Construction and selection of recombinant plasmids containing full-length complementary DNAs corresponding to rat insulins I and II.
Construction and selection of recombinant plasmids containing full-length complementary DNAs corresponding to rat insulins I and II.
复制标题
含有对应于大鼠胰岛素 I 和 II 的全长互补 DNA 的重组质粒的构建和选择。
DOI：
10.1073/pnas.76.10.5036
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发表时间：
1979
影响因子：
11.1
通讯作者：
D. Steiner
中科院分区：
文献类型：
--
作者：
S. Chan;B. E. Noyes;K. Agarwal;D. Steiner
我们已经使用了合成的脱氧癸核苷酸产生的胰岛素特异性cDNA探针，适合于选择转化体，含有几乎全长cDNA对应的mRNA编码的大鼠胰岛素I和II。从X射线诱导的大鼠胰岛素瘤poly（A）-RNA合成双链cDNA，通过均聚加尾技术插入到pBR 322质粒DNA中，并在大肠杆菌chi 1776中克隆。菌落杂交与阿托伐他汀引发的cDNA产生16个阳性克隆，其中7个对应于大鼠胰岛素I mRNA和9个对应于大鼠胰岛素II mRNA。每组代表性克隆的限制性内切酶图谱表明，这些克隆含有完整的编码序列，这一点通过大鼠胰岛素II克隆DNA 5'区的核苷酸序列分析得到证实。核苷酸序列分析还建立了大鼠前胰岛素原II的前肽的氨基酸序列。大鼠前胰岛素原I和II的前肽的氨基酸序列的比较表明，在该分子的该区域中发生了三个保守的氨基酸取代。
We have used a synthetic deoxydecanucleotide to generate an insulin-specific cDNA probe suitable for selecting transformants that contain nearly full-length cDNAs corresponding to the mRNAs coding for rat insulins I and II. Double-stranded cDNA was synthesized from x-ray-induced rat insulinoma poly(A)-RNA, inserted in pBR322 plasmid DNA by the homopolymeric tailing technique, and cloned in Escherichia coli chi 1776. Colony hybridization with oligonucleotide-primed cDNA yielded 16 positive clones of which 7 corresponded to rat insulin I mRNA and 9 to rat insulin II mRNA. Restriction endonuclease maps of representative clones of each group indicated that these contained the complete coding sequences, as was confirmed by nucleotide sequence analysis of the 5' region of the cloned DNA for rat insulin II. Nucleotide sequence analysis also established the amino acid sequence of the prepeptide of rat preproinsulin II. Comparison of the amino acid sequence of the prepeptides of rat preproinsulin I and II shows that three conservative amino acid substitutions have occurred in this region of the molecule.