Brunet L J, Babiss L E, Young C S, Mills D R
Mol Cell Biol. 1987 Mar;7(3):1091-100. doi: 10.1128/mcb.7.3.1091-1100.1987.
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.
我们开发了一个实验系统，用于研究在生产性感染期间，腺病毒主要晚期启动子在其正确基因组位置发生突变的影响。构建了一种病毒，其基因组可被消化以产生一个从22.9个图谱单位处的XhoI位点延伸的向右末端DNA片段，该片段可分别与包含从0到22.9或26.5个图谱单位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处的帽位点的碱基对-52和-12之间的区域产生突变，并将其转移到适当的质粒中进行病毒重建。在19个包含单个或多个G到A转换的突变体质粒序列中，14个可以置于病毒基因组中，且表型无明显变化。这些突变序列包括那些在TATA框下游紧邻的G残基串中包含四个转换的序列。主要晚期启动子的转录速率、转录起始位点或晚期mRNA的稳态水平均无改变。所有五个不能置于病毒中的突变序列在TATA框的上游和下游均包含多个转换。其中两个明显致死的突变序列用于启动子融合实验，以测试它们促进置于病毒可去除E1区域中的兔β-珠蛋白序列转录的能力。与野生型序列相比，这两个序列在此背景下促进转录的能力均降低。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残基串在促进主要晚期启动子转录方面发挥作用。总体数据还表明，主要晚期启动子该区域的特定核苷酸序列与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，并非严格受限，而是可以广泛突变而不丧失这些多种功能。
