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Gene disruptions indicate an essential function for the LmmCRK1 cdc2‐related kinase of Leishmania mexicana
Author(s) -
Mottram Jeremy C.,
McCready B. Philomena,
Brown Kathryn G.,
Grant Karen M.
Publication year - 1996
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1046/j.1365-2958.1996.00136.x
Subject(s) - biology , leishmania mexicana , cyclin dependent kinase 1 , kinase , function (biology) , gene , leishmania , genetics , microbiology and biotechnology , cell cycle , parasite hosting , world wide web , computer science
The generation of homozygous null mutants for the crk1 cdc2‐related kinase of Leishmania mexicana was attempted using targeted gene disruption. Promastigote mutants heterozygous for crk1 were readily isolated with a hyg ‐targeting fragment, but attempts to create null mutants by second‐round transfections with a ble ‐targeting fragment yielded two classes of mutant, neither of which was null. First, the transfected fragment formed an episome; second, the cloned transfectants were found to contain wild‐type crk1 alleles as well as hyg and ble integrations. DNA‐ content analysis revealed that these mutants were triploid or tetraploid. Plasticity in chromosome number following targeting has been proposed as a means by which Leishmania avoids deletion of essential genes. These data support this theory and implicate crk1 as an essential gene, validating CRK1 as a potential drug target. L. mexicana transfected with a Trypanosoma brucei homologue, tbcrk1 , was shown to be viable in an lmmcrk1 null background, thus showing complementation of function between these trypanosomatid genes. The expression of crk1 was further manipulated by engineering a six‐histidine tag at the C‐terminus of the kinase, allowing purification of the active complex by affinity selection on Ni 2+ –nitriloacetic acid (NTA) agarose.