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 majo r prolactin (PRL)-regulated protein (cp35). As one goal in understandi ng 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'-flanki ng region and the first six exons. The promoters of cp37 and cp35 are 84% identical over the first 210 bp upstream from the transcription st art point (tsp). Cell-free transcription demonstrated that this -210 r egion of cp37 contained an active promoter. Two nuclear protein-bindin g 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 sequen ces 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 plasm id and transfected into T-47D cells. Transient transfection analysis r evealed that the constructs containing cp35 5'-flanking regions were i nactive, whereas the cp37 promoter was constitutively active and conta ined both positive and negative cis-acting sequences.