ISOLATION, CHARACTERIZATION AND DISRUPTION OF THE CASEIN KINASE-II ALPHA-SUBUNIT GENE OF LEISHMANIA-CHAGASI

To elucidate the role played by casein kinase II in Leishmania surviva l, we have isolated and characterized the Leishmania chagasi casein ki nase II alpha subunit cDNA, (L.c CKII alpha). The 1083 bp coding regio n is flanked by 148 bp of 5' UTR and 1155 bp of 3' UTR. L.c CKII alpha shows a remarkable degree of similarity with other isolated casein ki nase II alpha subunit sequences. L.c CKII alpha protein is encoded by a single copy gene that transcribes a mRNA of 2.4 kb. The 41.2 kDa L.c CKII alpha protein expressed in vitro has been shown to be catalytica lly active. A single allele disruption of the L.c CKII alpha gene that removes 94 bp from the coding region which contains one of the 15 con served 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 alpha transcript levels over 14-fold and that of an iron supero xide dismutase mRNA by 5-fold. As well, the kinase activity of the sin gle allele disrupted cells showed a 3-fold reduction as compared to th e wild type cells suggesting a decrease in activity of the L.c CkII al pha enzyme. (C) 1998 Elsevier Science B.V. All rights reserved.