Cloning and characterization of bovine pyruvate carboxylase and phosphoenolpyruvate carboxykinase

Cansu Agca, Purdue University

Abstract

Our objective was to clone the bovine pyruvate carboxylase (PC) and the cytosolic (C) and mitochondrial (M) forms of phosphoenolpyruvate carboxykinase (PEPCK) and determine their expression during the immediate periparturient interval in dairy cows. Bovine PEPCK-C cDNA contains 2592 nucleotides and contains 84% similarity to the coding sequence of human PEPCK-C cDNA. A 449 nt partial clone of the 3′ end of PEPCK-M is 76% similar to the corresponding sequence of human PEPCKM. Northern blot analysis revealed single transcripts of 2.85 and 2.35 kb for PEPCK-C and PEPCK-M, respectively. The transition to lactation did not alter PEPCK-M transcript expression but expression of PEPCK-C mRNA is increased during lactation indicating that enhanced hepatic gluconeogenesis during lactation is partly due to a change in PEPCK-C. The coding region plus 3′ untranslated region (UTR) of PC mRNA is 3926 bases. A 5′ rapid amplification of cDNA ends protocol was used to clone the 5′ end of the mRNA. Six 5′ UTR which contain 68 (bPC5′ A), 263 (bPC5′B), 363 (bPC5 ′C), 89 (bPC5′D), 275 (bPC5′E), and 178 by (bPC5′ F) were cloned. All PC 5′ UTR variants contain a common coding sequence. The abundance of PC mRNA, determined by Northern blot analysis, indicates that PC is more abundant in gluconeogenic and lipogenic tissues where all PC variants are expressed compared with tissues that do not possess the full spectrum of PC transcripts. One day after parturition, all 5′ UTR variants were increased as was total PC mRNA. Transcripts bPC5′A and bPC5′ B are the most abundant, bPC5′ D, and bPC5′F are intermediate and bPC5′E and bPC5′ C are the least abundant. Results demonstrate regulation of PEPCK at the level of mRNA abundance that is specific for PEPCK-C and regulation of PC that may be coupled to changes in the spectrum of 5′UTR variants expressed.

Degree

Ph.D.

Advisors

Donkin, Purdue University.

Subject Area

Livestock

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