Structural and functional divergences of the columbid annexin I-encoding cp37 and cp35 genes

Annexin-I proteins in the pigeon are encoded by two genes. One encodes a constitutively expressed cp37 protein, and the other encodes a major prolactin (PRL)-regulated protein (cp35). As one goal in understanding the differential regulation of these genes, we have determined a 10.8-kb genomic sequence of the cp37 gene containing 1.3 kb of 5'-flanking region and the first six exons. The promoters of cp37 and cp35 are 84% identical over the first 210 bp upstream from the transcription start point (tsp). Cell-free transcription demonstrated that this -210 region of cp37 contained an active promoter. Two nuclear protein-binding sites, which may be involved in the constitutive expression of cp37, were localized between -205 and -106 bp. PRL-dependent transcription factors bind to the -73 to -8 region of cp35, but not cp37. The sequences of these regions contain six single-base differences, which may be responsible for the genes' divergent transcriptional regulation. The 5'-flanking regions of cp37 and cp35 were cloned into a reporter plasmid and transfected into T-47D cells. Transient transfection analysis revealed that the constructs containing cp35 5'-flanking regions were inactive, whereas the cp37 promoter was constitutively active and contained both positive and negative cis-acting sequences.