Cloning of a cDNA encoding an RNA binding protein by screening expression libraries using a northwestern strategy.
Cloning of a cDNA encoding an RNA binding protein by screening expression libraries using a northwestern strategy.
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使用西北策略筛选表达文库，克隆编码 RNA 结合蛋白的 cDNA。
DOI：
10.1006/abio.1993.1367
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发表时间：
1993
影响因子：
2.9
通讯作者：
Wilusz,J
中科院分区：
文献类型：
--
作者：
Qian,Z;Wilusz,J
利用cDNA表达文库的成功克隆涉及配体分子（探针）与表达的目的融合蛋白的相互作用。到目前为止，导致成功结果的那些配体分为三类：（i）抗体，（ii）与感兴趣的蛋白质相互作用的蛋白质配体，和（iii）被转录因子识别的DNA序列。我们以前已经确定了一个50 kDa的蛋白质（称为DSEF-1），它与功能上重要的14个碱基的富含G的RNA序列位于下游的猿猴病毒40晚聚腺苷酸化信号相互作用。通过使用含有DSEF-1结合位点的小RNA作为探针，分离cDNA克隆，其基因产物以序列特异性方式与DSEF-1结合位点相互作用。该RNA结合蛋白含有三个潜在的RNA识别基序。我们在这里提出了一个程序，以获得cDNA克隆的RNA结合蛋白的识别位点探针。
Successful cloning with cDNA expression libraries involves interaction of ligand molecules (probes) with expressed fusion proteins of interest. So far those ligands leading to successful results fall into three classes: (i) antibodies, (ii) protein ligands that interact with the protein of interest, and (iii) DNA sequences recognized by transcription factors. We have previously identified a 50-kDa protein (called DSEF-1) which interacts with a functionally important 14-base G-rich RNA sequence located downstream of the simian virus 40 late polyadenylation signal. By using small RNAs containing the DSEF-1 binding site as probes, a cDNA clone was isolated whose gene product interacted in a sequence-specific fashion with the DSEF-1 binding site. This RNA binding protein contains three potential RNA recognition motifs. We present here a procedure to obtain cDNA clones of RNA binding proteins using recognition site probes.