The effect of over-expression of ζ-crystallin on glutaminase mRNA stability

Abstract

During metabolic acidosis, increased renal catabolism of glutaminegenerates ammonium and bicarbonate ions to partially restore normal acid-basebalance. The remaining carbons derived from glutamine are then used to synthesizeglucose. This adaptive response is sustained in part by a pH-responsive increase inglutaminase (GA) that results from selective stabilization of the GA mRNA.Previous studies have shown that the 3’-UTR of the GA mRNA contains a pHresponseelement that consists of a direct repeat of an eight-base AU sequence andthat this element binds ζ-crystallin with high affinity and specificity. Increasedbinding of this protein during metabolic acidosis may initiate the pH-responsivestabilization of the GA mRNA. A tetracycline-responsive expression system (tet-off) was developed to test the effect of over-expression of ζ-crystallin on the expression and the stability of the GA mRNA. Two constructs, pcDNA 3.1-βG-GA–Hygro and pTRE2-ζ-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. Theconstruct, pTRE2-ζ-crystallin contains the tet-responsive element (TRE) that drivesthe expression of ζ-crystallin. The two plasmids were co-transfected into 8C cellsthat express high levels of the tTA protein that binds to and activates transcriptionfrom the TRE only in the absence of doxycycline (Dox). Clonal cell lines wereselected with hygromycin. These cells were grown in the presence and absence ofDox and screened with ζ-crystallin specific antibodies to identify clonal lines thatexhibit a large induction of ζ-crystallin when grown in the absence of Dox. RNAisolated from the selected line was quantified using Real-Time RT-PCR. Theresulting data demonstrate that over-expression of ζ-crystallin does not increase GAmRNA levels.