Structure of the RNA portion of the RNA-linked DNA pieces in bacteriophage T4-infected Escherichia coli cells.
Structure of the RNA portion of the RNA-linked DNA pieces in bacteriophage T4-infected Escherichia coli cells.
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噬菌体 T4 感染的大肠杆菌细胞中 RNA 连接的 DNA 片段的 RNA 部分的结构。
DOI：
10.1016/0022-2836(79)90515-1
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发表时间：
1979
影响因子：
5.6
通讯作者：
T. Okazaki
中科院分区：
文献类型：
--
作者：
Y. Kurosawa;T. Okazaki
已经开发了用于分离RNA连接的DNA片段的RNA部分的方法。该方法利用了与噬菌体T4 DNA聚合酶相关的3′至5′外切核酸酶对DNA的选择性降解。在DNA部分水解后，RNA连接的DNA的RNA大部分以脱氧核糖核苷酸末端的RNA形式回收，小部分以纯RNA形式回收。另一方面，无RNA的DNA的5′端大部分以二核苷酸形式回收，小部分以单核苷酸形式回收。从T4噬菌体感染的大肠杆菌细胞中分离出新生的短DNA片段，将其5′端脱磷酸化，然后用多核苷酸激酶和[γ-32 P]ATP磷酸化。在选择性降解DNA片段后，[5′-32 P]寡核苷酸（直至五核苷酸）在其3′端与脱氧单核苷酸共价结合。在所分离的寡核苷酸中，40%以上为5′-端二核苷酸带有pApC的五核苷酸。四核糖核苷酸的5′端核苷酸是AMP，但短链的5 ′端核苷酸不是唯一的。五核苷酸可以代表T4噬菌体DNA合成的完整引物RNA。
A method for the isolation of the RNA portion of RNA-linked DNA fragments has been developed. The method capitalizes on the selective degradation of DNA by the 3′ to 5′ exonuclease associated with bacteriophage T4 DNA polymerase. After hydrolysis of the DNA portion, the RNA of RNA-linked DNA is recovered mostly as RNA tipped with a deoxyribomononucleotide and a small fraction as pure RNA. On the other hand, the 5′ ends of RNA-free DNA are recovered mostly as dinucleotides and a small fraction as mononucleotides.Using this method, we have isolated the primer RNA for T4 phage DNA synthesis. Nascent short DNA pieces were isolated from T4 phage-infectedEscherichia colicells and the 5′ ends of the pieces were dephosphorylated and then phosphorylated with polynucleotide kinase and [γ-32P]ATP. After selective degradation of the DNA portions, [5′-32P]oligoribonucleotides (up to pentanucleotide) were obtained with covalently bound deoxymononucleotides at their 3′ ends. More than 40% of the oligoribonucleotides isolated were pentanucleotides with pApC at the 5′-terminal dinucleotide. The 5′-terminal nucleotide of the tetraribonucleotides was AMP, but that of the shorter chains was not unique. The pentanucleotide could represent the intact primer RNA for T4 phage DNA synthesis.