Isolation and characterization of the mouse cytosolic phosphoenolpyruvate carboxykinase (GTP) gene: evidence for tissue-specific hypersensitive sites

A 72 kilobase pair DNA fragment that contains the mouse phosphoenolpyruvate carboxykinase (PEPCK) gene locus, pck1, was isolated from a genomic bacter ial artificial chromosome library. The region from similar to -5.5 to +6.6 kilobase pairs relative to the pck1 transcription start site was sequenced and exhibits a high degree of homology to the rat and human genes. Addition ally, the chromatin structure of the PEPCK gene in mouse liver resembles th at seen in rat. Backcross panel analysis of a microsatellite sequence confi rms that the gene is located on chromosome 2. Hypersensitive site analysis was performed on nuclei isolated from the adipocyte cell line 3T3-F442A in the preadipose and adipose states. Several hypersensitive sites are present in the undifferentiated 3T3-F442A cells, before PEPCK mRNA is detected. Th e same sites are present after differentiation, however, the sensitivity of mHS 3 increases relative to the others. We conclude that the chromatin is open in 3T3-F442A cells and that factors are able to bind in the undifferen tiated state but that something else is required for transcription. (C) 199 9 Elsevier Science Ireland Ltd. All rights reserved.