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牛cAMP依赖性蛋白激酶I型调节亚基cDNA克隆的分离

Isolation of a cDNA clone for the type I regulatory subunit of bovine cAMP-dependent protein kinase.

作者信息

Lee D C, Carmichael D F, Krebs E G, McKnight G S

出版信息

Proc Natl Acad Sci U S A. 1983 Jun;80(12):3608-12. doi: 10.1073/pnas.80.12.3608.

DOI:10.1073/pnas.80.12.3608
PMID:6190178
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC394099/
Abstract

A cDNA clone for the type I regulatory subunit (RI) of cAMP-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) was isolated from bovine testis by a differential screening method. mRNA coding for RI was enriched 50- to 100-fold by polysome immunoadsorption chromatography with affinity-purified rabbit anti-RI and protein A-Sepharose. Poly(A)+ RNA from these polysomes was utilized to construct a cDNA library in pBR322, and this library was screened for hybridization to 32P-labeled cDNAs synthesized from either total or RI-enriched poly(A)+ RNA. Plasmids isolated from colonies showing preferential hybridization to the latter probe were further characterized by hybrid selection and DNA sequence analysis. One of these plasmids (designated 62C12) contains a 1,350-nucleotide insert that hybridized to RI mRNA; partial nucleotide sequence analysis confirmed its identity and indicated that it may contain the entire RI coding region. We also have identified a recombinant plasmid with a 1,550-nucleotide insert that selected through hybridization a mRNA coding for a 55,000-dalton protein that crossreacts with anti-RI antibodies. The function of this latter protein is unknown.

摘要

通过差异筛选法从牛睾丸中分离出一种编码环磷酸腺苷（cAMP）依赖性蛋白激酶（ATP：蛋白磷酸转移酶，EC 2.7.1.37）I型调节亚基（RI）的cDNA克隆。利用亲和纯化的兔抗RI抗体和蛋白A-琼脂糖通过多核糖体免疫吸附色谱法将编码RI的mRNA富集50至100倍。来自这些多核糖体的聚腺苷酸加尾（Poly(A)+）RNA被用于构建pBR322中的cDNA文库，并且筛选该文库以使其与由总聚腺苷酸加尾（Poly(A)+）RNA或RI富集的聚腺苷酸加尾（Poly(A)+）RNA合成的32P标记的cDNA杂交。从与后一种探针显示优先杂交的菌落中分离出的质粒通过杂交选择和DNA序列分析进一步表征。其中一个质粒（命名为62C12）含有一个1350个核苷酸的插入片段，该片段与RI mRNA杂交；部分核苷酸序列分析证实了它的身份，并表明它可能包含整个RI编码区。我们还鉴定出一个具有1550个核苷酸插入片段的重组质粒，该质粒通过杂交选择出一种编码与抗RI抗体发生交叉反应的55000道尔顿蛋白的mRNA。后一种蛋白的功能尚不清楚。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/20ad/394099/2bac1380570d/pnas00638-0077-c.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/20ad/394099/221236b18a0e/pnas00638-0075-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/20ad/394099/d727c7ebe080/pnas00638-0075-b.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/20ad/394099/f5ee9366a6b3/pnas00638-0076-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/20ad/394099/ad7a1bf9772c/pnas00638-0076-b.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/20ad/394099/f250fefa40cf/pnas00638-0076-c.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/20ad/394099/ad88e71ed9a0/pnas00638-0077-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/20ad/394099/9c1b1b9eee26/pnas00638-0077-b.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/20ad/394099/2bac1380570d/pnas00638-0077-c.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/20ad/394099/221236b18a0e/pnas00638-0075-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/20ad/394099/d727c7ebe080/pnas00638-0075-b.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/20ad/394099/f5ee9366a6b3/pnas00638-0076-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/20ad/394099/ad7a1bf9772c/pnas00638-0076-b.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/20ad/394099/f250fefa40cf/pnas00638-0076-c.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/20ad/394099/ad88e71ed9a0/pnas00638-0077-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/20ad/394099/9c1b1b9eee26/pnas00638-0077-b.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/20ad/394099/2bac1380570d/pnas00638-0077-c.jpg

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