Hunziker W, Siebert P D, King M W, Stucki P, Dugaiczyk A, Norman A W
Proc Natl Acad Sci U S A. 1983 Jul;80(14):4228-32. doi: 10.1073/pnas.80.14.4228.
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.
我们构建了一个重组cDNA文库，以促进对维生素D3及其激素活性代谢物1,25 - 二羟基维生素D3在雏鸡肠道中28,000道尔顿维生素D依赖性钙结合蛋白（CaBP）从头生物合成起始过程中的基因组作用的研究。重组质粒通过同聚物加尾和杂交方法制备，使用从维生素D充足（+D）雏鸡的肠黏膜获得的富含多聚腺苷酸（poly(A)）的mRNA作为起始模板。通过使用两种[32P]cDNA探针的比较原位菌落杂交技术对该文库中的9516个克隆进行筛选；这些探针是从维生素D缺乏（-D）雏鸡的肠黏膜总poly(A)-RNA以及通过免疫沉淀来自维生素D充足（+D）雏鸡的多核糖体而特异性富集的雏鸡肠CaBP mRNA的poly(A)-RNA制备的。当比较-D探针与富含CaBP的探针时，我们鉴定出26个克隆始终显示出显著增加的杂交信号。通过杂交选择翻译对这些克隆进行进一步评估，结果显示存在CaBP特异性序列。通过对poly(A)-RNA进行“RNA凝胶”分析，发现三种独立的mRNA种类与一个CaBP克隆杂交；在-D poly(A)-RNA中未发现这些RNA种类中的任何一种。通过这种比较菌落杂交程序，我们能够鉴定出与占总poly(A)-mRNA 0.1%的mRNA相对应的CaBP特异性克隆。使用富集探针与非富集探针的差异菌落杂交程序在筛选与稀有mRNA种类互补的其他cDNA克隆方面应该具有价值。
