免疫球蛋白轻链14S信使核糖核酸纯化产物互补DNA的酶促合成
Enzymatic synthesis of DNA complementary to purified 14S messenger RNA of immunoglobulin light chain.
作者信息
Digglemann H, Faust C H, Mach B
出版信息
Proc Natl Acad Sci U S A. 1973 Mar;70(3):693-6. doi: 10.1073/pnas.70.3.693.
The 14S messenger RNA, which contains poly(adenylic acid), of MOPC 41 (mouse plasmocytoma) immunoglobulin light chain, purified to a single peak as shown by polyacrylamide gel electrophoresis, was used to synthesize complementary DNA with the RNA-dependent DNA polymerase of avian myeloblastosis virus. DNA synthesis is entirely dependent on added RNA template and oligo(dT) primer. Both the size and the concentration of the primer affect the reaction. The product behaves similarly to DNA during centrifugation in cesium sulfate density gradients. It is shown by hybridization that the DNA made is complementary to the purified template, light-chain mRNA. The high specific activity of the complementary DNA should make it suitable for gene-dosage experiments. According to alkaline sucrose gradient analyses, some complete complementary DNA transcripts of the 14S mRNA seem to be made. Oligo(dG) can also function as a primer for DNA synthesis, possibly by annealing to an internal cluster of cytidines in the mRNA, that correspond to the bases coding for amino-acids 119 and 120 of the MOPC 41 light chain.
经聚丙烯酰胺凝胶电泳显示已纯化至单一峰的MOPC 41（小鼠浆细胞瘤）免疫球蛋白轻链的14S信使核糖核酸（含有聚腺苷酸），被用于利用禽成髓细胞瘤病毒的依赖RNA的DNA聚合酶合成互补DNA。DNA合成完全依赖于添加的RNA模板和寡聚（dT）引物。引物的大小和浓度都会影响反应。在硫酸铯密度梯度离心中，产物的行为与DNA相似。杂交结果表明，合成的DNA与纯化的模板轻链信使核糖核酸互补。互补DNA的高比活性应使其适用于基因剂量实验。根据碱性蔗糖梯度分析，似乎合成了一些14S信使核糖核酸的完整互补DNA转录本。寡聚（dG）也可作为DNA合成的引物，可能是通过与信使核糖核酸中胞嘧啶的内部簇退火，这些胞嘧啶对应于MOPC 41轻链第119和120位氨基酸的编码碱基。
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