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| Details of the target gene |
| Gene Model of Rodent Parasite |
PBANKA_0520400
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| Gene Model P. falciparum ortholog |
PF3D7_1037500
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| Gene product | dynamin-like protein |
| Gene product: Alternative name | DYN2; Dynamin 2 |
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| Details of the genetic modification |
| Inducable system used | No |
| Additional remarks inducable system |
|
| Type of plasmid/construct used | Plasmid double cross-over |
| PlasmoGEM (Sanger) construct/vector used | No |
| Modified PlasmoGEM construct/vector used | No
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| Plasmid/construct map |
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| Plasmid/construct sequence |
 
GATCCACTAGTTCTAGAGCGGCCGCCACCGCGGTGGAGCTCCAATTCGCCCTATAGTGAG
TCGTATTACGCGCGCTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGC
GTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAA
GAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGGACGCG
CCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACA
CTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTC
GCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCT
TTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCG
CCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTC
TTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGG
ATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCG
AATTTTAACAAAATATTAACGCTTACAATTTAGGTGGCACTTTTCGGGGAAATGTGCGCG
GAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAAT
AACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCC
GTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAA
CGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAAC
TGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGA
TGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAG
AGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCA
CAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCA
TGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAA
CCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGC
TGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAA
CGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAG
ACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCT
GGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCAC
TGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAA
CTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGT
AACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAAT
TTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTG
AGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATC
CTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGG
TTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAG
CGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACT
CTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTG
GCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGC
GGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCG
AACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGG
CGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAG
GGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTC
GATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCT
TTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCC
CTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCC
GAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAAC
CGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACT
GGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCC
AGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAAT
TTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTCGAAATTAACCCTCACTAAA
GGGAACAAAAGCTGGTACCGGGCCCCCCCTCGAGGTCGACGGTATCGATGAATTCTGCAG
CCACTTTGTGTAGCTTACTTCATTTCGCTTTATTACATCAGCGGATTGTCGTATTAATAT
TTACTTTTTTCCATGTTTTTGTGCGTATAATAGATATATACATAACTATATGGATAAATG
TGTATTTTTATCCATTTACCCATTCGTTTATTTTTAAGTACATATATATATTAACAGTTA
ATTGCATCCATACCTATACCTGATAATATTAAAGTAAGAGTTCGTGATAAACTCAGTTAT
ATACAAGTATACAATTCTTTTGGCATCAATAATTTATGTGTAATTATCCAAATCTAAATA
TTCTTTACCATTTGATTCAATGAGTAGTGAATAAAATTAAAAATTGGGTAAAGCACAAAA
TAAAATCAAGAAACGGTTATAGGCAATTCTCATTTTTACTATTTGCAGCGGAAACATACG
GAATATTTCAAGAAAATAATATATTTTTATAGAGATAAATAACGAATTAACCCACTTCTT
ATACAACAGATATACCACATTTTTATAAACCTCATATAAAATGGATAAACTAGTGCCCAT
TGTTAATAAATTACAAAATGTACTTTCGTCATTTATCAGTAGTGAAAGCTTGCATGCCTG
CAGGTCAACAATAAATAATAAATAAATATTGTGGAAATAAAATAACATATAATTATTTTT
AATACATTGATTTCCCTTTTATTTTTTTAAATTTCATTGATATAAAAATATATAATAATA
ACATATATGATTTCAAATTAATCTTTTCAAAAATGGTGTTTATTTTTGTATGTTTGTGTA
TGAATTAATCACATAACACATCTATTAAATTGAGTTGGTAATATAGACACAAATAAATAT
ATATATTTTTATAGCTTAAAAGTGTGTTATGAATATTTTAAGCATATTTTCTTTTTCTTT
GGATTGTGTAAAATGAACTCATATAATGCGTTTTTTTGTTTTTGTTATTTTGTCATTTTG
TTATTTTGCTATTTTATGGATTAATTTTTGTTTATAAAATGGGAAATAATTTTAACATAT
TTAAATAAATGGAGAAAAAATATAAAATAATTATAAAAAAAAGTTAATACACATTTTTTC
CTGTTATAGACCTTATATTTATTTATCCATATATATATATATATATATATATATATATAT
ATACATACCAAGTGAATTAAGAGGAAAGCTAATTTATTATTCAGAATAATATATGAACTA
TATATAATTTTTATTATTTTGGTGTATATTAATCTGTCTATATGCATACATGCAATAATT
TATCGACTTATATATCAAATAACATAAAATAGAAGTGTTTTAAATTATGGATATATGCTC
AATATTCATTTTTTTTAATAAGTTAGCTATATTTAAATTATACATTTTATATATGGTCTC
TTTTTTTTTTAAATATTATTTAAGTGATCATGAAAATATAAATAATTTTTTTTTATTTAA
TATCCTTTTGCTTGCATGTGGTAAATGGAAATTTGGATGTGTTTTGAARGTTCGGATATA
GTTGTATGGACATATAATATATTTTGTGAAAAATTGGTTTTATGTTTATACTTATGCCAA
TACTTTTTGAGTAAAACAAAGCAAGTGCTTATAAATAATTAAAGCCAATTTTATAATATA
TATTTTTTTATTTAATTTGAATTTAGTAGTATAATTTTTTATGGTAAGTGCTCAAAGAGA
GTTGCTTATAAAGTATGGTTTGTTTCTTTTTCGCCATTTTGAATTACACATTAAAAATAT
ATAGATACATATATTATAATATGAAATCATTAATAATTTAGGGAAATTCTACAAATTTAA
AAACGAATAAAATAATTGTTTTTCATCATGCCATAACACAATATTGATATATACATGTAC
AAACATTTTTTTTATTTGGAAAATATAAATTATATAAAAAAAAATGTATAGTATACAAAA
TGAGCATATTCACACGGGGTGGACGTTCATTTTTTCATTTTTCCCCTGTTTTTTATGAGT
ATATGATAAAATTTTATGAACATTTACACAAAATGAAAATGGATATATAGGAAAAATGGA
GCGGTATTTCATTTATCTTTGATTGTCATTTGGATATTATATTACCYTGGGTAGGCAATT
AAAAATGTTAAATAACAATTTAAGGAAATTATATTTTATATATTAAAATTAACACTGTAT
TATATGATTCGCTTATAAAAGCCACTCTTTCCCCATGCAAAGCTGTTTAATATCAATTTT
AACAAATTACACACATGTTAATATATTTATATATATAATTTATATATTTATAATTTATAT
ATTTATATTTTTATTATTTATATATTTATTATTTATTGTGTGTGTCAATTCGGGTAGGAT
ATACCTCTTTTTTATTGTTTAAAGCGATTTGTATTCTAAAATATAAAGRATTTGAAAAAG
AGAAAGATAGAATATGATCCCATCATATATAGCCCTATAATTTTTATTTAGCAGCGAATT
AATTTTTCTATTAAGTTTATGTGTAATTAAAATAACGGAATATATATAATACAATAAAAA
AGTGCATAAATTAAAATTTTTTCAATTAAATTTTTTTTTTTAAGGGGTTATATAATATTA
AATATATAAAATACGATTATATATTTTTGCTACAATTTTTTATATTAAGATATAAATAGT
AAATAAATGGTATTATATGGCATGTAATATATAAATTTTTTCCAATTTTTATTTTATATA
CACTTTTCCTTTTTTTGTCATAAAACTTAAACAATTTACACATTCATTTTAAAAATTGAC
TATTTGTTTCAACATTTTTTGAGTTTCCGTTTTATAATAGTATTTTCATTTGTATATTGC
TTATATATATAAATACACACCTAAATGTTACAAAGGATCAATGCATAAACCGGTGTGTCT
GGTCGTCGCGATGACCCCCAAGAGGGGCATCGGCATCAACAACGGCCTCCCGTGGCCCCA
CTTGACCACAGATTTCAAACACTTTTCTCGTGTGACAAAAACGACGCCCGAAGAAGCCAG
TCGCCTGAACGGGTGGCTTCCCAGGAAATTTGCAAAGACGGGCGACTCTGGACTTCCCTC
TCCATCAGTCGGCAAGAGATTCAACGCCGTTGTCATGGGACGGAAAACCTGGGAAAGCAT
GCCTCGAAAGTTTAGACCCCTCGTGGACAGATTGAACATCGTCGTTTCCTCTTCCCTCAA
AGAAGAAGACATTGCGGCGGAGAAGCCTCAAGCTGAAGGCCAGCAGCGCGTCCGAGTCTG
TGCTTCACTCCCAGCAGCTCTCAGCCTTCTGGAGGAAGAGTACAAGGATTCTGTCGACCA
GATTTTTGTCGTGGGAGGAGCGGGACTGTACGAGGCAGCGCTGTCTCTGGGCGTTGCCTC
TCACCTGTACATCACGCGTGTAGCCCGCGAGTTTCCGTGCGACGTTTTCTTCCCTGCGTT
CCCCGGAGATGACATTCTTTCAAACAAATCAACTGCTGCGCAGGCTGCAGCTCCTGCCGA
GTCTGTGTTCGTTCCCTTTTGTCCGGAGCTCGGAAGAGAGAAGGACAATGAAGCGACGTA
TCGACCCATCTTCATTTCCAAGACCTTCTCAGACAACGGGGTTCCCTACGACTTTGTGGT
TCTCGAGAAGAGAAGGAAGACTGACGACGCAGCCACTGCGGAACCGAGCAACGCAATGAG
CTCCTTGACGTCCACGAGGGAGACAACTCCCGTGCACGGGTTGCAGGCTCCTTCTTCGGC
CGCAGCCATTGCCCCGGTGTTGGCGTGGATGGACGAAGAAGACCGGAAAAAACGCGAGCA
AAAGGAACTGATTCGGGCCGTTCCGCATGTTCACTTTAGAGGCCATGAAGAGTTCCAGTA
CCTTGATCTCATTGCCGACATTATTAACAATGGAAGGACAATGGATGACCGAACGGGCGT
TGGTGTCATCTCCAAATTCGGCTGCACTATGCGCTACTCGCTGGATCAGGCCTTTCCACT
TCTCACCACAAAGCGTGTGTTCTGGAAAGGGGTCCTCGAAGAGTTGCTGTGGTTCATTCG
CGGCGACACGAACGCAAACCATCTTTCTGAGAAGGGCGTGAAGATCTGGGACAAGAATGT
GACACGCGAGTTCCTCGATTCGCGCAATCTCCCCCACCGAGAGGTCGGAGACATCGGCCC
GGGCTACGGCTTCCAGTGGAGACACTTCGGCGCGGCATACAAAGACATGCACACAGACTA
CACAGGGCAGGGCGTCGACCAGCTGAAGAATGTGATCCAGATGCTGAGAACGAATCCAAC
AGATCGTCGCATGCTCATGACTGCCTGGAATCCTGCAGCGCTGGACGAAATGGCGCTGCC
GCCTTGTCACTTGTTGTGCCAGTTCTACGTGAACGACCAGAAGGAGCTGTCGTGCATCAT
GTATCAGCGGTCGTGCGATGTCGGCCTCGGCGTCCCCTTCAACATCGCTTCCTATTCGCT
TTTGACGCTCATGGTTGCACACGTCTGCAACCTAAAACCTAAGGAGTTCATTCACTTCAT
GGGGAACACGCATGTCTACACGAACCATGTCGAGGCTTTAAAAGAGCAGCTGCGGAGAGA
ACCGAGACCGTTCCCCATTGTGAACATCCTCAACAAGGAACGCATCAAGGAAATCGACGA
TTTCACCGCCGAGGATTTTGAGGTCGTGGGCTACGTCCCGCACGGACGAATCCAGATGGA
GATGGCTGTCTAGCGGAAATACAGAAGCTAGCTTTGATCCCGTTTTTCTTACTTATATAT
TTATACCAATTGATTGTATTTATAACTGTAAAAATGTGTATGTTGTGTGCATATTTTTTT
TTGTGCATGCACATGCATGTAAATAGCTAAAATTATGAACATTTTATTTTTTGTTCAGAA
AAAAAAAACTTTACACACATAAAATGGCTAGTATGAATAGCCATATTTTATATAAATTAA
ATCCTATGAATTTATGACCATATTAAAAATTTAGATATTTATGGAACATAATATGTTTGA
AACAATAAGACAAAATTATTATTATTATTATTATTTTTACTGTTATAATTATGTTGTCTC
TTCAATGATTCATAAATAGTTGGACTTGATTTTTAAAATGTTTATAATATGATTAGCATA
GTTAAATAAAAAAAGTTGAAAAATTAAAAAAAAACATATAAACACAAATGATGTTTTTTC
CTTCAATTTCGATATCGCTATTAAAATGTTAGCAGAAATCAGAAACCAAGATATTTAGTT
AAAAATGAAATTAAGAATGTGAATGATTTTTTAAGAAAATTGAAAGGAGGGTTTTGAAAT
TTTTTTTTGTTTACTGTTTTGGATTTTGAGAGATAATGTAGTAATTTAAATGATGAACAA
CTGAACATAAACATATATACAATGGTACTTTTAATATATAATATGTCATATATATGCGTG
CATATCATATCTTATATTTGAATTTTTTATGAATACCAATTTTTTATAATTCTCAAAAAT
ATTATTCATTTTATCAAAGCGATATGAAAAATAATAAATGGTTTTATTTATTATATGTTG
AAATATCGCTTTTTTGTCTTTATTTTTTTTATTTTGTGTAATAAATTTACTTGCACATAT
TTGTGGGTGCAAACAAACTTCCTACTGTAACAATAATATATATGTAATATTTAATATATG
TAAACATTTAAATGAGATATTCATCATTTTTTCTTTTTTCTGACCCTTGAAACTTGGATG
AGCTTGATTATCATTATAACAAAACAAATAATCAATATAAGGATCTGTAAAAGGGTATG
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| Restriction sites to linearize plasmid |
|
| Partial or complete disruption of the gene | Complete |
| Additional remarks partial/complete disruption |
|
| Selectable marker used to select the mutant parasite | tgdhfr |
| Promoter of the selectable marker | pbdhfr |
| Selection (positive) procedure | pyrimethamine |
| Selection (negative) procedure | No |
| Additional remarks genetic modification | Disruption experiments were performed in the Leiden Malaria Research Group (exp. 673, 685, 696; plasmid pl1093 or mg049).
Other mutants:
RMgm-238: unsuccessful attempts to disrupt Dynamin-like protein, DYN1 (PBANKA_090360; Dynamin 1)
Information from Charneau S et al., Microbes Infect 9(7):797-805
The dynamin superfamily has been subdivided into several subgroups based on domain organization: classical dynamins, dynamin-like proteins (Dlps), Mx proteins, optic atrophy 1 protein (OPA1), Mitofusins, guanylate-binding proteins (GBP) and alastatins. All members display a common architecture: a large GTPase domain followed by a middle domain (dynamin_M domain) and a downstream GTPase effector domain (GED), these two latter domains being involved in enzyme-oligomerization and regulation of the GTPase activity. The GTPase domain contains four well conserved GTP-binding motifs required for guanine-nucleotide binding and hydrolysis. Most members contain additional domains that characterize the different subgroups. For example, classical dynamins contain a Pleckstrin-homology (PH) domain between the dynamin_M domain and the GED domain as well as a C-terminal proline-rich domain (PRD) while Dlps lack the PRD but have a PH domain, which may, however, be highly divergent. These various domains confer to dynamins a variety of biochemical properties and cellular localizations, that may explain the diversity of their biological implications in endocytosis, intracellular traffic, organelle fission and fusion, cytokinesis and pathogen resistance.
The amino acid sequence of P. falciparum DYN2 (PF10_0368;709 residues) displays features of dynamin superfamily members including (1) an N-terminal GTPase dynamin domain also called dynamin_N domain, with conserved GTP-binding and GTP-hydrolyzing consensus motifs (G1:G(x)4GKS/T, G2:T, G3:D(x)2G and G4:N/TKxD) (Fig. 1B); (2) a central region corresponding to the dynamin_M domain and (3) a GED at the C-terminal end (Fig. 1A). However, PfDYN2 lacks the C-terminal PRD and possesses a ∼100 aa divergent region in place of the PH domain.
Phylogenetic analysis of the tow P. falciparum dynamins, PfDYN2 and PfDYN1 (PF11_0465) revealed that, although PfDYN2 and PfDYN1 are Dlps, they were grouped in two distant clusters. PfDYN1, proposed to be implicated in vesicular trafficking, clustered with TgDYN1 in a group distinct from those already described. In contrast, PfDYN2 and TgDYN2 clustered with the vast group of Dlps mainly implicated in the division of the mitochondrial outer-membrane that contains Saccharomyces cerevisiae DNM1, human DRP1, Dictyostelium discoideum DYMA, Cyanidioschizon merolae DNM1 and also Trypanosoma brucei DLP.
Architecture and phylogenetic analysis of clearly places PfDYN2 in the Dlps subgroup of dynamins. PfDYN2 displays the main biochemical properties of dynamin superfamily members and localizes only in schizonts, in punctuate structures within the parasite cytoplasm. A partial co-localization was observed between PfDYN2 and markers of the endoplasmic reticulum (ER), the Golgi apparatus and the apicoplast, suggesting that PfDYN2 might be involved in vesicular trafficking as well as organelle segregation occurring during merozoite differentiation. |
| Additional remarks selection procedure | |
Primer information: Primers used for amplification of the target sequences Primer information: Primers used for amplification of the target sequences
| Sequence Primer 1 | aagaattcTGCAGCCACTTTGTGTAGC |
| Additional information primer 1 | 2434 (EcoRI); 5’- targeting region F |
| Sequence Primer 2 | aaaagcttTCACTACTGATAAATGACG |
| Additional information primer 2 | 2435 (HindIII); 5’- targeting region R |
| Sequence Primer 3 | aagatatcGCTATTAAAATGTTAGCAG |
| Additional information primer 3 | 2436 (EcoRV); 3’- targeting region F |
| Sequence Primer 4 | aaggatccAATACCCTTTTACAGATCC |
| Additional information primer 4 | 2437 (BamHI); 3’- targeting region R |
| Sequence Primer 5 | |
| Additional information primer 5 | |
| Sequence Primer 6 | |
| Additional information primer 6 | |
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*RMgm-765