Abstract
Multidrug resistance in human KB carcinoma cells selected for resistance to colchicine, vinblastine, or adriamycin results from overexpression, and frequently amplification, of a specific gene (mdr1). Overlapping cDNA clones representing a complete 4.7-kilobase mdr1 transcript have been obtained from multidrug-resistant KB cells. Primer extension and S1 nuclease protection experiments show that two transcripts initiate 136 and 140 bases upstream from the first ATG codon in all human multidrug-resistant cell lines. The mdr1 gene is expressed in human normal kidney cells and HepG2 liver cells as a poly(A)+ RNA which starts from the same sites. Less prominent transcripts were found to initiate 155-180 bases upstream from the first ATG codon in vinblastine- or adriamycin-selected cell lines and 480-630 bases upstream in colchicine-selected cell lines. Southern hybridization analyses with different portions of a full-length cDNA indicate that the human mdr1 gene encompasses at least 70 kilobases of DNA amplified in all highly multidrug-resistant cell lines.
Highlights
Institute, and the )I Center for Genetics, University of sion of the mdrl gene is responsible for development of the MDRphenotypes [23]
Multidrug resistance in human KB carcinoma cells We are interested in understandinghow expression of the selected for resistance to colchicine, vinblastine, or human mdrl gene is regulated
Using probes derived from lapping cDNA clones representing a complete 4.7-kil- this cDNA, major and minor initiation sites for mdrl tranobase mdrl transcript have been obtained from multi- scription have been mapped and thseize of the human mdrl drug-resistant KB cells
Summary
CATTTCTCAT TCTCCTAGGA GTACTCACTT CAGGAAGCAA CCAGATARAA GAGAGGTGCA tion was determined by hybridizing in vitro transcribed RNA fromclonespHDR5A, 5B, and 10 to poly(A)+ RNA from. GCATTCAGTC AATCCGGGCC GGGAGCAGTC ATCTGTGGTG AGGCTGATTG GCTGGGCAGG hybridized to poly(A)+ RNAfrom MDR cells, were identified. Together with restriction mapping data which gave the orit?.ACAGCGCCG GGGCGTGGG$ TGAGCACAGC GCTTCGCTCT CTTLGCCACA GGAAGCCTGA. -200 entation of 5Bwithrespectto 5A in XHDR5, thesedata allowed us to assign a direction of transcription with clone 5B 5' to 5A. Sequence analysis of clone 5A indicated that it did not contain a poly(A) stretch at its 3' end. The 0.63-kb 3'-end PuuII-EcoRI fragment (shownas probe B in Fig. 1) of GS?&TCA
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