The promoter region of the carbamoyl-phosphate synthetase III gene of Squalus acanthias

Abstract

Carbamoyl-phosphate synthetase III (CPSase III) of Squalus acanthias (spiny dogfish) is a nuclear-encoded mitochondrial enzyme that catalyzes glutamine-dependent formation of carbamoyl phosphate for urea synthesis. In this paper we report the results of cloning a 10-kb segment of genomic DNA which includes the region flanking the 5' end of the spiny dogfish CPSase III gene. A total of 1,295 base pairs of sequence straddling the start codon was obtained. Primer extension experiments revealed that the transcription start site is the G located 114 residues upstream of the translation start codon ATG. The first exon has 240 base pairs, including the 5' untranslated region, the coding sequence for the signal peptide (38 amino acids), and the four N-terminal amino acids of the mature enzyme. The boundary of the first exon and the first intron of the CPSase III gene is concordant with that of rat and frog (Rana catesbeiana) CPSase I, which have been suggested to have evolved from CPSase III. The putative TATA box sequence, TACAAA, is located at 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 of transcription by C/EBP-related proteins, as has been reported for rat CPSase I. The preparation and binding of a recombinant RcC/EBP-1 protein (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 region.