Characterisation of rat 9‐kDa cholecalcin (CaBP) messenger RNA using a complementary DNA

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Abstract
The rat posesses 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, 13 502–13 505]. Nucleotide sequence analysis of this cDNA shows the presence of two stop codons, TGA and TAG, at positions 207 and 271, respectively, of the 3â€Č 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‐hybridised mRNA from rat kidney and cerebellum was translated in a cell‐free system. Northern blots show that the cDNA pC109 sequence hybridises 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.