Ferbeyre G, Bratty J, Chen H, Cedergren R
Département de biochimie, Université de Montréal, Québec, Canada.
Gene. 1995 Mar 21;155(1):45-50. doi: 10.1016/0378-1119(94)00891-u.
To study factors that affect in vivo ribozyme (Rz) activity, a model system has been devised in Saccharomyces cerevisiae based on the inhibition of ADE1 gene expression. This gene was chosen because Rz action can be evaluated visually by the Red phenotype produced when the activity of the gene product is inhibited. Different plasmid constructs allowed the expression of the Rz either in cis or in trans with respect to ADE1. Rz-related inhibition of ADE1 expression was correlated with a Red phenotype and a diminution of ADE1 mRNA levels only when the Rz gene was linked 5' to ADE1. The presence of the expected 3' cleavage fragment was demonstrated using a technique combining RNA ligation and PCR. This yeast system and detection technique are suited to the investigation of general factors affecting Rz-catalyzed inhibition of gene expression under in vivo conditions.
为了研究影响体内核酶（Rz）活性的因素，基于对ADE1基因表达的抑制，在酿酒酵母中设计了一个模型系统。选择该基因是因为当基因产物的活性受到抑制时，可通过红色表型直观地评估Rz的作用。不同的质粒构建体允许Rz相对于ADE1顺式或反式表达。仅当Rz基因与ADE1基因5'端相连时，Rz对ADE1表达的相关抑制才与红色表型和ADE1 mRNA水平的降低相关。使用RNA连接和PCR相结合的技术证实了预期的3'切割片段的存在。该酵母系统和检测技术适用于研究体内条件下影响Rz催化基因表达抑制的一般因素。
