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[牛乳腺mRNA互补DNA的合成与克隆]

[Synthesis and cloning of DNA complementary to mRNA of the bovine mammary gland].

作者信息

Gorodetskiĭ S I, Kiarshulite D R, Ivanov V N, Kapelinskaia T V, Kaledin A S

出版信息

Genetika. 1985 Feb;21(2):191-200.

PMID:2859232
Abstract

Poly(A+)mRNA from bovine mammary glands was used to synthesize double-stranded cDNAs that were subsequently inserted into the plasmid vector pBR322 at the Pst1 site by means of oligo(dG)-oligo(dC) tailing. After transfection of Escherichia coli JC5183, recombinant plasmid library containing 5400 clones was screened by serial rounds of colony hybridization in situ to total [23P] poly(A+)mRNA and electrophoretically homogenious [32P]16SmRNA of mammary glands. Then hybrid selection of mRNA and subsequent in vitro translation of selected mRNAs were performed. In this manner, recombinant clones coding for alpha S1- beta-, kappa-casein were identified. cDNA clones range in size from 35% for beta-casein, 65% for alpha S1-casein to about 95% for kappa-casein, in comparison with their respective mRNAs.

摘要

来自牛乳腺的聚腺苷酸加尾(Poly(A+))mRNA被用于合成双链cDNA,随后通过寡聚(dG)-寡聚(dC)加尾法将其插入质粒载体pBR322的Pst1位点。在转染大肠杆菌JC5183后,通过对总的[23P]聚腺苷酸加尾(Poly(A+))mRNA和乳腺的电泳均一的[32P]16S mRNA进行连续几轮原位菌落杂交筛选,得到了包含5400个克隆的重组质粒文库。然后进行mRNA的杂交选择以及所选mRNA的后续体外翻译。通过这种方式,鉴定出了编码αS1-、β-、κ-酪蛋白的重组克隆。与各自的mRNA相比,cDNA克隆的大小范围为:β-酪蛋白为35%,αS1-酪蛋白为65%,κ-酪蛋白约为95%。

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