Lampson B C, Inouye M, Inouye S
Department of Biochemistry, Robert Wood Johnson Medical School at Rutgers University of Medicine and Dentistry of New Jersey, Piscataway 08854.
Cell. 1989 Feb 24;56(4):701-7. doi: 10.1016/0092-8674(89)90592-8.
msDNA is a peculiar molecule consisting of a branched RNA linked to single-stranded DNA via a 2',5' phosphodiester bond. A cell-free system, utilizing cells permeabilized with phenethyl alcohol, was established to study the synthesis of msDNA in M. xanthus. Permeablized cells labeled with [alpha-32P]dCTP in the presence of ddGTP, ddATP, or ddTTP produce a band that migrates at the same position as the full-sized msDNA in an polyacrylamide gel. However, when this band is treated with ribonuclease A prior to gel electrophoresis, it results in many different-sized bands. This indicates that during the labeling, intermediates are produced in which single-stranded DNAs of various lengths are associated with a compensatory length of RNA such that the total length for each intermediate is identical. These results provide evidence for the previously proposed model in which msDNA is synthesized by reverse transcriptase using a folded RNA precursor as a primer as well as a template. Furthermore, we found that there is a precise coupling mechanism of reverse transcriptase and ribonuclease H.
多顺反子DNA（msDNA）是一种特殊的分子，由通过2',5'-磷酸二酯键与单链DNA相连的分支RNA组成。建立了一种利用苯乙醇通透细胞的无细胞系统，以研究黄色粘球菌中msDNA的合成。在双脱氧鸟苷三磷酸（ddGTP）、双脱氧腺苷三磷酸（ddATP）或双脱氧胸苷三磷酸（ddTTP）存在的情况下，用[α-32P]脱氧胞苷三磷酸（dCTP）标记的通透细胞会产生一条在聚丙烯酰胺凝胶中与全长msDNA迁移到相同位置的条带。然而，当这条带在凝胶电泳前用核糖核酸酶A处理时，会产生许多不同大小的条带。这表明在标记过程中产生了中间体，其中各种长度的单链DNA与补偿长度的RNA相关联，使得每个中间体的总长度相同。这些结果为先前提出的模型提供了证据，该模型认为msDNA是由逆转录酶以折叠的RNA前体作为引物和模板合成的。此外，我们发现逆转录酶和核糖核酸酶H之间存在精确的偶联机制。
