STUDIES ON THE EFFECTS OF SOME FLAVONOIDS ON DNA TRANSCRIPTION AND CLONING OF LIPOAMIDE DEHYDROGENASE GENE
type: CreativeWork
description: An in-vitro run-off transcription system was utilised to study the effects of flavonoids on DNA transcriptional processes. The template DNA consists of a gene coding for the chicken lysozyme protein inserted in a vector plasmid. Following conventional molecular biology methodology, large quantities of the template DNA were obtained. In-vitro transcription was carried out in the presence of 32P-UTP. Labelled transcripts were acid-precipitated and quantitatively determined in scintillation cocktail. Upon the addition of luM quercetin, transcription activity was inhibited by 37.5%. Similarly, luM of morin inhibits 26% of the same process. In contrast, (+)-catechin at concentrations less than l0uM stimulates transcription. However, with increasing concentrations of 20, 30, 40 and 50 uM, respectively all the three flavonoids inhibited transcription. At the 50% inhibition level, the concentrations of quercetin, morin and (+)-catechin were B.5uM, 2luM and 50uM respectively. The presence of a carbonyl group at C4 positon together with a free hydroxyl group at c4 position appeared to be important for the inhibition of transcription. Possibility of DNA degradation by flavonoids was raised. However, incubation of DNA in the presence of 0.5 mM and 1 mM of quercetin, rutin or (+)-catechin did not cause degradation of DNA. In contrast, in the presence of an ascorbate-free radical generated system, quercetin was able to protect the degradation of the DNA. This is brought about by the free-radical scavenging properties of flavonoids. From these experiments, it was concluded that flavonoids particularly the flavonols inhibited transcription by SP6 RNA polymerase at the chain elongation step. The exact mechanism however remains to be elucidated. The interesting results obtained from the in-vitro run-off transcription experiments provided the impetus to clone a gene which could serve to illustrate these effects across a wider range of biological systems. The gene chosen was lipoamide …
abstract: An in-vitro run-off transcription system was utilised to study the effects of flavonoids on DNA transcriptional processes. The template DNA consists of a gene coding for the chicken lysozyme protein inserted in a vector plasmid. Following conventional molecular biology methodology, large quantities of the template DNA were obtained. In-vitro transcription was carried out in the presence of 32P-UTP. Labelled transcripts were acid-precipitated and quantitatively determined in scintillation cocktail. Upon the addition of luM quercetin, transcription activity was inhibited by 37.5%. Similarly, luM of morin inhibits 26% of the same process. In contrast, (+)-catechin at concentrations less than l0uM stimulates transcription. However, with increasing concentrations of 20, 30, 40 and 50 uM, respectively all the three flavonoids inhibited transcription. At the 50% inhibition level, the concentrations of quercetin, morin and (+)-catechin were B.5uM, 2luM and 50uM respectively. The presence of a carbonyl group at C4 positon together with a free hydroxyl group at c4 position appeared to be important for the inhibition of transcription. Possibility of DNA degradation by flavonoids was raised. However, incubation of DNA in the presence of 0.5 mM and 1 mM of quercetin, rutin or (+)-catechin did not cause degradation of DNA. In contrast, in the presence of an ascorbate-free radical generated system, quercetin was able to protect the degradation of the DNA. This is brought about by the free-radical scavenging properties of flavonoids. From these experiments, it was concluded that flavonoids particularly the flavonols inhibited transcription by SP6 RNA polymerase at the chain elongation step. The exact mechanism however remains to be elucidated. The interesting results obtained from the in-vitro run-off transcription experiments provided the impetus to clone a gene which could serve to illustrate these effects across a wider range of biological systems. The gene chosen was lipoamide …
author: LIM POH YAM
date published: 1990
date modified: 2026-07-24T03:30:47Z
provider: National University of Singapore
publisher: Networked Digital Library of Theses and Dissertations
type: WebPage
source organization: National University of Singapore