Destabilized green fluorescent protein detects rapid removal of transcription blocks after genotoxic exposure
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 modified a green fluorescent protein (GFP) gene within the episomal pMARS vector b...
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| Format: | Article |
| Language: | English |
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Taylor & Francis Group
2007-08-01
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| Series: | BioTechniques |
| Online Access: | https://www.future-science.com/doi/10.2144/000112479 |
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| author | Nataliya Kitsera Andriy Khobta Bernd Epe |
| author_facet | Nataliya Kitsera Andriy Khobta Bernd Epe |
| author_sort | Nataliya Kitsera |
| collection | DOAJ |
| description | 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 modified 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 1 h after inhibition of protein synthesis by cycloheximide, the number of fluorescent cells decreased more than 5-fold. Concordantly, treatments with transcription inhibitors α-amanitin and 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (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. |
| format | Article |
| id | doaj-art-b705fef153f342b89ce37cf7d169680f |
| institution | OA Journals |
| issn | 0736-6205 1940-9818 |
| language | English |
| publishDate | 2007-08-01 |
| publisher | Taylor & Francis Group |
| record_format | Article |
| series | BioTechniques |
| spelling | doaj-art-b705fef153f342b89ce37cf7d169680f2025-08-20T02:26:07ZengTaylor & Francis GroupBioTechniques0736-62051940-98182007-08-0143222222710.2144/000112479Destabilized green fluorescent protein detects rapid removal of transcription blocks after genotoxic exposureNataliya Kitsera0Andriy Khobta1Bernd Epe21Johannes Gutenberg University of Mainz, Mainz, Germany1Johannes Gutenberg University of Mainz, Mainz, Germany1Johannes Gutenberg University of Mainz, Mainz, GermanyHigh 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 modified 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 1 h after inhibition of protein synthesis by cycloheximide, the number of fluorescent cells decreased more than 5-fold. Concordantly, treatments with transcription inhibitors α-amanitin and 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (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.https://www.future-science.com/doi/10.2144/000112479 |
| spellingShingle | Nataliya Kitsera Andriy Khobta Bernd Epe Destabilized green fluorescent protein detects rapid removal of transcription blocks after genotoxic exposure BioTechniques |
| title | Destabilized green fluorescent protein detects rapid removal of transcription blocks after genotoxic exposure |
| title_full | Destabilized green fluorescent protein detects rapid removal of transcription blocks after genotoxic exposure |
| title_fullStr | Destabilized green fluorescent protein detects rapid removal of transcription blocks after genotoxic exposure |
| title_full_unstemmed | Destabilized green fluorescent protein detects rapid removal of transcription blocks after genotoxic exposure |
| title_short | Destabilized green fluorescent protein detects rapid removal of transcription blocks after genotoxic exposure |
| title_sort | destabilized green fluorescent protein detects rapid removal of transcription blocks after genotoxic exposure |
| url | https://www.future-science.com/doi/10.2144/000112479 |
| work_keys_str_mv | AT nataliyakitsera destabilizedgreenfluorescentproteindetectsrapidremovaloftranscriptionblocksaftergenotoxicexposure AT andriykhobta destabilizedgreenfluorescentproteindetectsrapidremovaloftranscriptionblocksaftergenotoxicexposure AT berndepe destabilizedgreenfluorescentproteindetectsrapidremovaloftranscriptionblocksaftergenotoxicexposure |