| top of page |
| Details of the target gene |
| Gene Model of Rodent Parasite |
PBANKA_1106500
|
| Gene Model P. falciparum ortholog |
PF3D7_0506900
|
| Gene product | rhomboid protease ROM4 |
| Gene product: Alternative name | ROM4 |
| top of page |
| 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 |
|
| Plasmid/construct sequence |
 
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 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 | The 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 Primer information: Primers used for amplification of the target sequences
| Sequence Primer 1 | aaagaattcTGCATGTATAAGTAATGTGC |
| Additional information primer 1 | L2408 (EcoRI); 5' |
| Sequence Primer 2 | aaaaagcttTCATTCAAGAGAGTTGGAAC |
| Additional information primer 2 | L2409 (HindIII); 5' |
| Sequence Primer 3 | aaagatatcCAATTAATTGAAGAAAAGGC |
| Additional information primer 3 | L2410 (EcoRV); 3' |
| Sequence Primer 4 | aaaggatccTTTATTATCTTAAAATGCGC |
| Additional information primer 4 | L2411 (BamHI); 3' |
| Sequence Primer 5 | |
| Additional information primer 5 | |
| Sequence Primer 6 | |
| Additional information primer 6 | |
|
| top of page |
*RMgm-187