Isolation and characterization of the gene coding for cytosolic phosphoenolpyruvate carboxykinase (GTP) from the rat.

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Abstract
The gene for cytosolic phosphoenolpyruvate carboxykinase (GTP) [GTP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.32] from the rat was isolated from a recombinant library containing the rat genome is phage .lambda. Charon 4A. The isolated clone, .lambda.PCK1, contains the complete gene for phosphoenolpyruvate carboxykinase and .apprxeq. 7 kilobases (kb) of flanking sequence at the 5'' end and 1 kb in at the 3'' terminus. Restriction endonuclease mapping. R-loop mapping and partial DNA sequence assay indicate that the gene is .apprxeq. 6.0 kb in length (coding for a mRNA of 2.8 kb) and contains 8 introns. Southern blotting of rat DNA digested with various restriction enzymes shows a pattern predicted from the restriction map of .lambda. PCK1. A control region at the 5'' end of the gene contained in a 1.2-kb restriction fragment was isolated and subcloned into pBR322. This segment of the gene contains the usual transcription start sequences and a 24-base sequence virtually identical to the sequence found in the 5''-flanking region of the human proopiomelanocortin gene, which is known to be regulated by glucocorticoids. The 1.2-kb fragment of the phosphoenolpyruvate carboxykinase gene can be transcribed into a unique RNA fragment of predicted size by an in vitro transcription assay.