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A fufl-length cDNA clone for the mouse mdrl gene can confer multidrug resistance when introduced by transfection into otherwise drug-sensitive cells. In the same assay, a full-length cDNA clone for a closely related member of the mouse mdr gene family, mdr2, fails to confer multidrug resistance. To identify the domains of mdrl which are essential for multidrug resistance and which may be functionally distinct in mdr2, we have constructed chimeric cDNA molecules in which discrete domains of mdr2 have been introduced into the homologous region of mdrl and analyzed these chimeras for their capacity to transfer drug resistance. The two predicted ATP-binding domains of mdr2 were found to be functional, as either could complement the biological activity of mdrl. Likewise, a chimeric molecule in which the highly sequence divergent linker domain of mdr2 had been introduced in mdrl could also confer drug resistance. However, the replacement of either the aminoor carboxy-terminus transmembrane (TM) domain regions of mdrl by the homologous segments of mdr2 resulted in inactive chimeras. The replacement of as few as two TM domains from either the amino ( or the carboxy (TM7-8) half of mdrl by the homologous mdr2 regions was sufficient to destroy the activity of mdrl. These results suggest that the functional differences detected between mdrl and mdr2 in our transfection assay reside within the predicted TM domains.The emergence of multidrug resistance in cultured cells is linked to the overexpression of a membrane phosphoglycoprotein termed P-glycoprotein, believed to act as an ATPdependent efflux pump to reduce the intracellular accumulation of antitumor drugs. P-glycoprotein has been shown to interact directly with a remarkably broad range of substrates; most are small hydrophobic lipid-soluble molecules of natural origin such as anthracyclines, vinca alkaloids, and colchicine (14, 22). P-glycoproteins are encoded by a small gene family, termed mdr or pgp, which comprises three members in rodents and two members in humans (40). Analysis of the nucleotide and predicted amino acid sequences of full-length cDNA clones corresponding to the three mouse (13,24,25,29) (30) origins. Both mdrl and mdr3 can confer drug resistance in transfection experiments, but the two proteins appear to have overlapping but distinct substrate specificities (13). In transfection experiments, mouse mdr2 fails to confer multidrug resistance (25).Analysis of the mouse mdr gene transcripts in normal tissues shows that expression of the three genes is controlled in a tissue-specific manner (11). mdrl is constitutively expressed in adrenal glands and kidney and is induced at high levels in the endometrial glands of the pregnant uterus (1, 2). mdr2 is primarily expressed in liver and muscle, and mdr3 is primarily expressed in intestine and lung.

Previously, it was reported that resistance and susceptibility to infection with virulent Mycobacterium intracellulare (Mino strain) among several inbred mouse strains was identical to the pattern observed with bacille Calmette-Gu6rin (BCG), Salmonella typhimurium, and Leishmania donovani (Goto et al. 1984). In the present study, we set out to test whether the Bcg gene controlled resistance and susceptibility to M. intracellulare. First, the growth kinetics of M. intracellulare were compared in the organs of congenic susceptible (Bcg s) and resistant (Bcg r) mice. One million colony-forming units (CFU) of M. intracellulare were injected into the BALB/c (Bcg ~) and the congenic resistant C.D2 (Bcg r) mice intravenously, and the number of viable bacteria in the spleens was measured after 24 h and at 1, 2, 3, 6, and 12 weeks. The results in Figure 1 show that there was no significant difference in the bacterial CFU recovered from spleens of BALB/c (Bcg ~) and C.D2 (Bcg r) mice 24 h post-infection. Thereafter, the bacteria multiplied continuously in susceptible BALB/c spleens throughout the 12-week observation period. In contrast, no growth was observed in the spleens of resistant C.D2 mice during the first 3 weeks, and only a marginal increase in the spleen CFU occurred between the 3rd and 12th week after infection.The growth of M. intracellulare was also measured in the livers and lungs of congenic BALB/c-Bcg r and Bcg s mice on day 1 and at 3 and 6 weeks post-infection

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In the mouse, innate resistance or susceptibility to infection with numerous mycobacteria is controlled by the Bcg host resistance locus located on the centromeric region of chromosome 1. The resistance/susceptibility phenotype is expressed by the mature tissue macrophage and Bcg has been identified as a locus that is involved in the regulation of macrophage activation and in the modulation of acquired immune responses to mycobacteria. Experiments aimed at the cloning of the Bcg gene via a "reverse genetics" approach have generated a detailed genetic map in the immediate vicinity of the locus, placing Bcg within the reach of long-range eukaryotic cloning techniques. The chromosomal segment around Bcg in the mouse is exactly conserved onto the long arm (q) of human chromosome 2. Linkage of genetic markers from human chromosome 2q with susceptibility to leprosy or tuberculosis would support both the existence of a susceptibility gene in humans and the contention that this susceptibility gene is a homologue of the mouse Bcg locus.

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A full-length cDNA clone for the mouse mdr1 gene can confer multidrug resistance when introduced by transfection into otherwise drug-sensitive cells. In the same assay, a full-length cDNA clone for a closely related member of the mouse mdr gene family, mdr2, fails to confer multidrug resistance. To identify the domains of mdr1 which are essential for multidrug resistance and which may be functionally distinct in mdr2, we have constructed chimeric cDNA molecules in which discrete domains of mdr2 have been introduced into the homologous region of mdr1 and analyzed these chimeras for their capacity to transfer drug resistance. The two predicted ATP-binding domains of mdr2 were found to be functional, as either could complement the biological activity of mdr1. Likewise, a chimeric molecule in which the highly sequence divergent linker domain of mdr2 had been introduced in mdr1 could also confer drug resistance. However, the replacement of either the amino- or carboxy-terminus transmembrane (TM) domain regions of mdr1 by the homologous segments of mdr2 resulted in inactive chimeras. The replacement of as few as two TM domains from either the amino (TM5-6) or the carboxy (TM7-8) half of mdr1 by the homologous mdr2 regions was sufficient to destroy the activity of mdr1. These results suggest that the functional differences detected between mdr1 and mdr2 in our transfection assay reside within the predicted TM domains.