Faculty Bibliography

The mRNA coding for rat intestinal calcium-binding protein, a vitamin D3-induced protein (Mr 7500), has been partially purified from growing rat duodenum. Double-stranded DNA synthesized from the purified mRNA preparation was inserted into the PstI site of pBR322, using the oligo(dG-dC) tailing procedure. Clones containing DNA complementary to vitamin D-dependent calcium-binding protein mRNA were selected by differential colony hybridization with [32P] cDNA synthesized from enriched or low vitamin D-dependent calcium-binding protein mRNA preparations. Plasmid DNAs from the selected clones were each verified by both a solution hybrid-arrest assay and a filter hybrid-selection assay. Four recombinant clones showed identical endonuclease restriction maps and contained inserts ranging from 250 to 380 base pairs.

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.