Neomycin and puromycin affect gene expression in Giardia lamblia stable transfection.
Two systems for stable transfection of Giardia have been established using selection either by neomycin or by puromycin. We asked if these selection systems themselves influenced expression of endogenous giardial genes. Northern blot analysis showed a approximately 1.4 to approximately 7-fold increase in the encystation-induced cyst wall protein 1 (cwp1), cwp2, and gmyb2 gene transcripts in the drug selected cell lines during vegetative growth, compared with untransfected cells. However, the levels of the constitutive ran, lrp3, or alpha2-tubulin gene transcripts decreased slightly or did not change in these stably transfected cell lines. Part of the effect could be due to drug selection, since treatment of untransfected cells with G418 or puromycin also had similar effects. Nuclear run-on assays showed that part of the effect comes from an increase in transcription initiation rate. The levels of CWP and cyst formation during vegetative growth also increased in the transfected cell lines. Using proteomic technologies, we identified eight genes whose expression is upregulated in neomycin selected cell lines, including phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase, ornithine carbamoyltransferase, carbamate kinase, orf 16424, cyclophilin, co-chaperone-like p21, and bip. Six of these are also upregulated in puromycin selected cell lines. Our results indicate that transfection and drug selection, per se, can alter expression of genes involved in metabolism, protein folding, and differentiation status in Giardia.
Su
Li-Hsin
LH
Department of Parasitology, College of Medicine, National Taiwan University, Taipei 100, Taiwan, ROC.
Lee
Gilbert A
GA
Huang
Yu-Chang
YC
Chen
Yi-Hsiu
YH
Sun
Chin-Hung
CH
D000818:Animals; D015152:Blotting, Northern; D015180:Electrophoresis, Gel, Two-Dimensional; D015870:Gene Expression / Q000187:drug effects*; D005786:Gene Expression Regulation; D016829:Giardia lamblia / Q000187:drug effects*; D009355:Neomycin / Q000494:pharmacology*; D020543:Proteome / Q000032:analysis; D015800:Protozoan Proteins / Q000096:biosynthesis; D011691:Puromycin / Q000494:pharmacology*; D016053:RNA, Protozoan / Q000096:biosynthesis; D014162:Transfection; D015854:Up-Regulation
D020543:Proteome; D015800:Protozoan Proteins; D016053:RNA, Protozoan; D011691:Puromycin; D009355:Neomycin