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mRNA extracted from rat duodenum, kidney and cerebellum was translated in a cell-free reticulocyte lysate system in the presence of L-[35S]methionine. Vitamin-D-dependent calcium-binding proteins (D-CaBPs) were identified by immunoprecipitation using antibodies specific to duodenal D-CaBP (7500 Mr) and cerebellar D-CaBP (28000 Mr). When duodenal mRNA was translated, the immunoprecipitated polypeptide, obtained using antibodies to duodenal D-CaBP, comigrated with the pure small D-CaBP. Only the addition of unlabeled small duodenal D-CaBP prevented the immunoprecipitation of the major protein. Likewise, when mRNA extracted from the kidney and cerebellum was translated, the product immunoprecipitated by antibodies specific to large mammalian D-CaBP was electrophoretically similar to pure 28000-Mr protein, being displaced only by the addition of unlabeled large D-CaBP. The yield of the duodenal D-CaBP synthesized in the reticulocyte lysate assay was remarkably high (about 10%) compared to that of the large D-CaBP with renal (1%) or cerebellar (0.4%) mRNA. In the absence or presence of microsomal membranes, proteins of similar molecular weight were synthesized, suggesting that the biosynthesis of both large and small D-CaBPs do not involve the processing of leader sequences. Moreover in our experimental conditions duodenal poly(A)-rich RNA was unable to direct the synthesis of large D-CaBP while the mRNAs extracted from kidney and cerebellum did not code for the small D-CaBP. Our data indicate that two distinct mRNAs, coding for small and for large vitamin-D-dependent CaBPs, are expressed in specific tissues of the rat. [ABSTRACT FROM AUTHOR]

The calbindin-D9k (CaBP9k) gene is mainly expressed in differentiated duodenal epithelial cells and is used as a model for studying the molecular mechanisms of intestine-specific transcription. The gene has been cloned, two major DNase-1-hypersensitive sites in the duodenum have been described, and a vitamin-D-response clement has been identified. We have now analysed the transcription factors and regulatory sequences involved in the transcription of the CaBP9k gene in the intestine in ex vivo and in vitro experiments. Transfection experiments in intestinal (CaCo-2) and non-intestinal (HeLa) cell lines defined two regions in the 5'-flanking sequences of the rat CaBP9k gene. A minimal proximal region (-117 to +20) promoted transcription in both intestinal expressing and non-expressing cell lines. Tissue specificity was conferred by the sequences situated further upstream. which are responsible for complete repression in the non-intestinal cells. Intestinal transcription was specified by the proximal region. containing a specialized TATA box, and a distal region, which contains a previously described intestinal DNase-1-hypersensitive site. In vitro DNase I footprinting, electrophoretic mobility shift assays and antibody supershift assays were used to examine the factors bound to the proximal promoter region (-800 to +80 bp). Rat duodenal nuclear extracts protected 12 sites. Sonic of them appear to be binding sites for ubiquitous (nuclear factor 1) or hepatic-enriched sites (hepatocyte nuclear factors 1 and 4, enhancer binding protein α and β) factors. DNA binding studies and transfection experiments indicated that an intestine-specific transcription factor, caudal homeobox-2, binds to the TATA box of the rat CaBP9k gene. These data contribute to our understanding of the control of the intestinal transcription of the CaBP9k gene and demonstrate that several transacting factors, other than the vitamin D receptor. may be factors for intestine-specific CaBP9k gene expression. [ABSTRACT FROM AUTHOR]

The structural organization of the entire rat vitamin-D-dependent calcium-binding protein (9-kDa CaBP) gene was determined by analysis of overlapping genomic clones isolated from a rat genomic library using the rat 9-kDa CaBP cDNA [Desplan C., Heidmann O., Lillie J., Auffray C, and Thomasset M. (1983) J. Biol. Chem. 258, 13502-13505]. These clones together span 30 kbp of rat genomic DNA, with the rat 9-kDa CaBP gene lying in the middle. The 9-kDa CaBP gene is 2.5 kbp long and contains three exons interrupted by two introns. The first exon contains almost the entire 5' untranslated region. The second exon codes for the calcium-binding site I, the third exon codes for site II and the 3' untranslated region. Therefore each of the calcium-binding domains is encoded by single, separate exons. The transcription initiation site was identified by S1 nuclease mapping and primer extension. A consensus sequence TATAAA is localized 31 bp upstream from the cap site and the 'CCAATbox' lies upstream from the transcription start. Single (AC)25 and (AG)23 repeats are present in the second intron together with an Alu-like sequence. Repetitive elements are present 5 kbp upstream from the cap site and in the 3' flanking region. Comparison of the known rat CaBP sequences (9-kDa CaBP, 28-kDa CaBP, S100 protein) shows that the 9-kDa CaBP is more closely related to the S100 protein than to the 28-kDa CaBP. There is no evidence to indicate that 9-kDa CaBP has arisen from the 28-kDa CaBP. [ABSTRACT FROM AUTHOR]

In view of the possible physiological importance of the 9-kDa cholecalcin (a 9000-Mr cholecalciferol-induced calcium-binding protein) in the intestinal transport of calcium in mammals, the gene expression of this protein has been analysed. Its regulation in the digestive tract of the growing rat by calcitriol (1.25-dihydroxycholecalciferol) was studied using a specific cloned [32P]cDNA to 9-kDa cholecalcin. Northern hybridisation studies show that the cDNA sequence hybridises to a single 500–600-nucleotide species throughout the digestive tract and therefore demonstrate identical 9-kDa-cholecalcin mRNA processing in the whole of the intestine and caecum. The highest concentrations of cholecalcin mRNA occur in the duodenum, proximal jejunum and caecum. The observed differences in 9-kDa-cholecalcin mRNA levels correlate well with both the in vivo variations in cholecalcin itself and with the known intestinal sites of calcium absorption. The whole intestine is able to respond to exogenous calcitriol but the response of the distal intestine and caecum, as measured by the increase in cholecalcin mRNA and corresponding protein, was proportionally higher than in the duodenum. The rapid production of fully functional cholecalcin mRNA, which was detectable as early as 1 h after a single dose of calcitriol to vitamin-D-deficient rats, provides convincing evidence that calcitriol increases 9-kDa cholecaicin production by increasing cholecalcin gene expression at the transcriptional level. [ABSTRACT FROM AUTHOR]