Destabilized green fluorescent protein detects rapid removal of transcription
Destabilized green fluorescent protein detects rapid removal of transcription
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DOI：
10.2144/000112479
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发表时间：
2007-08-01
期刊：
影响因子：
2.7
通讯作者：
Epe, Bernd
中科院分区：
文献类型：
--
作者：
Kitsera, Natahya;Khobta, Andriy;Epe, Bernd
报告蛋白及其信使 RNA (mRNA) 的高稳定性会干扰基因表达中快速瞬时变化的检测，例如由基因毒性 DNA 损伤引起的转录阻断。我们通过添加编码小鼠鸟氨酸脱羧酶 (ODC) 富含脯氨酸-谷氨酸-丝氨酸-苏氨酸 (PEST) 序列的片段，对附加型 pMARS 载体内的绿色荧光蛋白 (GFP) 基因进行了碘化，以便将该蛋白靶向蛋白酶体，并在永久转染的人类细胞中实现了前所未有的快速 GFP 周转。早在放线菌酮抑制蛋白质合成后 1 小时，荧光细胞的数量就减少了 5 倍以上。一致地，用转录抑制剂α-鹅膏蕈碱和5,6-二氯-1-,β-D-核糖基苯并咪唑(DRB)处理导致不稳定的GFP逐渐耗尽，检测为荧光下降，而稳定的蛋白质水平在相同条件下不受影响。此外，在紫外线-C (UVC) 照射诱导的瞬时转录阻断后，不稳定 GFP 的荧光以剂量依赖性方式强烈减弱，但正常 GFP 则没有。与转录偶联 DNA 修复导致的转录块的瞬时性质一致，几个小时后 GFP 荧光完全恢复。
High stabilities of reporter proteins and their messenger RNAs (mRNAs) interfere with the detection of rapid transient changes in gene expression, such as transcriptional blocks posed by genotoxic DNA lesions. We have niodified a green fluorescent protein (GFP) gene within the episomal pMARS vector by addition of a fragment encoding for mouse ornithine decarboxylase (ODC) proline-glutamate-serine-threonine-rich (PEST) sequence in order to target the protein to the proteasomes and achieved an unprecedentedly fast GFP turnover in permanently transfected human cells. As early as I h after inhibition of protein synthesis by cycloheximide, the number of fluorescent cells decreased more than 5-fold. Concordantly, treatments with transcription inhibitors alpha-amanitin and 5,6-dichloro-1-,beta-D-ribofurnosylbenzimidazole (DRB) resulted in progressive depletion of the destabilized GFP detected as fluorescence decline, while the stable protein levels were not affected under the same conditions. Moreover fluorescence of the destabilized but not of normal GFP decreased strongly, and in a dose-dependent manner following an instant transcription block induced by ultraviolet-C (UVC) irradiation. In agreement with the transient nature of the transcriptional block due to transcription-coupled DNA repair the GFP fluorescence fully recovered after several hours.