PROCYCLIC TRYPANOSOMA-BRUCEI CELL-LINES DEFICIENT IN ORNITHINE DECARBOXYLASE ACTIVITY

Ornithine decarboxylase (ODC) is a rate limiting enzyme in the biosynt hesis of polyamines. We report here the construction of ODC gene defic ient Trypanosoma brucei brucei cell lines by homologous recombination and disruption of the two alleles of the ODC gene. With our first stab le transfection vector, we replaced the 2.8 kb SacII ODC gene-containi ng fragment with a hygromycin-B-phosphotransferase gene (hph) cassette transcribed under the control of the endogenous promoter. For the sec ond ODC allele knock-out, we stably transfected similar constructs tha t contained either the phleomycin or G418 resistance gene cassette, an d included 1 mM putrescine in the media. These experiments resulted in two separate ODC- lines: one hygromycin and phleomycin resistant, the other hygromycin and G418 resistant. The two ODC gene knockout lines were verified by Southern and Northern hybridization, and confirmed by Western blot and enzymatic activity assay. There is no ODC expression in the two ODC- lines and the ODC messages in the single ODC gene kno ckouts were only half of that of the wild type. When grown in the pres ence of putrescine, the ODC- lines showed little difference, morpholog ically, from wild type trypanosomes. The growth rate of these lines va ried greatly, depending on the concentration of the putrescine. Intere stingly, when putrescine was completely withdrawn from the media, the ODC- trypanosomes soon reached a plateau phase and some cells remained viable for 7-8 weeks. The starved cells could be rescued by the addit ion of putrescine or introducing back the ODC gene. Cell cycle analysi s suggested that putrescine is required for G(1)-S transition in the p rocyclic form T. brucei.