Characterization of the 5' end of the growth-regulated Syrian hamster CAD gene

The carbamoylphosphate synthetase-aspartate transcarbamylase-dihydroorotase (CAD) gene encodes a tri-functional protein catalyzing the first three steps in de novo pyrimidine biosynthesis. Studies correlating CAD gene expression with cellular proliferation indicate the importance of understanding the regulation of the CAD gene. As a first step, the structure of the promoter region of the Syrian hamster CAD gene has been determined. Sequence analysis of 1671 base pairs of DNA revealed that the CAD promoter region is very GC rich. Primer extension analysis indicated that the transcription initiation site of the CAD gene is downstream from two GC boxes (consensus binding sites for the transcription factor Sp1). There is no TATA box appropriately spaced upstream from the transcription initiation site. Using RNase protection mapping, S1 nuclease analysis, and comparison to consensus splice donor/acceptor sites, the 5' end of the CAD gene has been determined to consist of a 241-base pair first exon, a 187-base pair first intron, a 140-base pair second exon, and a second intron that extends at least three kilobase pairs. Using conditions optimized for this GC-rich promoter, accurate transcription can be achieved in vitro. Analysis of CAD promoter deletions indicated that sequences extending only 114 base pairs upstream and 225 base pairs downstream from the transcription initiation site are sufficient for accurate and efficient transcription in vitro. DNase I footprinting reactions using this promoter fragment have identified three regions that bind proteins in a HeLa nuclear extract.