Isolation, characterization and disruption of the casein kinase II alpha subunit gene of Leishmania chagasi

Abstract

To elucidate the role played by casein kinase II in Leishmania survival, we have isolated and characterized the Leishmania chagasi casein kinase II alpha subunit cDNA, (L.c CKII α). The 1083 bp coding region is flanked by 148 bp of 5′ UTR and 1155 bp of 3′ UTR. L.c CKII α shows a remarkable degree of similarity with other isolated casein kinase II alpha subunit sequences. L.c CKII α protein is encoded by a single copy gene that transcribes a mRNA of 2.4 kb. The 41.2 kDa L.c CKII α protein expressed in vitro has been shown to be catalytically active. A single allele disruption of the L.c CKII α gene that removes 94 bp from the coding region which contains one of the 15 conserved amino acids closest to the carboxy-terminus of the protein has been generated. This mutant is viable and results in a reduction of L.c CKII α transcript levels over 14-fold and that of an iron superoxide dismutase mRNA by 5-fold. As well, the kinase activity of the single allele disrupted cells showed a 3-fold reduction as compared to the wild type cells suggesting a decrease in activity of the L.c CkII α enzyme.