Allen G C, Hall G, Michalowski S, Newman W, Spiker S, Weissinger A K, Thompson W F
Department of Botany, North Carolina State University, Raleigh 27695, USA.
Plant Cell. 1996 May;8(5):899-913. doi: 10.1105/tpc.8.5.899.
We have previously shown that yeast scaffold attachment regions (SARs) flanking a chimeric beta-glucuronidase (GUS) reporter gene increased per-copy expression levels by 24-fold in tobacco suspension cell lines stably transformed by microprojectile bombardment. In this study, we examined the effect of a DNA fragment originally identified in a tobacco genomic clone by its activity in an in vitro binding assay. The tobacco SAR has much greater scaffold binding affinity than does the yeast SAR, and tobacco cell lines stably transformed with constructs containing the tobacco SAR accumulated greater than fivefold more GUS enzyme activity than did lines transformed with the yeast SAR construct. Relative to the control construct, flanking the GUS gene with plant SARs increased overall expression per transgene copy by almost 140-fold. In transient expression assays, the same construct increased expression only approximately threefold relative to a control without SARs, indicating that the full SAR effect requires integration into chromosomal DNA. GUS activity in individual stable transformants was not simply proportional to transgene copy number, and the SAR effect was maximal in cell lines with fewer than approximately 10 transgene copies per tobacco genome. Lines with significantly higher copy numbers showed greatly greatly reduced expression relative to the low-copy-number lines. Our results indicate that strong SARs flanking a transgene greatly increases expression without eliminating variation between transformants. We propose that SARs dramatically reduce the severity or likelihood of homology-dependent gene silencing in cells with small numbers of transgenes but do not prevent silencing of transgenes present in many copies.
我们之前已经表明，在通过微粒轰击稳定转化的烟草悬浮细胞系中，嵌合β-葡萄糖醛酸酶（GUS）报告基因两侧的酵母支架附着区域（SAR）使单拷贝表达水平提高了24倍。在本研究中，我们检测了一个最初在烟草基因组克隆中通过体外结合试验活性鉴定的DNA片段的作用。烟草SAR比酵母SAR具有更高的支架结合亲和力，用含有烟草SAR的构建体稳定转化的烟草细胞系积累的GUS酶活性比用酵母SAR构建体转化的细胞系高五倍以上。相对于对照构建体，用植物SARs侧翼连接GUS基因使每个转基因拷贝的总体表达增加了近140倍。在瞬时表达试验中，相对于没有SARs的对照，相同的构建体仅使表达增加了约三倍，这表明完整的SAR效应需要整合到染色体DNA中。单个稳定转化体中的GUS活性与转基因拷贝数并非简单成正比，并且在每个烟草基因组中转基因拷贝数少于约10个的细胞系中，SAR效应最大。拷贝数明显更高的细胞系相对于低拷贝数细胞系显示出大大降低的表达。我们的结果表明，转基因两侧的强SARs可大大增加表达，而不会消除转化体之间的差异。我们提出，SARs可显著降低少量转基因细胞中同源依赖性基因沉默的严重程度或可能性，但不会阻止多拷贝存在的转基因的沉默。
