Atkins D, Patrikakis M, Izant J G
R.W. Johnson Pharmaceutical Research Institute, Sydney NSW, Australia.
Antisense Res Dev. 1995 Winter;5(4):295-305. doi: 10.1089/ard.1995.5.295.
A genetic system for the analysis of antisense and ribozyme mechanisms is a much needed experimental tool, and yeast represent a favorable organism on which to base such a system. We have shown previously that the fission yeast Schizosaccharomyces pombe has potential to satisfy the requirements of such a system. This report describes experiments designed to determine if antisense and ribozyme RNA-mediated gene suppression will be generally applicable to other genes in S. pombe. Antisense and ribozyme RNAs designed to suppress the ade6 gene were expressed at high levels from episomal expression vectors. The ade6 gene was chosen as a target as mutations within the gene confer adenine auxotrophy and a red colony phenotype, and it was expected that antisense or ribozyme RNA-mediated mutant phenocopies would exhibit the same readily detectable phenotype. No phenotypic indication of ade6 suppression was detected in transformed yeast, and ade6 target mRNA was analyzed by primer extension and Northern analysis. Initially, conflicting results were obtained from these techniques, which were determined to be due to duplex formation between antisense and target RNA in vitro. No detectable reduction in the ade6 mRNA levels was found, and it was concluded that the gene was not suppressed by the antisense or ribozyme RNAs tested. These results confirm that in S. pombe as with other organisms, the susceptibility of genes to RNA-mediated suppression may be gene specific and that design of antisense and ribozyme genes will be an empirical process.
用于分析反义及核酶机制的遗传系统是一种急需的实验工具，而酵母是构建此类系统的理想生物体。我们之前已经表明，裂殖酵母粟酒裂殖酵母有潜力满足此类系统的要求。本报告描述了旨在确定反义及核酶RNA介导的基因抑制是否普遍适用于粟酒裂殖酵母中其他基因的实验。设计用于抑制ade6基因的反义及核酶RNA从附加型表达载体中高水平表达。选择ade6基因作为靶点是因为该基因内的突变会导致腺嘌呤营养缺陷和红色菌落表型，并且预期反义或核酶RNA介导的突变拟表型会表现出相同的易于检测的表型。在转化的酵母中未检测到ade6抑制的表型迹象，并通过引物延伸和Northern分析对ade6靶mRNA进行了分析。最初，从这些技术中获得了相互矛盾的结果，确定这是由于体外反义RNA与靶RNA之间形成双链体所致。未发现ade6 mRNA水平有可检测到的降低，得出的结论是该基因未被测试的反义或核酶RNA抑制。这些结果证实，在粟酒裂殖酵母中与在其他生物体中一样，基因对RNA介导抑制的敏感性可能具有基因特异性，并且反义及核酶基因的设计将是一个经验性过程。
