Effect of Over Expression of ξ-Crystallin on Glutaminase mRNA Stability

Keri Propst
Colorado State University

During metabolic acidosis, increased renal catabolism of glutamine generates ammonium and bicarbonate ions to partially restore normal acid-base balance. The remaining carbons derived from glutamine are then used to synthesize glucose. This adaptive response is sustained in part by a pH-responsive increase in glutaminase (GA) that results from selective stabilization of the GA mRNA. Previous studies have shown that the 3??-UTR of the GA mRNA contains a pH-response element that consists of a direct repeat of an eight-base AU sequence and that this element binds ξ-crystallin with high affinity and specificity. Increased binding of this protein during metabolic acidosis may initiate the pH-responsive stabilization of the GA mRNA.

A tetracycline-responsive expression system (tet-off) was developed to test the effect of over expression of ξ-crystallin on the expression and the stability of the GA mRNA. Two constructs, pcDNA 3.1-βG-GA??Hygro and pTRE2-ξ-crystallin, were created. The pcDNA 3.1/Hygro vector is designed for high-level, constitutive expression in mammalian cell lines and contains the selectable marker, hygromycin. A chimeric βG-GA cDNA segment that encodes β-globin and the 3??-UTR of the GA mRNA was inserted into the pcDNA 3.1/Hygro vector. The construct, pTRE2-ξ-crystallin contains the tet-responsive element (TRE) that drives the expression of ξ-crystallin. The two plasmids were transfected into 8C cells that express high levels of the tTA protein that binds to and activates transcription from the TRE only in the absence of doxycycline (Dox). Clonal cell lines were selected with hygromycin. These cells will be grown in the presence and absence of Dox and screened with ξ-crystallin specific antibodies to identify clonal lines that exhibit a large induction of ξ-crystallin when grown in the absence of Dox. The selected line will be used to determine the effect of over expression of ξ-crystallin on the levels and the stability of GA mRNA that will be quantified using Real-Time RT-PCR
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