Isolation of cDNA clones coding for the catalytic subunit of mouse cAMP-dependent protein kinase.
Isolation of cDNA clones coding for the catalytic subunit of mouse cAMP-dependent protein kinase.
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DOI：
10.1073/pnas.83.5.1300
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发表时间：
1986-03
影响因子：
11.1
通讯作者：
Michael D. Uhler;D. Carmichael;D. C. Lee;John C. Chrivia;E. Krebs;G. McKnight
中科院分区：
文献类型：
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作者：
Michael D. Uhler;D. Carmichael;D. C. Lee;John C. Chrivia;E. Krebs;G. McKnight
通过多聚体免疫吸附和寡核苷酸(DT)层析，从牛睾丸中部分纯化了cAMP依赖蛋白激酶催化(C)亚单位(ATP：蛋白磷酸转移酶，EC 2.7.1.37)的mRNA。用这种浓缩的mRNA制备方法制备并差异筛选了一个cDNA库。其中一个选定的cDNA克隆被证明是杂交选择的，编码40 kDa蛋白的mRNA，该蛋白与C亚单位的抗体特异性地沉淀。然后用这个牛cDNA克隆来分离一系列与整个小鼠C亚单位mRNA互补的小鼠cDNA克隆。小鼠克隆的蛋白质编码351个氨基酸，与牛C亚单位有98%的同源性，并与小鼠心脏和大脑中2.4kb的单个mRNA杂交。对总基因组DNA的Southern杂交分析表明，只有一个小鼠基因编码C亚基。用SP6聚合酶制备的单链RNA探针对不同组织和细胞系中C亚基和I型调节亚基的mRNA水平进行了定量和比较。
mRNA coding for the catalytic (C) subunit of cAMP-dependent protein kinase (ATP: protein phosphotransferase, EC 2.7.1.37) was partially purified from bovine testis by polysome immunoadsorption and oligo(dT)-chromatography. This enriched mRNA preparation was used to prepare and differentially screen a cDNA library. One of the selected cDNA clones was shown to hybrid-select mRNA coding for a 40-kDa protein that was specifically precipitated with antibodies to the C subunit. This bovine cDNA clone was then used to isolate a series of mouse cDNA clones that are complementary to the entire mouse C subunit mRNA. The mouse clones code for a protein of 351 amino acids that shows 98% homology to the bovine C subunit and hybridize to a single mRNA of 2.4 kilobases in mouse heart and brain. Southern blot analysis of total genomic DNA suggests that there is a single mouse gene coding for the C subunit. mRNA levels for both the C subunit and the type I regulatory subunit in various mouse tissues and cell lines were quantitated and compared by using single-stranded RNA probes prepared with SP6 polymerase.