A genomic clone of the chicken osteopontin-encoding gene (opn) was iso
lated 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 glycosyla
tion. a second casein kinase II phosphorylation site; an exon containi
ng three functional regions, the poly-Asp sequence of seven consecutiv
e Asp residues, the RGD integrin recognition site and a potential N-li
nkage site for glycosylation; and a large C-terminal exon which also c
ontains a potential N-linkage site for glycosylation. Primer extension
analysis demonstrated only one strong transcriptional start point (ts
p) in mRNAs prepared from embryonic bone and cultured osteoblasts. Ana
lysis 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 p
utative vitamin-D-response element (VDRE) sequence at -474. Three plas
mid 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 o
ver background activity of the cat gene without a promoter. Each const
ruct 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 const
ructs, but was stimulatory for the 561-bp construct. In summary, these
results indicate that the functional domains of both the protein-codi
ng 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.