Characterisation of rat 9-kDa cholecalcin (CaBP) messenger RNA using a complementary DNA: absence of homology with 28-kDa cholecalcin mRNA

The rat possesses two cholecalciferol-induced calcium-binding proteins, the cholecalcins (CaBP). The 9-kDa CaBP is mainly concentrated in the duodenum while 28-kDa CaBP is located in the kidney and cerebellum. The mRNA encoding 9-kDa CaBP has been characterised using the cloned cDNA, pC109, synthesised from rat duodenal 9-kDa CaBP mRNA [Desplan et al. (1983) J. Biol. Chem. 258, 13502-13505]. Nucleotide sequence analysis of this cDNA shows the presence of two stop codons, TGA and TAG, at positions 207 and 271, respectively, of the 3' untranslated region. The cDNA-hybridised mRNA, isolated from rat duodenum, directs the cell-free synthesis of two proteins precipitable by antisera to 9-kDa intestinal CaBP. A major protein comigrates with 9-kDa CaBP whereas a minor product corresponds to a protein which is larger by 2000 Da. The minor protein appears to result from read-through of the 'leaky' UGA stop signal. No protein band which was immunoprecipitable with 28-kDa CaBP antiserum was detected when cDNA-hybridized mRNA from rat kidney and cerebellum was translated in a cell-free system. Northern blots show that the cDNA pC109 sequence hybridizes to a homogeneous mRNA species 500-600 nucleotides long from rat duodenum. Larger mRNA species encoding 28-kDa CaBP are undetectable in rat kidney and cerebellum even under low stringency conditions. All these findings demonstrate that there is no cross-hybridisation between 9-kDa and 28-kDa CaBP mRNAs. Southern blot analysis of rat genomic DNA, that shows only one homologous 9-kDa gene, is consistent with these findings. Thus, all our data indicate that there are distinct genes coding for each rat cholecalcin.