Molecular cloning of a vitamin D-dependent calcium-binding protein mRNA sequence from chick intestine.
We have constructed a recombinant cDNA library 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 calcium 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 9,516 clones in this library was effected by using a comparative in situ colony hybridization technique with two [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. We identified 26 clones that consistently displayed a significantly increased hybridization signal when comparing the -D vs. CaBP-enriched probe. Further evaluation of these clones by hybrid-selected translation showed the presence of CaBP-specific sequences. By "RNA gel" analysis of poly(A)-RNA, three 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, we were able to identify CaBP-specific clones corresponding to a mRNA that is 0.1% of the total poly(A)-mRNA. 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.
D000818:Animals; D001483:Base Sequence; D002135:Calcium-Binding Proteins / Q000235:genetics*; D002645:Chickens; D002762:Cholecalciferol / Q000494:pharmacology; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005796:Genes / Q000187:drug effects; D008297:Male; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics*; D064030:S100 Calcium Binding Protein G / Q000235:genetics* / Q000302:isolation & purification; D014808:Vitamin D Deficiency / Q000378:metabolism
D002135:Calcium-Binding Proteins; D012333:RNA, Messenger; D064030:S100 Calcium Binding Protein G; D002762:Cholecalciferol; D004247:DNA