Parnes J R, Velan B, Felsenfeld A, Ramanathan L, Ferrini U, Appella E, Seidman J G
Proc Natl Acad Sci U S A. 1981 Apr;78(4):2253-7. doi: 10.1073/pnas.78.4.2253.
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.
我们已经分离出了三个编码β2-微球蛋白（主要组织相容性抗原的小亚基）的cDNA克隆。β2-微球蛋白在小鼠肝脏蛋白质中所占比例不到0.1%，其mRNA约占肝脏多聚腺苷酸加尾（poly(A)+）mRNA的0.03%。通过筛选从9 - 10S小鼠肝脏多聚腺苷酸加尾mRNA制备的1400个cDNA克隆来鉴定这些cDNA克隆。筛选cDNA克隆的步骤包括将混合的质粒DNA与硝酸纤维素滤膜结合，并检测每个滤膜选择β2-微球蛋白mRNA的能力。对滤膜选择的mRNA在体外翻译反应中指导β2-微球蛋白合成的能力进行检测。通过核苷酸序列分析表明，分离出的克隆编码β2-微球蛋白。对阳性选择杂交试验进行了改进，以利于筛选大量的cDNA克隆，改进后的试验应该能够分离出与任何体外翻译产物可被免疫沉淀的mRNA相对应的cDNA。这些改进在分离与稀有mRNA种类相对应的cDNA克隆方面具有特别的价值。
