Molecular cloning of a vitamin D-dependent calcium-binding protein mRNA sequence from chick intestine.

Publisher Website
Google Scholar
Abstract
A recombinant cDNA [complementary DNA] library was constructed to facilitate study of the genomic actions of vitamin D3 and its hormonally active metabolite 1,25-dihydroxyvitamin D3 in initiation of the de novo biosynthesis of a 28,000-dalton vitamin D-dependent Ca binding protein (CaBP) present in chick intestine. The recombinant plasmids were prepared by the homopolymeric tailing and hybridization method using as a starting template poly(A)-enriched mRNA obtained from the intestinal mucosa of vitamin D3-replete (+D) chicks. Screening of 9516 clones in this library was effected by using a comparative in situ colony hybridization technique with 2 [32P]cDNA probes; these probes were prepared from total poly(A)-RNA from chick intestinal mucosa of vitamin D-deficient (-D) chicks and a poly(A)-RNA specifically enriched for chick intestinal CaBP mRNA by immunoprecipitation of polysomes derived from vitamin D-replete (+D) chicks. Clones (26) that consistently displayed a significantly increased hybridization signal when comparing the -D vs. CaBP-enriched probe were identified. Further evaluation of these clones by hybrid-selected translation showed the presence of CaBP-specific sequences. By RNA gel analysis of poly(A)-RNA, 3 independent mRNA species were found to hybridize to a CaBP clone; none of these RNA species were found in -D poly(A)-RNA. With this comparative colony hybridization procedure, CaBP-specific clones corresponding to a mRNA that is 0.1% of the total poly(A)-mRNA were identified. The differential colony hybridization procedure using an enriched vs. a nonenriched probe should be of value in screening for other cDNA clones complementary to rare mRNA species.