Consistent gene amplification has been observed in a number of independently established multidrug resistant (MDR) Chinese hamster ovary (CHO) cell lines. Amplified DNA from these resistant cells, about 100 kb long, has been isolated by molecular cloning methods. A transcript of 4.3-kb has been found to be overproduced in these MDR cells, and partial cDNA clone for a putative mRNA has been obtained. We propose here to clone the full-length cDNA to this transcript in an expressible vector for functional studies. Biochemical characterizations of the amplified gene will be performed by determining the nucleotide sequences of the cDNA, and from which to deduce the amino acid sequence. The full-length cDNA will be used to retrieve genomic DNA clones containing sequences encoding the 4.3-kb mRNA. The genomic organizations of the amplified gene will be determined. Tissue-specific expressions of this amplified gene in hamster animals will be determined by the Northern blot hybridizations and in situ hybridizations using radioactively labeled antisense RNA. These results may provide important information in understanding the functional role of this gene in normal cellular physiology. Furthermore, we have preliminary data showing a consistent DNA rearrangement is present in the amplified DNA domain in these MER CHO cell lines. We propose here to pursue DNA sequencing in determining the breakpoint for such rearrangement at the nucleotide level. We hope through these studies to gain new insights into the molecular genetic bases of MDR system.
