Capillary electrophoresis study on DNA-protein complex formation in the polymorphic 5' upstream region of the dopamine D4 receptor (DRD4) gene.
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 (2 x 120 bp) or single copy (1 x 120 bp) 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.
Ronai
Z
Z
Institute of Medical Chemistry, Molecular Biology and Pathobiochemistry, Semmelweis University, H-1088 Budapest, Puskin u.9., Hungary.
Guttman
A
A
Keszler
G
G
Sasvari-Szekely
M
M
D024506:5' Flanking Region / Q000502:physiology*; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D019075:Electrophoresis, Capillary / Q000379:methods; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods; D006367:HeLa Cells; D006801:Humans; D046911:Macromolecular Substances; D016133:Polymerase Chain Reaction / Q000379:methods; D011110:Polymorphism, Genetic / Q000235:genetics*; D017448:Receptors, Dopamine D2 / Q000235:genetics*; D050638:Receptors, Dopamine D4
D004268:DNA-Binding Proteins; C493599:DRD4 protein, human; D046911:Macromolecular Substances; D017448:Receptors, Dopamine D2; D050638:Receptors, Dopamine D4; D004247:DNA