Gooding P S, Batty N P, Goldsbrough A P, Mullineaux P M
John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, UK and Plant Breeding International,Maris Lane, Trumpington, Cambridge CB2 2LQ, UK.
Nucleic Acids Res. 1999 Apr 1;27(7):1709-18. doi: 10.1093/nar/27.7.1709.
We have used particle bombardment (biolistics) to deliver replication-competent wheat dwarf virus (WDV)-based constructs, carrying reporter gene sequences fused to the virion sense promoter (Pv) or the CaMV 35S promoter, to suspension culture cells and immature zygotic embryos of wheat. While the replication of WDV double-stranded DNA forms (replicons) was equivalent between wheat suspension culture cells and embryos, GUS reporter gene activity was 20-40 times higher in the embryo cultures. Maximum expression of WDV replicons occurred in the embryonic axis tissue of wheat embryos but their expression in suspension cells was compromised, compared with transiently maintained input plasmid DNA containing the same sequences. From these studies, we propose that WDV replicons are subject to a host cell-controlled competency for virion sense transcription. The term competency is used to distinguish between the phenomenon described here and control of gene expression by specific transcription factors. Control of competency is independent of Pv, the replacement 35S promoter and of the complementary sense control of virion sense expression involving specific sequences in Pv. We propose that factors controlling the competency for replicon expression may be present in cells which, as well as maintaining high rates of DNA synthesis, are totipotent. Cell type control of active chromatin, methylation of specific sequences in WDV minichromosomes and/or interaction of virus-encoded proteins with specific host factors are considered as possible mechanisms.
我们利用粒子轰击法（生物弹道技术）将携带与病毒粒子正义启动子（Pv）或花椰菜花叶病毒35S启动子融合的报告基因序列的具有复制能力的小麦矮缩病毒（WDV）构建体导入小麦悬浮培养细胞和未成熟合子胚中。虽然WDV双链DNA形式（复制子）在小麦悬浮培养细胞和胚中的复制情况相当，但GUS报告基因活性在胚培养物中要高20 - 40倍。WDV复制子的最大表达出现在小麦胚的胚轴组织中，但与含有相同序列的瞬时维持的输入质粒DNA相比，它们在悬浮细胞中的表达受到影响。从这些研究中，我们提出WDV复制子在病毒粒子正义转录方面受到宿主细胞控制的活性。“活性”一词用于区分此处描述的现象与特定转录因子对基因表达的控制。活性的控制独立于Pv、替代的35S启动子以及涉及Pv中特定序列的病毒粒子正义表达的互补链控制。我们提出控制复制子表达活性的因子可能存在于那些不仅维持高DNA合成速率而且具有全能性的细胞中。活性染色质的细胞类型控制、WDV微型染色体中特定序列的甲基化和/或病毒编码蛋白与特定宿主因子的相互作用被认为是可能的机制。
