Mehta N M, El-Gewely M R, Joshi J, Helling R B, Banerjee M R
Gene. 1981 Nov;15(2-3):285-8. doi: 10.1016/0378-1119(81)90138-4.
Casein messenger RNAs (mRNAcsn) were purified from lactating mammary glands of BALB/c mice and used as a starting material for cloning of casein gene sequences. Double-stranded casein cDNA (ds-cDNAcsn) was prepared and blunt-end ligated to HindIII-specific DNA linker molecules. After digestion with HindIII, the dsDNAcsn was inserted into the HindIII site of plasmid pBR322, using T4 DNA ligase. Escherichia coli strain RH202 was transformed with the hybrid plasmids, and transformants were selected for resistance to ampicillin. Electrophoresis of HindIII-digested hybrid plasmid DNAs, followed by Southern transfer and hybridization to [32P]cDNAcsn, revealed that one of the hybrid-plasmid-containing colonies, designated pCas51, contained a 400-bp insert which hybridized to the [32P]cDNAcsn. Purification of the individual casein mRNAs (mRNAcsn alpha, beta, and gamma) and solution hybridization of nick-translated insert DNA to each of these revealed that pCas51 contained sequences complementary primarily to mRNAcsn beta.
从BALB/c小鼠的泌乳乳腺中纯化酪蛋白信使核糖核酸（mRNAcsn），并将其用作克隆酪蛋白基因序列的起始材料。制备双链酪蛋白互补脱氧核糖核酸（ds-cDNAcsn），并将其平端连接到HindIII特异性DNA连接分子上。用HindIII消化后，使用T4 DNA连接酶将dsDNAcsn插入质粒pBR322的HindIII位点。用杂交质粒转化大肠杆菌菌株RH202，并选择对氨苄青霉素具有抗性的转化体。对经HindIII消化的杂交质粒DNA进行电泳，随后进行Southern转移并与[32P]cDNAcsn杂交，结果显示，其中一个含有杂交质粒的菌落（命名为pCas51）含有一个400碱基对的插入片段，该片段与[32P]cDNAcsn杂交。对单个酪蛋白mRNA（mRNAcsnα、β和γ）进行纯化，并将缺口平移的插入DNA与每种mRNA进行溶液杂交，结果显示pCas51包含主要与mRNAcsnβ互补的序列。
