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.
OBJECTIVE
The human ccn1 (hccn; hcyr61) gene has been identified previously at the mRNA and protein level as a 1,25-dihydroxyvitamin D(3) and growth factor regulated gene in human osteoblasts. This study aimed to analyse genomic clones containing the human ccn1 (cyr61) gene and to provide the 5' flanking region.


METHODS
Genomic clones were isolated by screening a lambda library and by array filter hybridisations of a genomic library. Sequencing was performed using the dye terminator method. Promoter activity was measured after transient transfection using a beta galactosidase assay. CA repeat motifs were studied by a combined PCR/fragment analysis protocol.


RESULTS
The human 5' flanking region of 870 nucleotides contains several stretches with high homology to the mouse promoter as well as CA repeat motifs. This first report on the human 5' flanking sequence of the hccn1 (hcyr61) gene provides important insights into regulation pathways for the expression of this 1,25-dihydroxyvitamin D(3) and growth factor responsive early gene. A genomic clone containing the hccn1 (hcyr61) gene region also yielded a CA sequence located 3' of the ccn1 (cyr61) gene. This CA repeat and one of the CA repeat motifs in the promoter were studied in detail and found to be polymorphic.


CONCLUSIONS
The 5' flanking sequence of the hccn1 (hcyr61) gene provides insights into the mechanisms of regulation of this immediate early gene product. The CA repeat polymorphisms within the gene region will be useful in the genetic study of disorders affecting bone metabolism.
Schütze
N
N
Orthopädische Klinik, König-Ludwig-Haus, Labor für Molekulare Experimentelle Orthopädie, Brettreichstrasse 11, 97074 Würzburg, Germany. n-schuetze.klh@mail.uni-wuerzburg.de
Rücker
N
N
Müller
J
J
Adamski
J
J
Jakob
F
F
D002874:Chromosome Mapping; D002999:Clone Cells; D016213:Cyclins; D005656:Fungal Proteins / Q000235:genetics*; D015698:Genomic Library; D006801:Humans; D010783:Photometry; D016133:Polymerase Chain Reaction / Q000379:methods; D011110:Polymorphism, Genetic; D011401:Promoter Regions, Genetic; D017422:Sequence Analysis, DNA; D014162:Transfection; D001616:beta-Galactosidase
D016213:Cyclins; D005656:Fungal Proteins; C077337:CLN1 protein, Candida albicans; D001616:beta-Galactosidase