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The mouse mdr gene family consists of three distinct genes (mdrl, mdr2, and mdr3), for which we have isolated full-length cDNA clones. cDNA subfragments corresponding to discrete regions showing little sequence conservation among the three mdr genes were used as gene-specific DNA probes in hybridization experiments. Long-range mapping by pulse-field gel electrophoresis indicated that the three mdr genes are closely linked on a genomic DNA segment of approximately 625 kilobases. The gene order and direction of transcription of the three genes were determined and indicate the arrangement (5') mdr3 (3')-(5') mdrl (3')-(3') mdr2 (5'). Southern blotting analyses of genomic DNA from a panel of independently derived multidrug-resistant cell lines identified mdr gene amplification in 10 of 12 cell lines studied. In individual cell lines showing gene amplification, the copy number of each of the three mdr genes was identical, suggesting that the three mdr genes became amplified as part of a single amplicon in these cells. Although increased expression of all three mdr genes was detected in 2 of 12 cell lines tested, multidrug resistance was associated in 10 of 12 lines with the independent overexpression of either mdrl (7 of 12) or mdr3 (3 of 12) but not mdr2. mdrl overexpression was consistently associated with gene amplification, while increased mdr3 expression was detected in certain cell lines that did not show gene amplification. Increased levels of mdrl mRNA were linked to the overexpression of a P glycoprotein of apparent molecular weight 180,000 to 200,000, whereas increased mdr3 expression resulted in increased expression of a P glycoprotein of molecular weight 160,000 to 180,000. Our results suggest that at least two members of the mouse mdr gene family, mdrl and mdr3, can independently confer multidrug resistance in the cell lines examined.In cultured cells, multidrug resistance is associated with the overexpression of a heterogeneous group of antigenically related membrane phosphoglycoproteins, termed P glycoproteins (12). P glycoproteins are encoded by a small family of closely related genes given the appellation pgp or mdr which often become amplified in highly drug-resistant cell lines (reviewed in references 8, 12, and 15). The mdrlpgp gene family is composed of three members in rodents (hamster and mouse) and two members in humans (24 suggest that P glycoprotein functions in drug-resistant cells as an ATP-dependent drug efflux pump.Sequence analysis of full-length cDNA clones encoding mouse mdrl, mdr2, and mdr3 indicates that the three encoded proteins are highly homologous (overall amino acid homology of 75 to 85%) and share the same predicted structural domains (10a, 19, 20

Large insert clone libraries have been the primary resource used for the physical mapping of the human genome. Research directions in the genome community now are shifting direction from purely mapping to large-scale sequencing, which in turn, require new standards to be met by physical maps and large insert libraries. Bacterial artificial chromosome libraries offer enormous potential as the chosen substrate for both mapping and sequencing studies. Physical mapping, however, has come under some scrutiny as being ''redundant'' in the age of large-scale automated sequencing. We report the development and applications of nonelectrophoretic, optical approaches for high-resolution mapping of bacterial artificial chromosome that offer the potential to complement and thereby advance large-scale sequencing projects.

The MDR1 P-glycoprotein (Pgp), responsible for a clinically important form of multidrug resistance in cancer, is an ATPase efflux pump for multiple lipophilic drugs. The G185V mutation near transmembrane domain 3 of human Pgp increases its relative ability to transport several drugs, including etoposide, but decreases the transport of other substrates. MDR1 cDNA with the G185V substitution was used in a function-based selection to identify mutations that would further increase Pgp-mediated resistance to etoposide. This selection yielded the I186N substitution, adjacent to G185V. Pgps with G185V, I186N, or both mutations were compared to the wild-type Pgp for their ability to confer resistance to different drugs in NIH 3T3 cells. In contrast to the differential effects of G185V, I186N mutation increased resistance to all the tested drugs and augmented the effect of G185V on etoposide resistance. The effects of the mutations on conformational transitions of Pgp induced by different drugs were investigated using a conformation-sensitive antibody UIC2. Ligand-binding analysis of the drug-induced increase in UIC2 reactivity was used to determine the K(m) value that reflects the apparent affinity of drugs for Pgp, and the Hill number reflecting the apparent number of drug-binding sites. Both mutations altered the magnitude of drug-induced increases in UIC2 immunoreactivity, the K(m) values, and the Hill numbers for individual drugs. Mutation-induced changes in the magnitude of UIC2 reactivity shift did not correlate with the effects of the mutations on resistance to the corresponding drugs. In contrast, an increase or a decrease in drug resistance relative to that of the wild type was accompanied by a corresponding increase or decrease in the K(m) or in both the K(m) and the Hill number. These results suggest that mutations that alter the ability of Pgp to transport individual drugs change the apparent affinity and the apparent number of drug-binding sites in Pgp.

The ability to amplify routinely long PCR products (5-25 kb) with high specificity and fidelity, regardless of target template sequence or structure, would provide significant benefits to genome mapping and sequencing endeavors. Although occasional reports have described the generation of long PCR products, (1-4) such results have been difficult to replicate and have frequently utilized probe hybridization to Identify the specific product from nonspecific amplified DNA. Production of specific PCR products has generally been limited to target templates of less than 3 kb. (s) To extend the effective range of standard PCR amplification, it may be necessary to utilize alternative reaction conditions and/or components, such as novel thermostable DNA polymerases or accessory proteins. We describe the use of a model system to evaluate systematically methodological changes that might enable efficient long-range PCR. Specifically, the transposon TnSsupF has been used to Introduce randomly identical, known primer binding sites within separate Isolates of phage clones carrying Identical Inserts. (6) Transposon-based PCR allows us to study amplification of DNA fragments that vary in size and sequence using only a single set of primers, in the present studies, we describe conditions that enable PCR amplification of specific DNA templates ranging in size up to 9 kb. Some of the key features of our methodology Include the use of recombinant Thermus thermophilus (rTth) DNA polymerase, the addition of gelatin to the reaction mixture, the use of wax-mediated "hot starts ''(7) and, lastly, the use of autosegment extension thermocycling. These results also provide Insights into additional approaches that might further enhance our ability to perform long-distance PCR.

The objectives of the Human Genome Project are to create high-resolution genetic and physical maps, and ultimately to determine the complete nucleotide sequence of the human genome. The result of this initiative will be to localize the estimated 50,000-100,000 human genes, and acquire information that will enable development of a better understanding of the relationship between genome structure and function. To achieve these goals, new methodologies that provide more rapid, efficient, and cost effective means of genomic analysis will be required. From both conceptual and practical perspectives, the polymerase chain reaction (PCR) represents a fundamental technology for genome mapping and sequencing. The availability of PCR has allowed definition of a technically credible form that the final composite map of the human genome will take, as described in the sequence-tagged site proposal. Moreover, applications of PCR have provided efficient approaches for identifying, isolating, mapping, and sequencing DNA, many of which are amenable to automation. The versatility and power provided by PCR have encouraged its involvement in almost every aspect of human genome research, with new applications of PCR being developed on a continual basis.

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