Kay R M, Harris R, Patient R K, Williams J G
Nucleic Acids Res. 1980 Jun 25;8(12):2691-707. doi: 10.1093/nar/8.12.2691.
This report describes the synthesis and cloning of almost complete DNA copies of the mRNAs encoding the major alpha-globin and major beta-globin of X. laevis. Double-stranded globin cDNA was inserted into the PstI site of the plasmid pBR322 and two cloned recombinants (designated pXG6C1 and pXG8D2) were selected. These were shown to contain almost complete copies of X. laevis globin mRNA. Restriction enzyme maps were determined for each cDNA sequence using the established method of partial digestion of end labelled DNA. However, this procedure was modified such that isolation of individual DNA fragments was no longer required. Each plasmid was shown, by both hybrid arrested translation and filter selection of complementary RNA, to contain a sequence coding for one or other of the two major globin polypeptides. Sufficient DNA sequence information has been determined from each cDNA clone to demonstrate that pXG8D2 contains a beta-globin sequence and pXG6C1 contains an alpha-globin sequence.
本报告描述了编码非洲爪蟾主要α-珠蛋白和主要β-珠蛋白的mRNA的几乎完整DNA拷贝的合成与克隆。双链珠蛋白cDNA被插入到质粒pBR322的PstI位点,并筛选出两个克隆重组体(命名为pXG6C1和pXG8D2)。结果表明,它们含有非洲爪蟾珠蛋白mRNA的几乎完整拷贝。使用已建立的末端标记DNA部分消化方法,确定了每个cDNA序列的限制性酶切图谱。不过,该方法有所改进,不再需要分离单个DNA片段。通过杂交抑制翻译和互补RNA的滤膜筛选,表明每个质粒都含有编码两种主要珠蛋白多肽中一种或另一种的序列。从每个cDNA克隆中已确定了足够的DNA序列信息,以证明pXG8D2含有β-珠蛋白序列,pXG6C1含有α-珠蛋白序列。