Mouse beta 2-microglobulin cDNA clones: a screening procedure for cDNA clones corresponding to rare mRNAs.
Mouse beta 2-microglobulin cDNA clones: a screening procedure for cDNA clones corresponding to rare mRNAs.
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小鼠 β2-微球蛋白 cDNA 克隆：与稀有 mRNA 相对应的 cDNA 克隆的筛选程序。
DOI：
10.1073/pnas.78.4.2253
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发表时间：
1981
影响因子：
11.1
通讯作者：
J. Seidman
中科院分区：
文献类型：
--
作者：
Jane R. Parnes;Baruch Velan;A. Felsenfeld;Lata RAMANATHANt;Umberto FERRINIt;Ettore APPELLAt;J. Seidman
我们已经分离出3个β 2-微球蛋白的cDNA克隆，β 2-微球蛋白是主要组织相容性抗原的小亚基。β 2-微球蛋白占小鼠肝脏蛋白的不到0.1%，其mRNA约为肝脏poly（A）+ mRNA的0.03%。从9- 10 S小鼠肝poly（A）+ mRNA中筛选出1400个cDNA克隆。筛选cDNA克隆的程序包括将合并的质粒DNA结合到硝酸纤维素滤膜上，并测试每个滤膜选择β 2-微球蛋白mRNA的能力。测定过滤选择的mRNA在体外翻译反应中指导β 2-微球蛋白合成的能力。核苷酸序列分析表明，分离的克隆编码β 2-微球蛋白.阳性选择-杂交试验已被修改，以方便大量的cDNA克隆的筛选，和修改后的测定应允许相应的任何mRNA的体外翻译产物可以免疫沉淀的cDNA的分离。这些修饰在分离对应于稀有mRNA种类的cDNA克隆中具有特别的价值。
We have isolated three cDNA clones for beta 2-microglobulin, the small subunit of the major histocompatibility antigens. beta 2-Microglobulin makes up less than 0.1% of mouse liver protein, and its mRNA is approximately 0.03% of liver poly(A)+ mRNA. The cDNA clones were identified by screening 1400 cDNA clones made from 9--10S mouse liver poly(A)+ mRNA. The procedure for screening the cDNA clones involved binding pooled plasmid DNA to nitrocellulose filters and testing the ability of each filter to select beta 2-microglobulin mRNA. The filter-selected mRNAs were assayed for their ability to direct the synthesis of beta 2-microglobulin in translation reactions in vitro. The isolated clones were shown by nucleotide sequence analysis to encode beta 2-microglobulin. The positive-selection--hybridization assay has been modified to facilitate the screening of large numbers of cDNA clones, and the modified assay should allow the isolation of cDNAs corresponding to any mRNA whose in vitro translation products can be immunoprecipitated. These modifications are of particular value in the isolation of cDNA clones corresponding to rare species of mRNA.