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标题：Optimal conditions for cell-free synthesis of citrus exocortis viroid and the question of specificity of RNA polymerase activity
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作者：J. S. Semancik ; K. L. Harper
期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：1984
卷号：81
期号：14
页码：4429-4433
DOI：10.1073/pnas.81.14.4429
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：Cell-free synthesis of citrus exocortis viroid (CEV) in nuclei-rich preparations from infected Gynura aurantiaca was optimum at 18-24{degrees}C. Incubation of reaction mixtures at higher temperatures (30-36{degrees}C) resulted in an increase of CEV linear molecules and the recovery of incomplete or nicked newly synthesized RNA species. Although the Mg2+ optimum (2.5-5 mM) for CEV synthesis was lower than that for total [32P]CMP incorporation (10 mM), the response to Mn2+ ion was distinctly different. Whereas maximum total activity was observed in 1 mM Mn2+ with a pronounced reduction (80%) in 5 mM Mn2+, CEV synthesis was maintained in 1-15 mM Mn2+. Inhibition of -amanitin-sensitive CEV synthesis in 200 mM (NH4)2SO4 resembles the reaction of RNA polymerase II on a free nucleic acid template. However, detection of trace levels of -amanitin-resistant CEV synthesis activity inhibited by low (NH4)2SO4 concentrations (25 mM) suggests the possible involvement of RNA polymerase I- and/or III-like activity.
关键词：nuclei-rich fraction ; Mn²⁺ resistance ; (NH₄)₂SO₄ inhibition ; α-amanitin ; denaturing polyacrylamide gel electrophoresis