5' flanking sequence of the human immediate early responsive gene ccn1*(cyr61) and mapping of polymorphic CA repeat sequence motifs in the human ccn1(cyr61) locus

Aims-The human ccnl (hccn; hcyr61) gene has been identified previously at t he mRNA and protein level as a 1,25-dihydroxyvitamin D, and growth factor r egulated gene in human osteoblasts. This study aimed to analyse genomic clo nes containing the human ccnl (cyr61) gene and to provide the 5 ' flanking region. Methods-Genomic clones were isolated by screening a lambda Library and by a rray filter hybridisations of a genomic library. Sequencing was performed u sing the dye terminator method. Promoter activity was measured after transi ent transfection using a p galactosidase assay. CA repeat motifs were studi ed by a combined PCR/fragment analysis protocol. Results-The human 5 ' flanking region of 870 nucleotides contains several s tretches with high homology to the mouse promoter as well as CA repeat moti fs. This first report on the human 5 ' flanking sequence of the hccn1 (hcyr 61) gene provides important insights into regulation pathways for the expre ssion of this 1,25-dihydroxyvitamin D, and growth factor responsive early g ene. A genomic clone containing the hccn1 (hcyr61) gene region also yielded a CA sequence located 3 ' of the ccnl (cyr61) gene. This CA repeat and one of the CA repeat motifs in the promoter were studied in detail and found t o be polymorphic. Conclusions-The 5 ' flanking sequence of the hccn1 (hcyr61) gene provides i nsights into the mechanisms of regulation of this immediate early gene prod uct. The GA repeat polymorphisms within the gene region will be useful in t he genetic study of disorders affecting bone metabolism.