Friez M, Hermansen R, Milavetz B
Department of Biochemistry and Molecular Biology, University of North Dakota School of Medicine, Grand Forks, North Dakota 58202, USA.
J Virol. 1999 Mar;73(3):1990-7. doi: 10.1128/JVI.73.3.1990-1997.1999.
The goal of this study was to determine the minimal sequence within the simian virus 40 (SV40) late promoter region, nucleotides (nt) 255 to 424, capable of phasing nucleosomes as measured by its ability to confer the greatest endonuclease sensitivity on adjacent DNA sequences. To identify the minimal sequence, a deletional analysis of the late region was performed by utilizing a SV40 recombinant reporter system. The reporter system consisted of a series of unique restriction sites introduced into SV40 at nt 2666. The unique restriction sites allowed the insertion of test sequences as well as measurement of conferred endonuclease sensitivity. The results of the deletional analysis demonstrated that constructs capable of conferring the greatest nuclease sensitivities consistently included nt 255 to 280. The activator protein 4 (AP-4) and GTIIC transcription factor binding sequences lie within this region and were analyzed individually. Their abilities to confer nuclease sensitivity upon the reporter nearly matched that of the entire late domain. These results suggest that transcription factors AP-4 and transcription-enhancing factor which binds the GTIIC sequence are able to confer significant levels of nuclease sensitivity and are likely involved in the formation of the SV40 nucleosome-free region.
本研究的目的是确定猿猴病毒40（SV40）晚期启动子区域（核苷酸（nt）255至424）内的最小序列，该序列能够通过赋予相邻DNA序列最大的核酸内切酶敏感性来确定核小体的相位。为了鉴定最小序列，利用SV40重组报告系统对晚期区域进行了缺失分析。报告系统由一系列在nt 2666处引入SV40的独特限制性酶切位点组成。这些独特的限制性酶切位点允许插入测试序列以及测量赋予的核酸内切酶敏感性。缺失分析的结果表明，能够赋予最大核酸酶敏感性的构建体始终包含nt 255至280。激活蛋白4（AP-4）和GTIIC转录因子结合序列位于该区域内，并分别进行了分析。它们赋予报告基因核酸酶敏感性的能力几乎与整个晚期结构域相当。这些结果表明，转录因子AP-4和与GTIIC序列结合的转录增强因子能够赋予显著水平的核酸酶敏感性，并且可能参与了SV40无核小体区域的形成。
