D'Souza Lisa M, Larios-Sanz Maia, Setterquist Robert A, Willson Richard C, Fox George E
Department of Biology and Biochemistry, University of Houston, 4800 Calhoun Road, Houston, Texas 77204-5513, USA.
Biotechnol Prog. 2003 May-Jun;19(3):734-8. doi: 10.1021/bp025755j.
This laboratory previously showed that an RNA derived from 5S ribosomal RNA could be used as a carrier to harbor a nucleic acid "tag" for monitoring genetically engineered or naturally occurring bacteria. The prototype system expressed a specific tagged RNA that was stable and accumulated to high levels. For such a system to be useful there should, however, be little limitation on the sequence composition and length of the insert. To test these limitations, a collection of insertion sequences were created and introduced into the artificial 5S rRNA cassette. This library consisted of random 13- and 50-base oligonucleotides that were inserted into the carrier RNA. We report here that essentially all of the insert-containing RNAs are stable and accumulate to detectable levels. Tagged RNAs were produced by both plasmid-borne and chromosomally integrated expression systems in E. coli and several Pseudomonas strains without obvious effect on the host cell. It is anticipated that in addition to its intended use in environmental monitoring, this system can be used for in vivo selection of useful artificial RNAs. Because the carrier lends stability to the RNAs, the system may also be useful in RNA production.
本实验室先前表明，源自5S核糖体RNA的一种RNA可作为载体，用于携带核酸“标签”，以监测基因工程改造的或天然存在的细菌。该原型系统表达一种特定的带标签RNA，它稳定且能积累到高水平。然而，要使这样一个系统有用，对插入片段的序列组成和长度应几乎没有限制。为测试这些限制，构建了一系列插入序列并将其引入人工5S rRNA盒中。该文库由随机的13个碱基和50个碱基的寡核苷酸组成，它们被插入到载体RNA中。我们在此报告，基本上所有含插入片段的RNA都是稳定的，并能积累到可检测水平。带标签的RNA由大肠杆菌和几种假单胞菌菌株中的质粒携带表达系统和染色体整合表达系统产生，对宿主细胞没有明显影响。预计除了其在环境监测中的预期用途外，该系统还可用于体内筛选有用的人工RNA。由于载体赋予RNA稳定性，该系统在RNA生产中可能也有用。
