Summary

RMgm-59
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_0616700; Gene model (P.falciparum): PF3D7_0719200; Gene product: NIMA related kinase 4 (serine/threonine protein kinase, Pfnek-4)
Phenotype Fertilization and ookinete;
Last modified: 21 December 2011, 12:39
  *RMgm-59
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene disruption
Reference (PubMed-PMID number) Reference 1 (PMID number) : 15935755
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 cl15cy1
Other information parent lineA reference wild type clone from the ANKA strain of P. berghei (PubMed: PMID: 17406255).
The mutant parasite was generated by
Name PI/ResearcherS.M. Khan, C.J. Janse, A.P. Waters
Name Group/DepartmentLeiden Malaria Research Group
Name InstituteLeiden University Medical Center
CityLeiden
CountryThe Netherlands
Name of the mutant parasite
RMgm numberRMgm-59
Principal name545cl1, 545cl2
Alternative nameNEK4−
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteNot different from wild type
Fertilization and ookineteGamete formation is not affected. Fertilisation occurs, but development is arrested at 2N zygote stage. The defect is female gamete specific; male gametes are fertile and able to fertilize wild-type female gametes.
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of NEK4 protein.

Protein (function)
NEK4 is a serine/threonine NIMA-related kinase; these kinases are mostly described as being involved in controlling/coordinating signalling processes in mitosis.

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).

Phenotype
Cross-fertilization studies with fertile female gametes from an the parasite line P48/45− (that produce defective males and fertile females; RMgm-15) demonstrated that mutant male gametes are fertile and the defect is exclusively female gamete specific.
In the mutant, fertilsation occurs but parasites arrest during zygote development. Flow cytometric analysis of the DNA content of the arrested mutant  zygotes revealed a clear diploid DNA content without meiotic DNA replication after female and male nucleus fusion. This observation indicates that NEK4 is important either just before or during meiosis.

Additional information
Disruption of the P. falciparum ortholog has been succesful (Solyakov et al., 2011, Nat Commun, 2:565) indicating that this gene is not essential for asexual proliferation.
 

Other mutants
An independent P. berghei nek4- mutant (RMgm-60) has been generated that also lacks NEK4 expression and has essentially the same phenotype.


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0616700
Gene Model P. falciparum ortholog PF3D7_0719200
Gene productNIMA related kinase 4
Gene product: Alternative nameserine/threonine protein kinase, Pfnek-4
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct usedPlasmid double cross-over
PlasmoGEM (Sanger) construct/vector usedNo
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
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Plasmid/construct sequence
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"ATCTTATTTTCATAATATATGCACCATATTAATTAACAATTCTATTATTAGATCCCCGAT
TTACCAAATATTTATTCAAAAGAACTCAACAATATATATAAAAGTATGTTAATAAGAGAG
CCCAATTATCGAGTTACTGTTCAGCAATTATTAGTTTCGGATATTGTTCAGGTAATATAT
ATGTATGCTCCTTAAAAAAAAAAAAAATATTCTCAGTAGAAATAAAAACATAGTAACCAA
TTTTAAATTCATTAAAATTGAAACATATATATTTGTTGATTTGTGTATTTATTATTTTTT
AAAATTAATCAGAGACAACTTAAGTTGTTGATTGAAGAAAAAATCAGAGAGAAGCAAAGT
ATGAAAAAACCTTTAAAAGAAAAACAAACTATAGAAAATGATAACTTAGGAGCAAATGAA
CAAGAAGTTAAAACATTATTATTGGATATTGTTGATGTTTGATATGTTATTCTTCCACAA
AAAAAAAGAATATAATATTACAGTCCATACACAGGCATATATATATATATATATATATAT
ACAAATGATTGATAAAAAATATAAATAACAGTATAGATTTCCAAATTTGCAAATCGAAAA
AACTTTCCAGATTTAAGTCATGGAAGAAGGATCCACTAGTTCTAGAGCGGCCGCCACCGC
GGTGGAGCTCCAATTCGCCCTATAGTGAGTCGTATTACGCGCGCTCACTGGCCGTCGTTT
TACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATC
CCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGT
TGCGCAGCCTGAATGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTG
TGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCG
CTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGG
GGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATT
AGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGT
TGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTA
TCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAA
ATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGCTTACAATTT
AGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACA
TTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAA
AAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATT
TTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCA
GTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAG
TTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGC
GGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCA
GAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGT
AAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCT
GACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGT
AACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGA
CACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACT
TACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACC
ACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGA
GCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGT
AGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGA
GATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACT
TTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGA
TAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGT
AGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCA
AACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCT
TTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTA
GCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCT
AATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTC
AAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACA
GCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGA
AAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGG
AACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGT
CGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAG
CCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTT
TGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTT
TGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGA
GGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTA
ATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAA
TGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTAT
GTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTA
CGCCAAGCTCGAAATTAACCCTCACTAAAGGGAACAAAAGCTGGTACCGGGCCCCCCCTC
GAGGTCGACGGTATCGATGAATTCAAGAGTCTTTTGAAAGCCGAGCAAAAGTGTTAAAAT
TGATTAAATATATTTTTTAAAAAAGTGTAAAAAATATATGGGGGTAAGCAAAAGGGAATA
TACATAATATAAATATAGAAACAACATTTGTGAAATGTAACAACCAATAAGTATATTAAG
AAATACTCATTAATAAGAATAAAAAAAAATTTTAGTTCAAAAAAAAAAAATGAAAGATAT
TTTTTAGTATATACACCTAATATTAGGAAACGCCATAAACTTTATCATCAAATCGCTACA
CCGCTTTTTAGGAAAAAAAAATATAATAAATAGAAAGTATGGAAAATGGACAAAAAAAAA
AAATCATAAACTTAATATTATGACTGAAAAATATTTTTTAATAAATTTATATACAAATAT
GTATACTAATTTTTTTGTATTATACAATAATTAATTTTTACTAAACACAATCTATTTAAT
CTACAAAATGTAGTATTATTTTTTATTCACCATATTCATTAATTTTCTCTCTCTATATAT
ATGTAACTATAATATACACACAAAAAAAACAGTATATTTCAACATTGTATTTTTATTCCT
TGGATTGTGTATTGCTCATTCTTTATTATACATATTCTCACAAAATTTTTTTTTATTAAC
TAATATAATTTCCTGTAACCTTACAATTTGAAATTCCTAAAAGCTTGCATGCCTGCAGGT
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
ATTTCGAT
Restriction sites to linearize plasmid ClaI, XbaI
Partial or complete disruption of the geneComplete
Additional remarks partial/complete disruption
Selectable marker used to select the mutant parasitetgdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationIn the same study an independent uncloned nek4- mutant was generated using the same sequences (line 563).
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 1aaagaattcAAGAGTCTTTTGAAAGCCGAGC
Additional information primer 12210EcoRI; Primer F 5'
Sequence Primer 2aaaaagcttTTAGGAATTTCAAATTGTAAGG
Additional information primer 22211HindIII; Primer R 5'
Sequence Primer 3aaagatatcTTATTTTCATAATATATGCACC
Additional information primer 32212EcoRV; Primer F 3'
Sequence Primer 4aaaggatccTTCTTCCATGACTTAAATCTGG
Additional information primer 42213BamHI; Primer R 3'
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6