THE PROMOTER REGION OF THE CARBAMOYL-PHOSPHATE SYNTHETASE-III GENE OFSQUALUS-ACANTHIAS

Carbamoyl-phosphate synthetase III (CPSase III) of Squalus acanthias ( spiny dogfish) is a nuclear-encoded mitochondrial enzyme that catalyze s glutamine-dependent formation of carbamoyl phosphate for urea synthe sis. In this paper we report the results of cloning a IO-kb segment of genomic DNA which includes the region flanking the 5' end of the spin y dogfish CPSase III gene. A total of 1,295 base pairs of sequence str addling the start codon was obtained. Primer extension experiments rev ealed that the transcription start site is the G located 114 residues upstream of the translation start codon ATG. The first exon has 240 ba se pairs, including the 5' untranslated region, the coding sequence fo r the signal peptide (38 amino acids), and the four N-terminal amino a cids of the mature enzyme. The boundary of the first exon and the firs t intron of the CPSase III gene is concordant with that of rat and fro g (Rana catesbeiana) CPSase I, which have been suggested to have evolv ed from CPSase m. The putative TATA box sequence, TACAAA, is located a t position -31 with an uncommonly found C at the third position. Two C /EBP binding site sequences, ATTCTGCAAG (-405 to -397) and GTGCAGTAAG (-168 to -160), were identified in the promoter region, which suggests that spiny dogfish CPSase III might be subjected to transactivation o f transcription by C/EBP-related proteins, as has been reported for ra t CPSase I. The preparation and binding of a recombinant RcC/EBP-1 pro tein (the R. catesbeiana homolog of the mammalian C/EBP alpha) to the two spiny dogfish C/EBP binding sequences are described. Two putative heat-shock binding elements were also identified in the promoter regio n.