Localization of sequences that influence basal and cell type-specific activity of the murine mdr2 promoter
| Autor: | C P, Yang, L S, Kirschner, L, Yu, S B, Horwitz |
|---|---|
| Rok vydání: | 1996 |
| Předmět: |
ATP Binding Cassette Transporter
Subfamily B Binding Sites Base Sequence Macrophages Recombinant Fusion Proteins Molecular Sequence Data DNA Footprinting Sequence Analysis DNA DNA Methylation Vinblastine Drug Resistance Multiple Cell Line Mice Gene Expression Regulation Liver Genes Reporter Animals ATP-Binding Cassette Transporters ATP Binding Cassette Transporter Subfamily B Member 1 Cloning Molecular Genes MDR Luciferases Promoter Regions Genetic Sequence Deletion |
| Zdroj: | Cell growthdifferentiation : the molecular biology journal of the American Association for Cancer Research. 7(9) |
| ISSN: | 1044-9523 |
| Popis: | 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 CCAAT 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 the downstream sequence may be involved in cell type-specific expression of the mdr2 gene. 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. |
| Databáze: | OpenAIRE |
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