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| Details of the target gene |
| Gene Model of Rodent Parasite |
PBANKA_1443000
|
| Gene Model P. falciparum ortholog |
PF3D7_1228300
|
| Gene product | NIMA related kinase 1 |
| Gene product: Alternative name | nek-1 |
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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
|
| Plasmid/construct map |
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| Plasmid/construct sequence |
 
AATTCTTAATTTACTCTCATTTTATCGTTTTATACATTTTTAATAAAAGCCATTGAATTA
TTATACATATTTTTATATAGTTTTTTAAATTTAATTAATATATTCCATTTATTCTTAAAA
AGCGAGATAGAGAAAAAAAAATCTAGCTATATTTTAATCAAACAACTATAATACACTTTT
TTTTTAACTCATTTTAGCTTTTCTTAATAATGCATATCATTAAAGTGGCGTAAAACATTT
TTAGAGTTTTAAAATATTTATTTTTGTATATAACGAATTGTTTTTTACACAAAGTATTTT
ATTTTAAGGGAAAATAATATTACAAATATATATTTTTTTATTCAGTTTAAAAAAAAATAA
ACAAGTTTCCCCAAAAGATGATGAAAATTAACATAAAAATTACAGTATAAGGCATAAACA
ACAAAGTTATTATTTAATCAATCTCCATTACTATATGGGACATAACACATGCTTGATACA
CAATTATGACCATGTGCTTGGAATAATTCGTAAAAAACAAATAATAGTGAAAAATTAAAA
AAAAATATGAGTAAAACTTAATAAAGCATAATATTCCAAAAAATAACCAAAAATTTTCGA
AAGATAAAAAAATGATAAAGAACAAATTTGAGAGAACATAAAAGTAATAATATTAAAGCA
TAAAACCCCTTATTTAAACATATGTCTCAAATGTGTACATAATATTATGTATTTTAATTG
AGTCAAGTTATACCATCTTTCACAAGTTATATGCTTTTGACTATATTTGTTAAACATTTA
TATATATAGCTTTATTTCATTTTAATTAATATAATATTAAAAAAAAATTGTCTTTTAAAA
AATAAAACGTATATTTTTTTGTTGTTTTTTTAATGTATGTATGCATACTTGTAATGGCAT
ATTAATATTTAATTTATTCCTACAATTTACGGTACATATAAAGCTTGCATGCCTGCAGGT
CAACAATAAATAATAAATAAATATTGTGGAAATAAAATAACATATAATTATTTTTAATAC
ATTGATTTCCCTTTTATTTTTTTAAATTTCATTGATATAAAAATATATAATAATAACATA
TATGATTTCAAATTAATCTTTTCAAAAATGGTGTTTATTTTTGTATGTTTGTGTATGAAT
TAATCACATAACACATCTATTAAATTGAGTTGGTAATATAGACACAAATAAATATATATA
TTTTTATAGCTTAAAAGTGTGTTATGAATATTTTAAGCATATTTTCTTTTTCTTTGGATT
GTGTAAAATGAACTCATATAATGCGTTTTTTTGTTTTTGTTATTTTGTCATTTTGTTATT
TTGCTATTTTATGGATTAATTTTTGTTTATAAAATGGGAAATAATTTTAACATATTTAAA
TAAATGGAGAAAAAATATAAAATAATTATAAAAAAAAGTTAATACACATTTTTTCCTGTT
ATAGACCTTATATTTATTTATCCATATATATATATATATATATATATATATATATATACA
TACCAAGTGAATTAAGAGGAAAGCTAATTTATTATTCAGAATAATATATGAACTATATAT
AATTTTTATTATTTTGGTGTATATTAATCTGTCTATATGCATACATGCAATAATTTATCG
ACTTATATATCAAATAACATAAAATAGAAGTGTTTTAAATTATGGATATATGCTCAATAT
TCATTTTTTTTAATAAGTTAGCTATATTTAAATTATACATTTTATATATGGTCTCTTTTT
TTTTTAAATATTATTTAAGTGATCATGAAAATATAAATAATTTTTTTTTATTTAATATCC
TTTTGCTTGCATGTGGTAAATGGAAATTTGGATGTGTTTTGAARGTTCGGATATAGTTGT
ATGGACATATAATATATTTTGTGAAAAATTGGTTTTATGTTTATACTTATGCCAATACTT
TTTGAGTAAAACAAAGCAAGTGCTTATAAATAATTAAAGCCAATTTTATAATATATATTT
TTTTATTTAATTTGAATTTAGTAGTATAATTTTTTATGGTAAGTGCTCAAAGAGAGTTGC
TTATAAAGTATGGTTTGTTTCTTTTTCGCCATTTTGAATTACACATTAAAAATATATAGA
TACATATATTATAATATGAAATCATTAATAATTTAGGGAAATTCTACAAATTTAAAAACG
AATAAAATAATTGTTTTTCATCATGCCATAACACAATATTGATATATACATGTACAAACA
TTTTTTTTATTTGGAAAATATAAATTATATAAAAAAAAATGTATAGTATACAAAATGAGC
ATATTCACACGGGGTGGACGTTCATTTTTTCATTTTTCCCCTGTTTTTTATGAGTATATG
ATAAAATTTTATGAACATTTACACAAAATGAAAATGGATATATAGGAAAAATGGAGCGGT
ATTTCATTTATCTTTGATTGTCATTTGGATATTATATTACCYTGGGTAGGCAATTAAAAA
TGTTAAATAACAATTTAAGGAAATTATATTTTATATATTAAAATTAACACTGTATTATAT
GATTCGCTTATAAAAGCCACTCTTTCCCCATGCAAAGCTGTTTAATATCAATTTTAACAA
ATTACACACATGTTAATATATTTATATATATAATTTATATATTTATAATTTATATATTTA
TATTTTTATTATTTATATATTTATTATTTATTGTGTGTGTCAATTCGGGTAGGATATACC
TCTTTTTTATTGTTTAAAGCGATTTGTATTCTAAAATATAAAGRATTTGAAAAAGAGAAA
GATAGAATATGATCCCATCATATATAGCCCTATAATTTTTATTTAGCAGCGAATTAATTT
TTCTATTAAGTTTATGTGTAATTAAAATAACGGAATATATATAATACAATAAAAAAGTGC
ATAAATTAAAATTTTTTCAATTAAATTTTTTTTTTTAAGGGGTTATATAATATTAAATAT
ATAAAATACGATTATATATTTTTGCTACAATTTTTTATATTAAGATATAAATAGTAAATA
AATGGTATTATATGGCATGTAATATATAAATTTTTTCCAATTTTTATTTTATATACACTT
TTCCTTTTTTTGTCATAAAACTTAAACAATTTACACATTCATTTTAAAAATTGACTATTT
GTTTCAACATTTTTTGAGTTTCCGTTTTATAATAGTATTTTCATTTGTATATTGCTTATA
TATATAAATACACACCTAAATGTTACAAAGGATCAATGCATAAACCGGTGTGTCTGGTCG
TCGCGATGACCCCCAAGAGGGGCATCGGCATCAACAACGGCCTCCCGTGGCCCCACTTGA
CCACAGATTTCAAACACTTTTCTCGTGTGACAAAAACGACGCCCGAAGAAGCCAGTCGCC
TGAACGGGTGGCTTCCCAGGAAATTTGCAAAGACGGGCGACTCTGGACTTCCCTCTCCAT
CAGTCGGCAAGAGATTCAACGCCGTTGTCATGGGACGGAAAACCTGGGAAAGCATGCCTC
GAAAGTTTAGACCCCTCGTGGACAGATTGAACATCGTCGTTTCCTCTTCCCTCAAAGAAG
AAGACATTGCGGCGGAGAAGCCTCAAGCTGAAGGCCAGCAGCGCGTCCGAGTCTGTGCTT
CACTCCCAGCAGCTCTCAGCCTTCTGGAGGAAGAGTACAAGGATTCTGTCGACCAGATTT
TTGTCGTGGGAGGAGCGGGACTGTACGAGGCAGCGCTGTCTCTGGGCGTTGCCTCTCACC
TGTACATCACGCGTGTAGCCCGCGAGTTTCCGTGCGACGTTTTCTTCCCTGCGTTCCCCG
GAGATGACATTCTTTCAAACAAATCAACTGCTGCGCAGGCTGCAGCTCCTGCCGAGTCTG
TGTTCGTTCCCTTTTGTCCGGAGCTCGGAAGAGAGAAGGACAATGAAGCGACGTATCGAC
CCATCTTCATTTCCAAGACCTTCTCAGACAACGGGGTTCCCTACGACTTTGTGGTTCTCG
AGAAGAGAAGGAAGACTGACGACGCAGCCACTGCGGAACCGAGCAACGCAATGAGCTCCT
TGACGTCCACGAGGGAGACAACTCCCGTGCACGGGTTGCAGGCTCCTTCTTCGGCCGCAG
CCATTGCCCCGGTGTTGGCGTGGATGGACGAAGAAGACCGGAAAAAACGCGAGCAAAAGG
AACTGATTCGGGCCGTTCCGCATGTTCACTTTAGAGGCCATGAAGAGTTCCAGTACCTTG
ATCTCATTGCCGACATTATTAACAATGGAAGGACAATGGATGACCGAACGGGCGTTGGTG
TCATCTCCAAATTCGGCTGCACTATGCGCTACTCGCTGGATCAGGCCTTTCCACTTCTCA
CCACAAAGCGTGTGTTCTGGAAAGGGGTCCTCGAAGAGTTGCTGTGGTTCATTCGCGGCG
ACACGAACGCAAACCATCTTTCTGAGAAGGGCGTGAAGATCTGGGACAAGAATGTGACAC
GCGAGTTCCTCGATTCGCGCAATCTCCCCCACCGAGAGGTCGGAGACATCGGCCCGGGCT
ACGGCTTCCAGTGGAGACACTTCGGCGCGGCATACAAAGACATGCACACAGACTACACAG
GGCAGGGCGTCGACCAGCTGAAGAATGTGATCCAGATGCTGAGAACGAATCCAACAGATC
GTCGCATGCTCATGACTGCCTGGAATCCTGCAGCGCTGGACGAAATGGCGCTGCCGCCTT
GTCACTTGTTGTGCCAGTTCTACGTGAACGACCAGAAGGAGCTGTCGTGCATCATGTATC
AGCGGTCGTGCGATGTCGGCCTCGGCGTCCCCTTCAACATCGCTTCCTATTCGCTTTTGA
CGCTCATGGTTGCACACGTCTGCAACCTAAAACCTAAGGAGTTCATTCACTTCATGGGGA
ACACGCATGTCTACACGAACCATGTCGAGGCTTTAAAAGAGCAGCTGCGGAGAGAACCGA
GACCGTTCCCCATTGTGAACATCCTCAACAAGGAACGCATCAAGGAAATCGACGATTTCA
CCGCCGAGGATTTTGAGGTCGTGGGCTACGTCCCGCACGGACGAATCCAGATGGAGATGG
CTGTCTAGCGGAAATACAGAAGCTAGCTTTGATCCCGTTTTTCTTACTTATATATTTATA
CCAATTGATTGTATTTATAACTGTAAAAATGTGTATGTTGTGTGCATATTTTTTTTTGTG
CATGCACATGCATGTAAATAGCTAAAATTATGAACATTTTATTTTTTGTTCAGAAAAAAA
AAACTTTACACACATAAAATGGCTAGTATGAATAGCCATATTTTATATAAATTAAATCCT
ATGAATTTATGACCATATTAAAAATTTAGATATTTATGGAACATAATATGTTTGAAACAA
TAAGACAAAATTATTATTATTATTATTATTTTTACTGTTATAATTATGTTGTCTCTTCAA
TGATTCATAAATAGTTGGACTTGATTTTTAAAATGTTTATAATATGATTAGCATAGTTAA
ATAAAAAAAGTTGAAAAATTAAAAAAAAACATATAAACACAAATGATGTTTTTTCCTTCA
ATTTCGATGGGTGAGAAAATATGGGGAATACAGAAAATTATTTTGATTCTTTCCCCTTTA
TTTCCCTTTTTGCCATTTAATTGTTATTATTATATATATTTTTTTTTCTGTTCATTAAAT
GGCTTAATTATGACATAAAATATTTCATTATGTGGCATTTATTATACTCTATCATTATTA
TATATATTTAGATATCGCTACCCACGATATTATATGAAATAGTTTTGCTTCATTCTCTAT
TTTTTCTTCTTGTTATTACACATTTTATCATATATATATTAATATTGTTATTTACATATT
ATGGAAATATAAATGCACTTAATTTTTGGAACGAATGCGAAAAATATCTATGATATATAT
ATGCATATCGTTCCATATATACACACATACACATGTATGTATGTATGTATACTTGCCCGT
GGTACATAATATTTACATATTTGTGCATGCTAATATAAATTCTTCTAGTTTAATTTGTTT
TGGTTTCATAATAAAATGTTGTTTATTTTATTAAACATAATTTTTCCCATTTTATGCTTA
ACATTTTTATTTTATACTATTTATTTTATTTTTAATTTGGCGATGTGGCAACAGAGATTC
TTAAGTTAAACACGTATTATAATGGTATGTGATTTTCAAATTTCATTTTTCATAGTTCAA
TTCCTTTTTGCGGTTTTTTAAGCTTAGTTTTTCTGACTGATTAATTTTAGCGTGCATATA
TAATGTATGCAGGATCCACTAGTTCTAGAGCGGCCGCCACCGCGGTGGAGCTCCAATTCG
CCCTATAGTGAGTCGTATTACGCGCGCTCACTGGCCGTCGTTTTACAACGTCGTGACTGG
GAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGG
CGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGC
GAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGC
GTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTT
CTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTC
CGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGT
AGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTT
AATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTT
GATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAA
AAATTTAACGCGAATTTTAACAAAATATTAACGCTTACAATTTAGGTGGCACTTTTCGGG
GAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGC
TCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTA
TTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTG
CTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGG
GTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAAC
GTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTG
ACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGT
ACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTG
CTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGAC
CGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTT
GGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAG
CAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGC
AACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCC
TTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTA
TCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGG
GGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGA
TTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAAC
TTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAA
TCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGAT
CTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGC
TACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTG
GCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACC
ACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGG
CTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGG
ATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAA
CGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCG
AAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGA
GGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCT
GACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCA
GCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTC
CTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCG
CTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCC
CAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACA
GGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTC
ATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGA
GCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTCGAAATTA
ACCCTCACTAAAGGGAACAAAAGCTGGTACCGGGCCCCCCCTCGAGGTCGACGGTATCGA
TG
|
| 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. 583, 607, 628, 660; pl1024).
The NIMA-related protein kinases (Neks) constitute an extended family of eukaryotic mitotic serine/threonine kinases. The best characterized members of the Nek family include NIMA (never in mitosis/Aspergillus), the founding member from the fungus Aspergillus nidulans, and its closest homologue in mammals, Nek2. Initially identified as a kinase essential for mitotic entry in Aspergillus, NIMA has been also shown to participate in nuclear membrane fission. Eleven members of the NIMA kinase family (Nek1-11) have now been identified in various human tissues, and together fulfil a number of cell cycle-related functions in centrosome separation, mitosis, meiosis and checkpoint control. The P. falciparum kinome includes four NIMA-related serine/threonine kinases. Pfnek-1 (PlasmoDB identifier PFL1370w) clusters within the Aspergillus NIMA/human Nek2 branch in phylogenetic trees, while clear orthology to mammalian or yeast Neks could not be assigned for the three other P. falciparum sequences (Pfnek-2, -3 and -4, PlasmoDB identifiers PFE1290w, PFL0080c and MAL7P1.100,respectively).
The unsuccessful attempts to disrupt this gene indicates an essential function during blood stage development.
See also RMgm-564 for unsuccessful attempts to disrupt PBANKA_144300
Disruption of the P. falciparum ortholog has been attempted (Solyakov et al., 2011, Nat Commun, 2:565).
The gene is likely essential for asexual proliferation as integration of the KO vector was not achieved. Accesibility of the locus to recombination was verified by C-terminal tagging. |
| 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 | L2218 (EcoRI); 5' |
| Additional information primer 1 | aaagaattcTTAATTTACTCTCATTTTATCG |
| Sequence Primer 2 | L2219 (HindIII); 5' |
| Additional information primer 2 | aaaaagcttTATATGTACCGTAAATTGTAGG |
| Sequence Primer 3 | L2220 (XmaI); 3' |
| Additional information primer 3 | aaacccgggTGAGAAAATATGGGGAATACAG |
| Sequence Primer 4 | L2221 (KpnI); 3' |
| Additional information primer 4 | aaaggatccTGCATACATTATATATGCACGC |
| Sequence Primer 5 | |
| Additional information primer 5 | |
| Sequence Primer 6 | |
| Additional information primer 6 | |
|
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*RMgm-247