Episomally driven antisense mRNA abrogates the hyperinducible expression and function of a unique cell surface class I nuclease in the primitive trypanosomatid parasite, Crithidia luciliae.
Here, we show that Crithidia luciliae, a primitive trypanosomatid, purine auxotroph, up-expressed its unique, bi-functional, surface membrane 3'-nucleotidase/nuclease (Cl 3'NT/NU) activity by approximately 1000-fold in response to purine starvation. A second surface membrane phospho-monoesterase, i.e. a tartrate-resistant acid phosphatase (Cl MAcP) was also found to be up-expressed in such purine-starved cells. Here, we used homologous episomal-expression of an antisense construct of the Cl3'NT/NU to dissect the functional expression of these two surface membrane enzymes. In antisense transfected cells, a large excess of the antisense transcript was produced and no trace of any endogenous Cl3'NT/NU sense message was detected. Further, the purine-starvation hyper-induced levels of 3'NT/NU enzyme activity were completely abrogated in these transfected cells versus controls. Moreover, such antisense transcription completely abolished the ability of these transfectants to grow in poly(A)-containing medium demonstrating the essential nature of the 3'NT/NU for the growth/survival of this parasite. In contrast, antisense transcription had no apparent deleterious effects on either endogenous or purine-starvation-induced levels of MAcP enzyme activity, its steady-state mRNA levels, or the constitutive expression of house-keeping genes (e.g. Cl alpha-tubulin) in these transfectants. Cumulatively, results of our antisense experiments demonstrated that the functional nuclease activity of the surface membrane Cl 3'NT/NU was, in fact, critical/essential for the growth and development of these primitive parasites.
Yamage
Mat
M
Cell Biology Section, Laboratory of Parasitic Diseases, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892-0425, USA.
Joshi
Manju B
MB
Dwyer
Dennis M
DM
D000135:Acid Phosphatase / Q000378:metabolism; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D003421:Crithidia / Q000201:enzymology* / Q000378:metabolism; D004790:Enzyme Induction; D009708:Nucleotidases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D016372:RNA, Antisense / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D016053:RNA, Protozoan / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection
D016372:RNA, Antisense; D012333:RNA, Messenger; D016053:RNA, Protozoan; D009708:Nucleotidases; D000135:Acid Phosphatase