Summary

RMgm-187
Malaria parasiteP. berghei
Genotype
Genetic modification not successful
DisruptedGene model (rodent): PBANKA_1106500; Gene model (P.falciparum): PF3D7_0506900; Gene product: rhomboid protease ROM4 (ROM4)
PhenotypeNo phenotype has been described
Last modified: 16 March 2013, 12:06
  *RMgm-187
Successful modificationThe gene/parasite could not be changed/generated by the genetic modification.
The following genetic modifications were attempted Gene disruption
Number of attempts to introduce the genetic modification 3
Reference (PubMed-PMID number) Reference 1 (PMID number) : 23490234
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).
Attempts to generate the mutant parasite were performed by
Name PI/ResearcherJ. Lin, G.R. Mair, C.J. Janse, S.M. Khan
Name Group/DepartmentLeiden Malaria Research Group
Name InstituteLeiden University Medical Center (LUMC)
CityLeiden
CountryThe Netherlands

  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1106500
Gene Model P. falciparum ortholog PF3D7_0506900
Gene productrhomboid protease ROM4
Gene product: Alternative nameROM4
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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ATCCAATTAATTGAAGAAAAGGCTCGAGAAAAAAAAATAAAAGGTGAAAATTATCGAAAA
AAACAAATAGCTAATAAAGTACATAAAACAGATGCATTACATACTATTATGTCTATTATG
AAGAACAGAATAAATGATGAAGGAAGGCCATCATGTAAAATGAAACCAAGAGAGTGGATT
GTACGTATCACATCTGCGTCCATTTTAATTATATTATGGATCATCTTATTTATATACTTA
CTAGATGAAAGTGCTTATCGATCTTATACACCAATGGGGCAAATTAAATTTAGTGGAGTT
CATTCTTGTTTTTGTTGTGATATTGTTAAAAAAATTCCTTTAACAAAAAATGGAAAATTA
TATTGGTGTTTTAGCAATCAAGAAGCATTTGATTATTATTGCAAGGAATAAACATAAAAT
TTCGGATATATTTATTTCTATTCTTTTTATTCATATATTTGGAATTGTTTATCTAAATTA
TTGTTTCAAGTCAGCAAACAAAAGTAGAATGGAAGTACCATATTAAAATATAAACATGTT
GGTAATATATATCATTTTTATTATGTTGTATGAGAGTACATTGAAGTATATACTATGCTG
GCGCATTTTAAGATAATAAAGGATCCACTAGTTCTAGAGCGGCCGCCACCGCGGTGGAGC
TCCAATTCGCCCTATAGTGAGTCGTATTACGCGCGCTCACTGGCCGTCGTTTTACAACGT
CGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTC
GCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGC
CTGAATGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTT
ACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTC
CCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCT
TTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGAT
GGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCC
ACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTC
TATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTG
ATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGCTTACAATTTAGGTGGCA
CTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATA
TGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGA
GTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTC
CTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTG
CACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCC
CCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTAT
CCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACT
TGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAAT
TATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGA
TCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCC
TTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGA
TGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAG
CTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGC
GCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGT
CTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCT
ACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTG
CCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTG
ATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCA
TGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGA
TCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAA
AACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGA
AGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGT
TAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGT
TACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGAT
AGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCT
TGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCA
CGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAG
AGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTC
GCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGA
AAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACA
TGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAG
CTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGG
AAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCT
GGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTT
AGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTG
GAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGC
TCGAAATTAACCCTCACTAAAGGGAACAAAAGCTGGTACCGGGCCCCCCCTCGAGGTCGA
CGGTATCGATGAATTCTGCATGTATAAGTAATGTGCACATTTTTTGTAATTATTTTTTTT
GAAAATCGATCTTTAAAATTTTATAAAAATCCAAATAATAAGAAAAAATAATTAAAATTA
ATTTTCCAAAAATGCATACTCTTAATTATGTATCTCACTTATTAAGGAATAATTATTTTT
TTTATGTATTCATGAGAGAGTGTACATGATCACAGTTCGTCTTTGTTTTTTTTGTGCAAT
GTAATATATTTAATATATTTTTTTTCCTTATTTCCTTTTTATATATATCCCCATTTTGTT
GGCTTATTCATGTCAATCACTTAGGTCTAACATTTACACAATTTTAAGTAATTTATTGTT
AACATAATTCGGAATATTATTATTGTAATTTTACAATTTTCATTCTGAATATATCTATTT
TGACTTGTTTTGGGCTAATTTTTATCTCATTATATATATATATATATTTACCCTCTGATT
TATCACAATAGTGAAATGAGGGATCAAATATACGCACAATTTTGGTAATAAAAATTAAGG
TAGATTAGGCAAAGCAAATACATTGATAATAAAAAAAAGTGTACACATATATATATATAT
ATATACGTGTGTGTATATAACTACTTGTACACATATTAAGAGTGTAACAGGTTCCGAACG
CAAACATTTATTTGTCTTTATGATATAATGTTCCAACTCTCTTGAATGAAAGCTTGCATG
CCTGCAGGTCAACAATAAATAATAAATAAATATTGTGGAAATAAAATAACATATAATTAT
TTTTAATACATTGATTTCCCTTTTATTTTTTTAAATTTCATTGATATAAAAATATATAAT
AATAACATATATGATTTCAAATTAATCTTTTCAAAAATGGTGTTTATTTTTGTATGTTTG
TGTATGAATTAATCACATAACACATCTATTAAATTGAGTTGGTAATATAGACACAAATAA
ATATATATATTTTTATAGCTTAAAAGTGTGTTATGAATATTTTAAGCATATTTTCTTTTT
CTTTGGATTGTGTAAAATGAACTCATATAATGCGTTTTTTTGTTTTTGTTATTTTGTCAT
TTTGTTATTTTGCTATTTTATGGATTAATTTTTGTTTATAAAATGGGAAATAATTTTAAC
ATATTTAAATAAATGGAGAAAAAATATAAAATAATTATAAAAAAAAGTTAATACACATTT
TTTCCTGTTATAGACCTTATATTTATTTATCCATATATATATATATATATATATATATAT
ATATATACATACCAAGTGAATTAAGAGGAAAGCTAATTTATTATTCAGAATAATATATGA
ACTATATATAATTTTTATTATTTTGGTGTATATTAATCTGTCTATATGCATACATGCAAT
AATTTATCGACTTATATATCAAATAACATAAAATAGAAGTGTTTTAAATTATGGATATAT
GCTCAATATTCATTTTTTTTAATAAGTTAGCTATATTTAAATTATACATTTTATATATGG
TCTCTTTTTTTTTTAAATATTATTTAAGTGATCATGAAAATATAAATAATTTTTTTTTAT
TTAATATCCTTTTGCTTGCATGTGGTAAATGGAAATTTGGATGTGTTTTGAARGTTCGGA
TATAGTTGTATGGACATATAATATATTTTGTGAAAAATTGGTTTTATGTTTATACTTATG
CCAATACTTTTTGAGTAAAACAAAGCAAGTGCTTATAAATAATTAAAGCCAATTTTATAA
TATATATTTTTTTATTTAATTTGAATTTAGTAGTATAATTTTTTATGGTAAGTGCTCAAA
GAGAGTTGCTTATAAAGTATGGTTTGTTTCTTTTTCGCCATTTTGAATTACACATTAAAA
ATATATAGATACATATATTATAATATGAAATCATTAATAATTTAGGGAAATTCTACAAAT
TTAAAAACGAATAAAATAATTGTTTTTCATCATGCCATAACACAATATTGATATATACAT
GTACAAACATTTTTTTTATTTGGAAAATATAAATTATATAAAAAAAAATGTATAGTATAC
AAAATGAGCATATTCACACGGGGTGGACGTTCATTTTTTCATTTTTCCCCTGTTTTTTAT
GAGTATATGATAAAATTTTATGAACATTTACACAAAATGAAAATGGATATATAGGAAAAA
TGGAGCGGTATTTCATTTATCTTTGATTGTCATTTGGATATTATATTACCYTGGGTAGGC
AATTAAAAATGTTAAATAACAATTTAAGGAAATTATATTTTATATATTAAAATTAACACT
GTATTATATGATTCGCTTATAAAAGCCACTCTTTCCCCATGCAAAGCTGTTTAATATCAA
TTTTAACAAATTACACACATGTTAATATATTTATATATATAATTTATATATTTATAATTT
ATATATTTATATTTTTATTATTTATATATTTATTATTTATTGTGTGTGTCAATTCGGGTA
GGATATACCTCTTTTTTATTGTTTAAAGCGATTTGTATTCTAAAATATAAAGRATTTGAA
AAAGAGAAAGATAGAATATGATCCCATCATATATAGCCCTATAATTTTTATTTAGCAGCG
AATTAATTTTTCTATTAAGTTTATGTGTAATTAAAATAACGGAATATATATAATACAATA
AAAAAGTGCATAAATTAAAATTTTTTCAATTAAATTTTTTTTTTTAAGGGGTTATATAAT
ATTAAATATATAAAATACGATTATATATTTTTGCTACAATTTTTTATATTAAGATATAAA
TAGTAAATAAATGGTATTATATGGCATGTAATATATAAATTTTTTCCAATTTTTATTTTA
TATACACTTTTCCTTTTTTTGTCATAAAACTTAAACAATTTACACATTCATTTTAAAAAT
TGACTATTTGTTTCAACATTTTTTGAGTTTCCGTTTTATAATAGTATTTTCATTTGTATA
TTGCTTATATATATAAATACACACCTAAATGTTACAAAGGATCAATGCATAAACCGGTGT
GTCTGGTCGTCGCGATGACCCCCAAGAGGGGCATCGGCATCAACAACGGCCTCCCGTGGC
CCCACTTGACCACAGATTTCAAACACTTTTCTCGTGTGACAAAAACGACGCCCGAAGAAG
CCAGTCGCCTGAACGGGTGGCTTCCCAGGAAATTTGCAAAGACGGGCGACTCTGGACTTC
CCTCTCCATCAGTCGGCAAGAGATTCAACGCCGTTGTCATGGGACGGAAAACCTGGGAAA
GCATGCCTCGAAAGTTTAGACCCCTCGTGGACAGATTGAACATCGTCGTTTCCTCTTCCC
TCAAAGAAGAAGACATTGCGGCGGAGAAGCCTCAAGCTGAAGGCCAGCAGCGCGTCCGAG
TCTGTGCTTCACTCCCAGCAGCTCTCAGCCTTCTGGAGGAAGAGTACAAGGATTCTGTCG
ACCAGATTTTTGTCGTGGGAGGAGCGGGACTGTACGAGGCAGCGCTGTCTCTGGGCGTTG
CCTCTCACCTGTACATCACGCGTGTAGCCCGCGAGTTTCCGTGCGACGTTTTCTTCCCTG
CGTTCCCCGGAGATGACATTCTTTCAAACAAATCAACTGCTGCGCAGGCTGCAGCTCCTG
CCGAGTCTGTGTTCGTTCCCTTTTGTCCGGAGCTCGGAAGAGAGAAGGACAATGAAGCGA
CGTATCGACCCATCTTCATTTCCAAGACCTTCTCAGACAACGGGGTTCCCTACGACTTTG
TGGTTCTCGAGAAGAGAAGGAAGACTGACGACGCAGCCACTGCGGAACCGAGCAACGCAA
TGAGCTCCTTGACGTCCACGAGGGAGACAACTCCCGTGCACGGGTTGCAGGCTCCTTCTT
CGGCCGCAGCCATTGCCCCGGTGTTGGCGTGGATGGACGAAGAAGACCGGAAAAAACGCG
AGCAAAAGGAACTGATTCGGGCCGTTCCGCATGTTCACTTTAGAGGCCATGAAGAGTTCC
AGTACCTTGATCTCATTGCCGACATTATTAACAATGGAAGGACAATGGATGACCGAACGG
GCGTTGGTGTCATCTCCAAATTCGGCTGCACTATGCGCTACTCGCTGGATCAGGCCTTTC
CACTTCTCACCACAAAGCGTGTGTTCTGGAAAGGGGTCCTCGAAGAGTTGCTGTGGTTCA
TTCGCGGCGACACGAACGCAAACCATCTTTCTGAGAAGGGCGTGAAGATCTGGGACAAGA
ATGTGACACGCGAGTTCCTCGATTCGCGCAATCTCCCCCACCGAGAGGTCGGAGACATCG
GCCCGGGCTACGGCTTCCAGTGGAGACACTTCGGCGCGGCATACAAAGACATGCACACAG
ACTACACAGGGCAGGGCGTCGACCAGCTGAAGAATGTGATCCAGATGCTGAGAACGAATC
CAACAGATCGTCGCATGCTCATGACTGCCTGGAATCCTGCAGCGCTGGACGAAATGGCGC
TGCCGCCTTGTCACTTGTTGTGCCAGTTCTACGTGAACGACCAGAAGGAGCTGTCGTGCA
TCATGTATCAGCGGTCGTGCGATGTCGGCCTCGGCGTCCCCTTCAACATCGCTTCCTATT
CGCTTTTGACGCTCATGGTTGCACACGTCTGCAACCTAAAACCTAAGGAGTTCATTCACT
TCATGGGGAACACGCATGTCTACACGAACCATGTCGAGGCTTTAAAAGAGCAGCTGCGGA
GAGAACCGAGACCGTTCCCCATTGTGAACATCCTCAACAAGGAACGCATCAAGGAAATCG
ACGATTTCACCGCCGAGGATTTTGAGGTCGTGGGCTACGTCCCGCACGGACGAATCCAGA
TGGAGATGGCTGTCTAGCGGAAATACAGAAGCTAGCTTTGATCCCGTTTTTCTTACTTAT
ATATTTATACCAATTGATTGTATTTATAACTGTAAAAATGTGTATGTTGTGTGCATATTT
TTTTTTGTGCATGCACATGCATGTAAATAGCTAAAATTATGAACATTTTATTTTTTGTTC
AGAAAAAAAAAACTTTACACACATAAAATGGCTAGTATGAATAGCCATATTTTATATAAA
TTAAATCCTATGAATTTATGACCATATTAAAAATTTAGATATTTATGGAACATAATATGT
TTGAAACAATAAGACAAAATTATTATTATTATTATTATTTTTACTGTTATAATTATGTTG
TCTCTTCAATGATTCATAAATAGTTGGACTTGATTTTTAAAATGTTTATAATATGATTAG
CATAGTTAAATAAAAAAAGTTGAAAAATTAAAAAAAAACATATAAACACAAATGATGTTT
TTTCCTTCAATTTCGAT
Restriction sites to linearize plasmid
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 modificationThe unsuccessful attempts to disrupt the rom4 gene suggest that ROM4 is essential for asexual blood stage development (exp. 653, 684, 695 using DNA construct pL1078)

Rhomboid proteins are intra-membrane proteases that play a role in multiple processes. They belong to a family of serine proteases that cleave cell-surface proteins within their transmembrane domains. The Plasmodium genome encodes a total of 8 rhomboid proteases (ROM1, 3, 4, 6, 7, 8, 9 and 10).

P. falciparum ROM4 has been detected in proteome analyses of asexual blood stages, gametocytes and sporozoites.

In vitro substrates of P. falciparum ROM4 include the merozoite-specific proteins AMA1 (apical membrane antigen 1) and proteins of the EBL (erythrocyte binding ligands) and RBL (reticulocyte binding ligands) families as well as several proteins of the invasive ookinete- and sporozoite-stages, such as TRAP (thrombospondin-related anonymous protein), CTRP (circumsporozoite- and TRAP-related protein) and MAEBL (merozoite adhesive erythrocytic binding protein).

Evidence has been presented for cleavage of of the erythrocyte binding antigen 175 (EBA175) by P. falciparum ROM4 during merozoite invasion.

It has been suggested that ROM4 plays a role in processing of thrombospondin-related anonymous protein (TRAP) at the surface of sporozoites


See RMgm-764 for a P. berghei mutant expressing a (C-terminal) mCherry-tagged version of rhomboid protease ROM4
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 1aaagaattcTGCATGTATAAGTAATGTGC
Additional information primer 1L2408 (EcoRI); 5'
Sequence Primer 2aaaaagcttTCATTCAAGAGAGTTGGAAC
Additional information primer 2L2409 (HindIII); 5'
Sequence Primer 3aaagatatcCAATTAATTGAAGAAAAGGC
Additional information primer 3L2410 (EcoRV); 3'
Sequence Primer 4aaaggatccTTTATTATCTTAAAATGCGC
Additional information primer 4L2411 (BamHI); 3'
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6