STRUCTURE OF THE CANINE PANCREATIC COLIPASE GENE INCLUDES 2 PROTEIN-BINDING SITES IN THE PROMOTER REGION

Characterization of a lambda phage genomic clone, CL5A, which encodes the canine pancreatic colipase gene, revealed the primary structure of 987 nucleotides (nt) of 5'-flanking sequence, 2066 nt defining the pr imary transcriptional unit, which is organized into three exon sequenc es, and 130 nt of 3'-flanking sequence. Exon 1 encodes the amino-termi nal signal peptide, the propeptide (Val1-Pro-Asp-Pro-Arg), and the hyd rophobic lipid-binding region (Gly6-Ile-Ile-Ile) at the amino terminus of the mature coenzyme. Exon 2 encodes carboxylate residues (Glu12 an d Glu15) likely to be involved in binding of pancreatic lipase to coli pase at the aqueous-lipid interface. Exon 3 encodes the hydrophobic se quence (Leu54-Tyr-Gly-Tyr-Tyr) that is essential for binding the centr al tightly structured disulfide-bonded region of the coenzyme to lipid . Southern blot analysis was consistent with the presence of a single- copy colipase gene and a potential colipase gene homologue. Among 16 t issues examined by Northern blot analysis, colipase expression was det ected only in pancreas. Proteins contained in nuclear extracts prepare d from dog pancreas conferred two regions of DNase I protection coinci dent for both coding and noncoding strands (positions -62 to -44 (CL-I site) and -128 to -106 (CL-II site) in the coding strand). Competitio n gel mobility shift experiments indicated that protein-DNA interactio ns that occur at colipase sites I and II are sequence- and protein-spe cific and unrelated to the PAN-binding sequence described in the 5'-en hancer region of the rat chymotrypsin B gene (Nelson, C., Shen, L.P., Meister, A., Fodor, E., and Rutter, W. J. (1990) Genes & Dev. 4, 1035- 1043). Nuclear extracts from pancreas and brain, but not liver, contai n similar CL-I and CL-II-binding proteins. CL-I and CL-II represent pr otein-binding elements that may participate as additional promoter reg ions in regulated expression of the colipase gene. CL-I contains a cen tral homopolymeric d(G) sequence. CL-II shows a GC-rich region on the noncoding strand (5' GGGGGCGTGT 3') that is similar (8/9 match) to the Sp1-binding sequence.