Molecular Cloning of Smooth Muscle Calponin h1 in SHeep.

It was found that the level of Calponin h1 (CaP h1) mRNA was significantly up-regulated by Estrogen in the myometrium of sheep towards the end pregnancy. Although the CaP h1 has been widely used as a reference gene to observe the changes of expression level of other genes, the full-length gene in sheep has not been obtained. With the oligo nucleotide primers according to human, mouse and pig CaP h1 mRNA, the full-length cDNA of CaP h1 was cloned by 5′- and 3′-RACE (GenBank accession number = AY327118). The cDNA was 1499 bp in length and contained a complete open reading frame of 891 bp, encoding a protein of 297 amino acid residues. 5′-and 3′-UTR was 79 bp and 529 bp, respectively. With PCR-SSP approaches,the genomic DNA of sheep CaP h1 was obtained.It showed that the gene has 7 exons and 6 introns, spanning over 8 kb(GenBank accession number of introns : AY771807,AY771808, AY771809, AY771810.) Homologous comparison indicated that the cDNA sequences are highly conserved across the species. The highest homology was found in wild pig (92%), followed by human (88%), rat (81%), mouse (81%) and chicken (79%). The intron sequence and length showed a large variation among species((50%).)