CHARACTERIZATION OF THE CHICKEN OSTEOPONTIN-ENCODING GENE

A genomic clone of the chicken osteopontin-encoding gene (opn) was isolated and found to be organized as follows: an untranslated 5' exon, a signal peptide; a recognition sequence for phosphorylation by casein kinase II; a domain containing a possible O-linkage site for glycosylation. a second casein kinase II phosphorylation site; an exon containing three functional regions, the poly-Asp sequence of seven consecutive Asp residues, the RGD integrin recognition site and a potential N-linkage site for glycosylation; and a large C-terminal exon which also contains a potential N-linkage site for glycosylation. Primer extension analysis demonstrated only one strong transcriptional start point (tsp) in mRNAs prepared from embryonic bone and cultured osteoblasts. Analysis of the 5' flanking region identified a TATA sequence at -31. an inverted CAAT motif at -57. an AP1-recognition sequence at -84 and a putative vitamin-D-response element (VDRE) sequence at -474. Three plasmid constructs containing 963, 561 and 368 bp of 5' flanking sequence of the avian promoter were used to drive expression of bacterial cat. Comparison of the relative promoter activities of these constructs was carried out in MC3T3/E1 cells, a murine osteoblast cell line. All of the constructs showed approximately 20-fold the levels of expression over background activity of the cat gene without a promoter. Each construct also demonstrated a strong induction with phorbol-12-myristyl-13-acetate (PMA). In contrast, dihydroxycholecalciferol [1.25(OH)2D3] had neither a positive nor a negative effect on the 368- and 936-bp constructs, but was stimulatory for the 561-bp construct. In summary, these results indicate that the functional domains of both the protein-coding exons and the cassette of regulatory sequences of the promoter for the avian opn gene have been conserved and are functional over widely divergent species.