Procyclic Trypanosoma brucei cell lines deficient in ornithine decarboxylase activity.
Ornithine decarboxylase (ODC) is a rate limiting enzyme in the biosynthesis of polyamines. We report here the construction of ODC gene deficient Trypanosoma brucei brucei cell lines by homologous recombination and disruption of the two alleles of the ODC gene. With our first stable transfection vector, we replaced the 2.8 kb SacII ODC gene-containing fragment with a hygromycin-B-phosphotransferase gene (hph) cassette transcribed under the control of the endogenous promoter. For the second ODC allele knock-out, we stably transfected similar constructs that contained either the phleomycin or G418 resistance gene cassette, and 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 knockouts were only half of that of the wild type. When grown in the presence of putrescine, the ODC- lines showed little difference, morphologically, from wild type trypanosomes. The growth rate of these lines varied greatly, depending on the concentration of the putrescine. Interestingly, when putrescine was completely withdrawn from the media, the ODC- trypanosomes soon reached a plateau phase and some cells remained viable for 7-8 weeks.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 addition of putrescine or introducing back the ODC gene. Cell cycle analysis suggested that putrescine is required for G1-S transition in the procyclic form T. brucei.
Li
F
F
Department of Medicine, Columbia College of Physicians and Surgeons, New York, NY 10032, USA.
Hua
S B
SB
Wang
C C
CC
Gottesdiener
K M
KM
D000483:Alleles; D000818:Animals; D002453:Cell Cycle; D003470:Culture Media; D017353:Gene Deletion; D015870:Gene Expression; D017125:Genes, Protozoan; D016254:Mutagenesis, Insertional; D009955:Ornithine Decarboxylase / Q000235:genetics / Q000378:metabolism*; D011073:Polyamines / Q000378:metabolism; D011700:Putrescine / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D016053:RNA, Protozoan / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic; D014162:Transfection; D014346:Trypanosoma brucei brucei / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development
D003470:Culture Media; D011073:Polyamines; D012333:RNA, Messenger; D016053:RNA, Protozoan; D009955:Ornithine Decarboxylase; D011700:Putrescine