Tissue-specific enhancer of the human multidrug-resistance (MDR1) gene.
Identification of cis-regulatory sequences is a first step in analyzing the regulation of the human multidrug-resistant 1 (MDR1) gene which encodes the 170-kilodalton membrane P-glycoprotein in normal tissues and tumor cells. We have studied several overlapping genomic clones containing the 5'-flanking region of the gene. These clones span about 30 kilobases (kb) of contiguous DNA containing 10 kb of the gene and 20 kb of the 5'-flanking sequence. The nucleotide sequence of the first exon and the 2 kb preceding the exon were determined. DNA sequences containing the 5'-flanking regions were linked to the chloramphenicol acetyltransferase (CAT) gene. For transient CAT assay, we have employed six cell lines, including human cancer KB, vincristine-resistant VJ-300 derived from KB, mouse adrenal tumor Y-1, African green monkey kidney CV-1, mouse fibroblast NIH3T3, and human adrenal carcinoma SW-13 cells. Promoter activity was very weak regardless of the length of the promoter region in mouse adrenal tumor Y-1 and monkey kidney CV-1 cells, in which endogenous P-glycoprotein was expressed. Introduction of a 700-base genomic DNA fragment from a site located at 10 kb far upstream of the initiation site increased the transcription of the CAT gene in Y-1, CV-1, and SW-13 cells. However, no significant increase in the CAT activity could be observed in NIH3T3, KB, and VJ-300 cells. This fragment markedly augmented the expression of the CAT gene regardless of orientation or position, and it acted in a cell type-specific manner even with heterogenous promoters. Our present study suggests that the 700-base pair fragment may carry a tissue-specific transcriptional enhancer that is active in at least some adrenal and kidney-derived cell lines.
Kohno
K
K
Department of Biochemistry, Oita Medical School, Japan.
Sato
S
S
Uchiumi
T
T
Takano
H
H
Kato
S
S
Kuwano
M
M
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification; D015342:DNA Probes; D004351:Drug Resistance / Q000235:genetics; D004742:Enhancer Elements, Genetic / Q000235:genetics*; D005091:Exons; D006801:Humans; D008562:Membrane Glycoproteins / Q000235:genetics*; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D011401:Promoter Regions, Genetic / Q000235:genetics; D012045:Regulatory Sequences, Nucleic Acid; D015183:Restriction Mapping; D014162:Transfection; D014407:Tumor Cells, Cultured
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D015342:DNA Probes; D008562:Membrane Glycoproteins; D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase