Cloning and characterization of the cDNA and gene encoding Xenopus laevis osteocalcin

Abstract

A full length Xenopus laevis osteocalcin (bone Gla protein, BGP) has been cloned by a combination of reverse transcription and amplification by the polymerase chain reaction, sequenced, and found to encode a polypeptide with 101 amino acid residues, including a 52-residue prepro-region and a 49-residue mature protein. The N-terminal region of the mature Xenopus BGP (xBGP), as deduced from the cDNA, is in full agreement with the sequence of the BGP previously purified from Xenopus long bones. This cDNA was used to clone the xBGP gene and its promoter region. The xBGP gene spans 3727 bp from the site of transcription initiation corresponding to the 5′end of the cDNA to the site of insertion of the poly-A+ tail, and it contains four exons. This structure is similar to the one obtained for both fish and mammalian BGP genes and indicates that the molecular organization of this gene has been conserved throughout vertebrate evolution. Also similar to other known vertebrate systems, xBGP gene expression is restricted to bone, with no signal for xBGP messenger RNA (mRNA) detected in all other tissues analyzed. The availability of the xBGP promoter will permit to analyze its regulation in a widely used non-mammalian model system for vertebrate development, taking advantage of the availability of sequences for various Xenopus steroid hormone receptors and transcription factors known to affect BGP expression in the mammalian system.