Capillary electrophoresis study on DNA-protein complex formation in the polymorphic 5′ upstream region of the dopamine D4 receptor (DRD4) gene

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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.

Original languageEnglish
Pages (from-to)1023-1029
Number of pages7
JournalCurrent Medicinal Chemistry
Volume11
Issue number8
DOIs
Publication statusPublished - Apr 2004

Fingerprint

Dopamine D4 Receptors
Capillary electrophoresis
Capillary Electrophoresis
Assays
Genes
DNA
Electrophoretic mobility
Proteins
Electrophoretic Mobility Shift Assay
Oligonucleotides
Base Pairing
Dilution

Keywords

  • 120 bp repeat
  • Capillary electrophoresis
  • Dopamine D4 receptor (DRD4)
  • Electrophoretic mobility shift assay
  • Polymorphism
  • Sp 1
  • Transcription

ASJC Scopus subject areas

  • Organic Chemistry
  • Biochemistry, Genetics and Molecular Biology(all)
  • Biochemistry
  • Pharmacology

Cite this

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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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AU - Guttman, A.

AU - Keszler, G.

AU - Sasvári, M.

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N2 - 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.

AB - 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.

KW - 120 bp repeat

KW - Capillary electrophoresis

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KW - Electrophoretic mobility shift assay

KW - Polymorphism

KW - Sp 1

KW - Transcription

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