STUDIES ON THE EFFECTS OF SOME FLAVONOIDS ON DNA TRANSCRIPTION AND CLONING OF LIPOAMIDE DEHYDROGENASE GENE
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 dehydrogenase(lpd). This gene was chosen because it appears to be conserved throughout evolution. Isolation of the gene from a Bacillus subtilis genomic library and a human cDNA library were attempted simultaneously. However the Bacillus system was complicated by high reversion frequencies. Several isolates from the human cDNA library were obtained following Southern hybridisation of the library with a 32-P labelled 27 mer synthetic probe. Enzymological studies indicated the presence of a correctly inserted gene which is expressed following IPTG induction. Double digestion with restriction enzymes KpnI and SstI showed the presence of a 1.5 kb insert. Further investigation can be carried out resulting from the present report to characterize this insert. The original contributions from this study include: 1. The demonstration that flavonols (quercetin and morin) and the flavan, (+)-catechin could inhibit DNA transcription in-vitro in a dose dependent manner. 2. The mechanism of inhibition of transcription of DNA in the presence of the flavonoids was not due to degradation of the DNA template. 3. Flavonoid quercetin was were able to inhibit the degradation of DNA by the ascorbate-free radical system through their free-radical scavenging action. 4. Isolation and partial characterisation of the mammalian cDNA coding for lipoamide dehydrogenase was carried out.
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