Adelaide University Centre for Gene Technology, Department of Biochemistry, University of Adelaide, 5000, Adelaide, South Australia, Australia.
Plant Mol Biol. 1985 Sep;4(5):293-304. doi: 10.1007/BF02418248.
In order to investigate the mechanism of replication of viroids and virusoids, we have compared the replication intermediates of three members of each group in nucleic acid extracts of infected plants. Viroids were avocado sunblotch viroid (ASBV), citrus exocortis viroid (CEV) and coconut cadang cadang viroid (CCCV). Virusoids were from velvet tobacco mottle virus (VTMoV), solanum nodiflorum mottle virus (SNMV) and lucerne transient streak virus (LTSV). Analysis of intermediates was by the Northern hybridization technique with single-strand DNA and RNA probes prepared from recombinant DNA clones. The results obtained are discussed in terms of current models of viroid and virusoid replication.The plus RNA species consisted of an oligomeric series up to decamers based on the unit of full-length viroid or virusoid, which was always the major component, except for CEV where only monomer and dimer species were found. In the case of ASBV and the virusoids of VTMoV and SNMV, a minor, multimeric series of components (X-bands) was superimposed on the main oligomeric series.The complementary minus species proved more difficult to detect and characterise, with each viroid and virusoid exhibiting a unique pattern on Northern hybridization. However, they all had greater than unit-length minus species. In addition, minus species analogous to the plus X-bands were found in ASBV and CEV. The experimental difficulties encountered in this work are discussed in terms of the problem of detecting minus species by Northern analysis in the presence of excess complementary plus species.
为了研究类病毒和类病毒的复制机制，我们比较了感染植物核酸提取物中三种成员的复制中间体。类病毒为鳄梨日灼病类病毒（ASBV）、柑橘外果皮坏死类病毒（CEV）和椰子死亡椰枣树类病毒（CCCV）。类病毒为 velvet tobacco mottle virus（VTMoV）、solanum nodiflorum mottle virus（SNMV）和 lucerne transient streak virus（LTSV）。中间产物的分析采用 Northern 杂交技术，使用从重组 DNA 克隆制备的单链 DNA 和 RNA 探针。根据当前的类病毒和类病毒复制模型讨论了获得的结果。正链 RNA 由基于全长类病毒或类病毒的寡聚体系列组成，直至十聚体，这始终是主要成分，除了 CEV 仅发现单体和二聚体。对于 ASBV 和 VTMoV 和 SNMV 的病毒，在主要寡聚体系列上叠加了一个较小的、多聚体系列的成分（X 带）。互补的负链种更难以检测和表征，每种类病毒和类病毒在 Northern 杂交中表现出独特的模式。然而，它们都有大于单位长度的负链种。此外，在 ASBV 和 CEV 中发现了类似于正 X 带的负链种。本文讨论了在存在过量互补正链种的情况下通过 Northern 分析检测负链种所遇到的实验困难。
