. Similarly, another fusion protein plasmid was made with the same bait protein but with a 13X GGGGS linker between the C-terminus of the protein and BioID2. TurboID-3XHA (Addgene #107171) was fused in-frame to the C-terminal end of the Nup138 using the plasmid pLIS0574; likewise mini-TurboID-3XHA (Addgene #107172) and BirA* (12). As a wild-type control, we used the 2.34 P. berghei clone containing none of the biotin ligases. To ensure that the nuclear periphery-specific signal was from the biotin ligase fused to Nup138, we constructed a plasmid with the promoter sequence of Nup138 and TurboID as a negative control. All cloning was carried out either with T4 DNA ligase or NEBuilder HiFi (NEB). | ||||||||||||||||||||||||||
Additional remarks selection procedure | |||||||||||||||||||||||||||
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