Arndt G M, Atkins D, Patrikakis M, Izant J G
R. W. Johnson Pharmaceutical Research Institute-Sydney, Johnson and Johnson Research Pty Limited, NSW, Australia.
Mol Gen Genet. 1995 Aug 21;248(3):293-300. doi: 10.1007/BF02191596.
This report describes experiments designed to demonstrate the suitability of the fission yeast Schizosaccharomyces pombe as a host for antisense RNA regulation. A lacZ gene-expressing yeast strain was constructed and used as a host for the expression of a series of antisense RNAs complementary to various regions of the target lacZ mRNA. All lacZ antisense genes were placed under control of the thiamine-repressible nmt1 promoter of S. pombe and expressed from episomal plasmids. For each antisense plasmid a corresponding sense control plasmid was constructed. All lacZ antisense genes were shown to express antisense RNAs of the expected size at equivalent steady-state levels. beta-Galactosidase activity in transformed cells expressing the long, short 5' or short 3' lacZ antisense RNAs was shown to be reduced by 45%, 20%, and 10%, respectively, relative to control transformants. Further experiments indicated that antisense RNA regulation in this system was conditional and reversible, with the observed reduction of beta-galactosidase activity being dependent on the transcription of lacZ antisense RNA. Our results represent the first successful example of antisense RNA regulation of gene expression in yeast and establish S. pombe as an experimental model for the biochemical analysis of antisense RNA regulation.
本报告描述了旨在证明裂殖酵母粟酒裂殖酵母作为反义RNA调控宿主的适用性的实验。构建了一个表达lacZ基因的酵母菌株，并将其用作表达一系列与靶标lacZ mRNA不同区域互补的反义RNA的宿主。所有lacZ反义基因都置于粟酒裂殖酵母硫胺素可抑制的nmt1启动子的控制下，并从附加体质粒表达。对于每个反义质粒，构建了相应的正义对照质粒。所有lacZ反义基因均显示在等效的稳态水平下表达预期大小的反义RNA。相对于对照转化体，表达长、短5'或短3'lacZ反义RNA的转化细胞中的β-半乳糖苷酶活性分别降低了45%、20%和10%。进一步的实验表明，该系统中的反义RNA调控是有条件的且可逆的，观察到的β-半乳糖苷酶活性降低取决于lacZ反义RNA的转录。我们的结果代表了酵母中基因表达的反义RNA调控的第一个成功例子，并将粟酒裂殖酵母确立为反义RNA调控生化分析的实验模型。
