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2005 Projects

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Browsing 2005 Projects by Subject "Messenger RNA"

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    Effect of dominant negative MKK expression on pH-induced increases in PEPCK mRNA levels
    (Colorado State University. Libraries, 2005) O'Hayre, Morgan, author; Taylor, Lynn, author; Curthoys, Norman P., author
    Acid-base homeostasis is essential for survival. When metabolic acidosis is induced by factors such as prolonged starvation, severe shock, high protein diet, or uncontrolled type I diabetes, the kidneys act to compensate for the decreasing pH. Renal catabolism of glutamine, which is sustained through increased expression of phosphoenolpyruvate carboxykinase (PEPCK) and glutaminase (GA), is activated during metabolic acidosis. Mitogen activated protein kinase kinases three and six (MKK3 and MKK6) are thought to play roles in the signal transduction pathway that lead to enhanced PEPCK and glutaminase activity. To examine the potential roles of MKK3 and MKK6, LLC-PK1-FBPase+ cells were stably transfected with dominant negative (dn) forms of either or both kinases. Expression of the transgenes was controlled by a Tetracycline-responsive promoter element (TRE). Doxycycline (dox) is used to inhibit transcription by preventing the tTA transcription factor from binding to the TRE. The absence of dox then enables transcription and turns on expression of the mutated kinase. Western blots were performed on extracts of clonal cell lines to determine the levels of the MKK isoforms as well as the levels of p38 and phosphorylated p38 in LLC-PK1-FBPase+ cells grown in both the absence and presence of dox. Northern blots were also performed to determine the effect of dnMKK expression on levels of PEPCK mRNA. Expression of both dominant negative kinases, but not the expression of either dnMKK3 or dnMKK6 alone, blocked the acid-induced increases in the levels of PEPCK mRNA and the Anisomycin stimulated increases in levels of phosphorylated p38.
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    The effect of over-expression of ζ-crystallin on glutaminase mRNA stability
    (Colorado State University. Libraries, 2005) Propst, Keri J., author; Taylor, Lynn, author; Lee, Yeon, author; Curthoys, Norman P., author
    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.