Molecular cloning and characterization of the human dihydropyrimidine dehydrogenase promoter

Svetlana A. Shestopal, Martin R. Johnson, Robert B Diasio

Research output: Contribution to journalArticle

31 Citations (Scopus)

Abstract

Several studies have demonstrated that dihydropyrimidine dehydrogenase (EC 1.3.1.2) has a critical role in the pharmacokinetics of the anticancer agent 5-fluorouracil. We previously reported the structural organization of the human DPYD gene. In this article, we describe the molecular cloning and functional characterization of 1.2 kb of the 5' flanking region of the DPYD gene. Sequence analysis demonstrated that this region of the DPYD gene lacks the typical TATA or CCAAT boxes with several GC-rich regions containing potential cis-regulatory elements. Progressive 5' deletions of the 5' flanking region were fused to the luciferase reporter gene and transient expression measured following transfection into HeLa and 293 cells. Comparative analysis of luciferase activity revealed that a 208 bp region of the DPYD gene (-121/+86) contained equivalent transcriptional activity to the complete 1.2 kb 5' flanking region of the DPYD gene. Site-directed mutagenesis of the luciferase reporter constructs demonstrated that the -72/-23 sequence contained two regulatory regions (designated elements I and II) essential for promoter activity. Gel shift experiments demonstrated that both regulatory elements specifically bind with protein(s) from nuclear extracts of 293 cells. Competitive binding experiments with 293 nuclear extracts and radiolabeled oligonucleotides (corresponding to elements I and II) suggest that the same protein(s) bind to both regulatory elements. We conclude that constitutive expression of the DPYD gene involves a limited GC-rich region of the 5' flanking sequence of the DPYD gene which contains two regulatory elements. Copyright (C) 2000 Elsevier Science B.V.

Original languageEnglish (US)
Pages (from-to)162-169
Number of pages8
JournalBiochimica et Biophysica Acta - Gene Structure and Expression
Volume1494
Issue number1-2
DOIs
StatePublished - Nov 15 2000
Externally publishedYes

Fingerprint

Dihydrouracil Dehydrogenase (NADP)
Cloning
Molecular Cloning
Genes
5' Flanking Region
Luciferases
GC Rich Sequence
Gene Expression
Competitive Binding
Nucleic Acid Regulatory Sequences
Nuclear Proteins
Site-Directed Mutagenesis
Cell Extracts
Reporter Genes
HeLa Cells
Oligonucleotides
Fluorouracil
Antineoplastic Agents
Mutagenesis
Pharmacokinetics

Keywords

  • Cloning
  • Dihydropyrimidine dehydrogenase
  • Human
  • Promoter

ASJC Scopus subject areas

  • Biochemistry
  • Genetics
  • Structural Biology
  • Biophysics

Cite this

Molecular cloning and characterization of the human dihydropyrimidine dehydrogenase promoter. / Shestopal, Svetlana A.; Johnson, Martin R.; Diasio, Robert B.

In: Biochimica et Biophysica Acta - Gene Structure and Expression, Vol. 1494, No. 1-2, 15.11.2000, p. 162-169.

Research output: Contribution to journalArticle

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