Cargando…
Heavy Water Reduces GFP Expression in Prokaryotic Cell-Free Assays at the Translation Level While Stimulating Its Transcription
The in vitro proliferation of prokaryotic and eukaryotic cells is remarkably hampered in the presence of heavy water (D(2)O). Impairment of gene expression at the transcription or translation level can be the base for this effect. However, insights into the underlying mechanisms are lacking. Here, w...
Autores principales: | , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Hindawi Publishing Corporation
2013
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3886400/ https://www.ncbi.nlm.nih.gov/pubmed/24455706 http://dx.doi.org/10.1155/2013/592745 |
_version_ | 1782478891061870592 |
---|---|
author | Hohlefelder, Luisa S. Stögbauer, Tobias Opitz, Madeleine Bayerl, Thomas M. Rädler, Joachim O. |
author_facet | Hohlefelder, Luisa S. Stögbauer, Tobias Opitz, Madeleine Bayerl, Thomas M. Rädler, Joachim O. |
author_sort | Hohlefelder, Luisa S. |
collection | PubMed |
description | The in vitro proliferation of prokaryotic and eukaryotic cells is remarkably hampered in the presence of heavy water (D(2)O). Impairment of gene expression at the transcription or translation level can be the base for this effect. However, insights into the underlying mechanisms are lacking. Here, we employ a cell-free expression system for the quantitative analysis of the effect of increasing percentages of D(2)O on the kinetics of in-vitro GFP expression. Experiments are designed to discriminate the rates of transcription, translation, and protein folding using pDNA and mRNA vectors, respectively. We find that D(2)O significantly stimulates GFP expression at the transcription level but acts as a suppressor at translation and maturation (folding) in a linear dose-dependent manner. At a D(2)O concentration of 60%, the GFP expression rate was reduced to 40% of an undisturbed sample. We observed a similar inhibition of GFP expression by D(2)O in a recombinant Escherichia coli strain, although the inhibitory effect is less pronounced. These results demonstrate the suitability of cell-free systems for quantifying the impact of heavy water on gene expression and establish a platform to further assess the potential therapeutic use of heavy water as antiproliferative agent. |
format | Online Article Text |
id | pubmed-3886400 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2013 |
publisher | Hindawi Publishing Corporation |
record_format | MEDLINE/PubMed |
spelling | pubmed-38864002014-01-21 Heavy Water Reduces GFP Expression in Prokaryotic Cell-Free Assays at the Translation Level While Stimulating Its Transcription Hohlefelder, Luisa S. Stögbauer, Tobias Opitz, Madeleine Bayerl, Thomas M. Rädler, Joachim O. Biomed Res Int Research Article The in vitro proliferation of prokaryotic and eukaryotic cells is remarkably hampered in the presence of heavy water (D(2)O). Impairment of gene expression at the transcription or translation level can be the base for this effect. However, insights into the underlying mechanisms are lacking. Here, we employ a cell-free expression system for the quantitative analysis of the effect of increasing percentages of D(2)O on the kinetics of in-vitro GFP expression. Experiments are designed to discriminate the rates of transcription, translation, and protein folding using pDNA and mRNA vectors, respectively. We find that D(2)O significantly stimulates GFP expression at the transcription level but acts as a suppressor at translation and maturation (folding) in a linear dose-dependent manner. At a D(2)O concentration of 60%, the GFP expression rate was reduced to 40% of an undisturbed sample. We observed a similar inhibition of GFP expression by D(2)O in a recombinant Escherichia coli strain, although the inhibitory effect is less pronounced. These results demonstrate the suitability of cell-free systems for quantifying the impact of heavy water on gene expression and establish a platform to further assess the potential therapeutic use of heavy water as antiproliferative agent. Hindawi Publishing Corporation 2013 2013-12-25 /pmc/articles/PMC3886400/ /pubmed/24455706 http://dx.doi.org/10.1155/2013/592745 Text en Copyright © 2013 Luisa S. Hohlefelder et al. https://creativecommons.org/licenses/by/3.0/ This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Research Article Hohlefelder, Luisa S. Stögbauer, Tobias Opitz, Madeleine Bayerl, Thomas M. Rädler, Joachim O. Heavy Water Reduces GFP Expression in Prokaryotic Cell-Free Assays at the Translation Level While Stimulating Its Transcription |
title | Heavy Water Reduces GFP Expression in Prokaryotic Cell-Free Assays at the Translation Level While Stimulating Its Transcription |
title_full | Heavy Water Reduces GFP Expression in Prokaryotic Cell-Free Assays at the Translation Level While Stimulating Its Transcription |
title_fullStr | Heavy Water Reduces GFP Expression in Prokaryotic Cell-Free Assays at the Translation Level While Stimulating Its Transcription |
title_full_unstemmed | Heavy Water Reduces GFP Expression in Prokaryotic Cell-Free Assays at the Translation Level While Stimulating Its Transcription |
title_short | Heavy Water Reduces GFP Expression in Prokaryotic Cell-Free Assays at the Translation Level While Stimulating Its Transcription |
title_sort | heavy water reduces gfp expression in prokaryotic cell-free assays at the translation level while stimulating its transcription |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3886400/ https://www.ncbi.nlm.nih.gov/pubmed/24455706 http://dx.doi.org/10.1155/2013/592745 |
work_keys_str_mv | AT hohlefelderluisas heavywaterreducesgfpexpressioninprokaryoticcellfreeassaysatthetranslationlevelwhilestimulatingitstranscription AT stogbauertobias heavywaterreducesgfpexpressioninprokaryoticcellfreeassaysatthetranslationlevelwhilestimulatingitstranscription AT opitzmadeleine heavywaterreducesgfpexpressioninprokaryoticcellfreeassaysatthetranslationlevelwhilestimulatingitstranscription AT bayerlthomasm heavywaterreducesgfpexpressioninprokaryoticcellfreeassaysatthetranslationlevelwhilestimulatingitstranscription AT radlerjoachimo heavywaterreducesgfpexpressioninprokaryoticcellfreeassaysatthetranslationlevelwhilestimulatingitstranscription |