Abstract
DNA-protein interaction in the 5 upstream polymorphic region of the dopamine D4 receptor (DRD4) gene was analyzed by capillary electrophoretic mobility shift assay (CEMSA). The sequence of interest was amplified using a fluorescent primer and applied as a probe in the binding assays with HeLa nuclear extract. Serial dilution of the probe resulted in a concentration dependent DNA-protein complex formation. Sp 1 specific oligonucleotide competitor significantly inhibited the DNA-protein complex formation. A non-specific competitor, differing only in three base pairs, showed weaker effect pointing to the contribution of the Sp 1 recognition sequence in the complex. Polymorphic competitors were also prepared from homozygous individuals possessing either duplicated (2x120 bp) or single copy (1x120bp) of the 120 bp repeat sequence and were used against the Sp 1 specific probe in competition assays. Our data provide experimental evidence for the binding of Sp 1 to the 120 bp duplicated sequence of the DRD4 5 upstream region and suggest enhanced binding capacity of the duplicated form.
Keywords: capillary electrophoresis, electrophoretic mobility shift assay, dopamine d4 receptor
Current Medicinal Chemistry
Title: Capillary Electrophoresis Study on DNA-Protein Complex Formation in the Polymorphic 5 Upstream Region of the Dopamine D4 Receptor (DRD4) Gene
Volume: 11 Issue: 8
Author(s): Z. Ronai, A. Guttman, G. Keszler and M. Sasvari-Szekely
Affiliation:
Keywords: capillary electrophoresis, electrophoretic mobility shift assay, dopamine d4 receptor
Abstract: DNA-protein interaction in the 5 upstream polymorphic region of the dopamine D4 receptor (DRD4) gene was analyzed by capillary electrophoretic mobility shift assay (CEMSA). The sequence of interest was amplified using a fluorescent primer and applied as a probe in the binding assays with HeLa nuclear extract. Serial dilution of the probe resulted in a concentration dependent DNA-protein complex formation. Sp 1 specific oligonucleotide competitor significantly inhibited the DNA-protein complex formation. A non-specific competitor, differing only in three base pairs, showed weaker effect pointing to the contribution of the Sp 1 recognition sequence in the complex. Polymorphic competitors were also prepared from homozygous individuals possessing either duplicated (2x120 bp) or single copy (1x120bp) of the 120 bp repeat sequence and were used against the Sp 1 specific probe in competition assays. Our data provide experimental evidence for the binding of Sp 1 to the 120 bp duplicated sequence of the DRD4 5 upstream region and suggest enhanced binding capacity of the duplicated form.
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Ronai Z., Guttman A., Keszler G. and Sasvari-Szekely M., Capillary Electrophoresis Study on DNA-Protein Complex Formation in the Polymorphic 5 Upstream Region of the Dopamine D4 Receptor (DRD4) Gene, Current Medicinal Chemistry 2004; 11 (8) . https://dx.doi.org/10.2174/0929867043455503
DOI https://dx.doi.org/10.2174/0929867043455503 |
Print ISSN 0929-8673 |
Publisher Name Bentham Science Publisher |
Online ISSN 1875-533X |
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