In vivo DNA expression of functional brome mosaic virus RNA replicons in Saccharomyces cerevisiae
In vivo DNA expression of functional brome mosaic virus RNA replicons in Saccharomyces cerevisiae
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DOI：
10.1128/jvi.71.10.7781-7790.1997
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发表时间：
1997-10-01
影响因子：
5.4
通讯作者：
Ahlquist, P
中科院分区：
文献类型：
--
作者：
Ishikawa, M;Janda, M;Ahlquist, P
为了促进雀麦花叶病毒（BMV）RNA复制子在酿酒酵母中的操作以及用于BMV RNA复制、基因表达和宿主相互作用的酵母遗传分析，我们构建了DNA质粒，从该DNA质粒中BMV RNA 3和RNA 3衍生物可以在体内从半乳糖诱导的酵母GAL 1启动子转录，并在其天然3'末端或其天然3'末端附近被自切割核酶终止，在携带这种质粒的半乳糖诱导的酵母中，BMV RNA复制蛋白Ia和2a的表达导致负链RNA 3的合成，正链RNA 3的扩增至比DNA衍生的RNA 3转录物高45倍以上的水平，以及RNA 3编码的外壳蛋白的亚基因组mRNA的合成，尽管GAL 1启动子从多个位点启动转录，但Ia和2a选择性地扩增具有真实病毒5'末端的RNA 3。正如预期的，取代3'-近端外壳蛋白基因的报告基因不能直接从DNA衍生的RNA 3转录物翻译，因此它们的表达依赖于Ia和2a指导的亚基因组mRNA合成。在诱导B3 CAT（氯霉素乙酰转移酶（CAT）基因取代外壳基因的RNA 3衍生物）的DNA转录的酵母中，CAT活性在不存在Ia和2a时保持在接近背景水平，但增加超过500，类似地，编码B3 URA 3的质粒，即酵母URA 3基因取代外壳基因的RNA 3衍生物，仅在Ia和2a表达和半乳糖诱导后才赋予ura 3（-）酵母尿嘧啶非依赖性生长。一旦它的Ia和2a依赖性复制开始，B3 URA 3作为游离RNA复制子维持在分裂的酵母中，即使在GAL 1启动子被抑制或B3 URA 3 cDNA质粒丢失之后，这些发现对于许多实验目的应该是有用的。
To facilitate manipulation of brome mosaic virus (BMV) RNA replicons in Saccharomyces cerevisiae and for yeast genetic analysis of BMV RNA replication, gene expression, and host interactions, we constructed DNA plasmids from which BMV RNA3 and RNA3 derivatives can be transcribed in vivo from the galactose-inducible yeast GAL1 promoter and terminated by a self-cleaving ribozyme at or near their natural 3' ends, In galactose-induced yeast harboring such plasmids, expression of BMV RNA replication proteins la and 2a led to synthesis of negative-strand RNA3, amplification of positive-strand RNA3 to levels over 45-fold higher than those of DNA-derived RNA3 transcripts, and synthesis of the RNA3-encoded subgenomic mRNA for coat protein, Although the GAL1 promoter initiated transcription from multiple sites, la and 2a selectively amplified RNA3 with the authentic viral 5' end, As expected, reporter genes substituted for the 3'-proximal coat protein gene could not be translated directly from DNA-derived RNA3 transcripts, so their expression depended on la-and 2a-directed subgenomic mRNA synthesis, In yeast in which DNA transcription of B3CAT, an RNA3 derivative with the chloramphenicol acetyltransferase (CAT) gene replacing the coat gene, was induced, CAT activity remained near background levels in the absence of la and 2a but increased over 500,000-fold when la and 2a were expressed, Similarly, a plasmid encoding B3URA3, an RNA3 derivative with the yeast URA3 gene replacing the coat gene, conferred uracil-independent growth to ura3(-) yeast only after la and 2a expression and galactose induction. Once its la-and 2a-dependent replication was initiated, B3URA3 was maintained in dividing yeast as a free RNA replicon, even after repression of the GAL1 promoter or the loss of the B3URA3 cDNA plasmid, These findings should be useful for many experimental purposes.