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首页> 外文期刊>Molecular and Cellular Biology >Mutations in the adenovirus major late promoter: effects on viability and transcription during infection.
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Mutations in the adenovirus major late promoter: effects on viability and transcription during infection.

机译：腺病毒主要晚期启动子中的突变：感染期间对生存力和转录的影响。

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We developed an experimental system to examine the effects of mutations in the adenovirus major late promoter in its correct genomic location during a productive infection. A virus was constructed whose genome could be digested to give a rightward terminal DNA fragment extending from the XhoI site at 22.9 map units, which can be ligated or recombined with plasmid DNA containing adenovirus sequences extending from 0 to 22.9 or 26.5 map units, respectively. Mutations were made by bisulfite mutagenesis in the region between base pairs -52 and -12 with respect to the cap site at +1 and transferred to the appropriate plasmids for viral reconstruction. Of 19 mutant plasmid sequences containing single or multiple G-to-A transitions, 14 could be placed in the viral genome with no apparent change in phenotype. These mutant sequences included those which contained four transitions in the string of G residues immediately downstream of the TATA box. There were no alterations in rates of transcription from the major late promoter, sites of transcription initiation, or steady-state levels of late mRNAs. All of the five mutant sequences which could not be placed in virus contained multiple transitions both up- and downstream of the TATA box. Two of these apparently lethal mutant sequences were used in promoter fusion experiments to test their ability to promote transcription of rabbit beta-globin sequences placed in the dispensable E1 region of the virus. Both sequences showed diminished ability compared with wild-type sequences to promote transcription in this context. Comparisons between these two sequences and the viable mutant sequences suggest a role for the string of G residues located between -38 and -33 in promoting transcription from the major late promoter. The data as a whole also demonstrate that the specific nucleotide sequence of this region of the major late promoter, which overlaps transcription elements of the divergent IVa2 transcription unit and coding sequences of the adenovirus DNA polymerase, is not rigidly constrained but can mutate extensively without loss of these several functions.
机译：我们开发了一个实验系统，以检查生产性感染期间腺病毒主要晚期启动子中突变在其正确基因组位置中的作用。构建了一种病毒，其基因组可以被消化以产生从XhoI位点延伸到22.9个图谱单元的向右末端DNA片段，可以将其与含有分别从0到22.9或26.5个图谱单元延伸的腺病毒序列的质粒DNA连接或重组。通过亚硫酸氢盐诱变在相对于+1位的帽位在碱基对-52和-12之间的区域中进行突变，并转移至用于病毒重建的合适质粒。在包含单个或多个G到A转换的19个突变质粒序列中，有14个可以放置在病毒基因组中，但表型没有明显变化。这些突变序列包括那些在TATA盒的紧下游的G残基串中包含四个转变的序列。主要晚期启动子的转录速率，转录起始位点或晚期mRNA的稳态水平没有变化。不能置于病毒中的所有五个突变序列在TATA盒的上下游都包含多个转变。这些明显致命的突变序列中的两个被用于启动子融合实验中，以测试其促进放置在病毒可分配E1区的兔β-珠蛋白序列转录的能力。在这种情况下，与野生型序列相比，两种序列均显示出减弱的促进转录的能力。这两个序列与可行突变序列之间的比较表明，位于-38和-33之间的G残基串可促进主要晚期启动子的转录。整体数据还表明，主要晚期启动子该区域的特定核苷酸序列不受严格限制，但可以广泛地突变而不会丢失，该核苷酸与异源IVa2转录单元的转录元件和腺病毒DNA聚合酶的编码序列重叠。这几个功能中的一个

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