Cloning and analysis of calbindin-D28K cDNA and its expression in the central nervous system

Abstract

The vitamin D-dependent calcium-binding protein (CaBP), calbindin-D28K (CaBP28K), is present in the central nervous system (CNS), the sensory system, and kidneys of mammals and birds. Recent studies have indicated that several other CaBPs of very similar M r s are also present in the CNS. This study was carried out to establish the relationship between CaBP28K and other CaBP, particularly spot 35, to provide a basis for further studies on the tissue-specific regulation and distribution of CaBP28K. A cloned pC28 cDNA was isolated from a rat brain expression library using synthetic oligodeoxyribonucleotides (oligos) complementary to rat spot-35 mRNA. This pC28 cDNA had an open reading frame (ORF) of 783 nucleotides (nt) coding for a 261-aa, 30-kDa protein. There was 100% homology between the pC28 sequence and that of the CaBP28K isolated from rat brain cDNA library using a chicken intestinal CaBP28K probe (Hunziker and Schrickel, 1988). Thus the aa and nt sequences of rat CaBP28K and spot 35 are identical. Primer extension studies and Northern analyses show that the major species of CaBP28K mRNA contains a 5'-untranslated region of 132 nt, a coding region of 261 codons and a 3' -untranslated region of 804 nt without the poly(A) tail. The rat CaBP28K probe hybridizes to one major RNA species (1.9 kb) and two minor ones (2.8 and 3.2 kb) in the cerebellum, hippocampus, retina and kidney. This distribution correlates well with the distribution of CaBP28K itself in these organs. Comparison of the genomic organization of the CaBP28K gene with that of other members of the ‘EF-hand’ CaBP family emphasizes that the CaBP28K gene diverged from the others at the first duplication of the gene encoding one CaBP domain. All the members of the ‘EF-hand’ gene CaBP family evolved by exon shuffling and specific genomic rearrangements.