Localization of sequences that influence basal and cell type-specific activity of the murine mdr2 promoter

Chia-Ping H. Yang, Lawrence S. Kirschner, Lijia Yu, Susan Band Horwitz

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

The mdr2 gene is highly expressed in liver and is involved in the translocation of phospholipid. To study the regulation of mdr2 expression, the promoter of the mdr2 gene has been isolated from a murine vinblastine- resistant cell line, J7.V2-1, and characterized. The 5' flanking region of this gene is GC-rich, has multiple transcription initiation sites as mapped by primer extension, and does not contain either TATA or 5CCAAT boxes. To test promoter activity, a 1.9-kb (-1867 to +37) DNA fragment was cloned in front of the luciferase reporter gene and transient transfection assays were done in a variety of cell lines. The promoter-luciferase construct displayed a 20- to 120-fold increase in activity compared to the promoterless vector. 5' and 3' deletion analysis using transient transfections revealed two major regulatory regions in the promoter, one located upstream and one situated downstream of the transcription start sites. The upstream region may be involved in basal expression and rite downstream sequence may be involved in cell type-specific expression of the mdr2 gena. Gel mobility shift and DNA footprinting assays have identified a 29-bp sequence (-78 to -50) to which nuclear protein binds. Methylation interference analysis using this fragment has further determined that CTGGCAGCTCGCCC, within the 29-mer, contains the core sequence with which nuclear protein directly interacts. Mutation of the core sequence reduced basal promoter activity, indicating that it is involved in the basal expression of the mdr2 gene. Mutagenesis studies also suggested that the upstream and downstream sequences act independently in regulation of cell type-specific mdr2 expression.

Original languageEnglish (US)
Pages (from-to)1227-1237
Number of pages11
JournalCell Growth and Differentiation
Volume7
Issue number9
StatePublished - 1996

Fingerprint

Transcription Initiation Site
Nuclear Proteins
Luciferases
Transfection
DNA Footprinting
Genes
Cell Line
Vinblastine
5' Flanking Region
Nucleic Acid Regulatory Sequences
Reporter Genes
Mutagenesis
Methylation
Phospholipids
Gels
Gene Expression
Mutation
Liver
DNA

ASJC Scopus subject areas

  • Cell Biology
  • Molecular Biology

Cite this

Localization of sequences that influence basal and cell type-specific activity of the murine mdr2 promoter. / Yang, Chia-Ping H.; Kirschner, Lawrence S.; Yu, Lijia; Band Horwitz, Susan.

In: Cell Growth and Differentiation, Vol. 7, No. 9, 1996, p. 1227-1237.

Research output: Contribution to journalArticle

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