MULTIPLE DRUG RESISTANCE GENE OF ASPERGILLUS FUMIGATUS

Abstract: The invention provides isolated nucleic acid compounds encoding a multiple drug resistance protein of Aspergillus fumigatus. Vectors and transformed host cells comprising the multiple drug resistance-encoding DNA of Aspergillus fumigatus MDR-1 are also provided. The invention further provides assays which utilize these transformed host cells.

Description: MULTIPLE DRUG RESISTANCE GENE OF ASPERGILLUS FUMIGATUS This invention relates to recombinant DNA technology. In particular, the invention concerns the cloning of nucleic acid encoding a multiple drug resistance protein of Aspergillus fumigat s . Multiple drug resistance (MDR) mediated by the human .mdr-1 gene product was initially recognized during the course of developing regimens for cancer chemotherapy (Fojo et al . , 1987, Journal of Clinical Oncology 5:1922-1927) . A multiple drug resistant cancer cell line exhibits resistance to high levels of a large variety of cytotoxic compounds. Frequently these cytotoxic compounds will have no common structural features nor will they interact with a common target within the cell. Resistance to these cytotoxic agents is mediated by an outward directed, ATP- dependent pump encoded by the mdr-1 gene. By this mechanism, toxic levels of a particular cytotoxic compound are not allowed to accumulate within the cell. MDR-like genes have been identified in a number of divergent organisms including numerous bacterial species, the fruit fly Drosophila melanogaster, Plasmodiu fal ciparum, the yeast Saccharomyces cerevisiae, Caenorhabdi tis elegans, Leishmania donovanii , marine sponges, the plant Arabidopεis thaliana , as well as Homo sapiens . Extensive searches have revealed several classes of compounds that are able to reverse the MDR phenotype of multiple drug resistant human cancer cell lines rendering them susceptible to the effects of cytotoxic compounds. These compounds, referred to herein as "MDR inhibitors", include for example, calcium channel blockers, anti- arrhythmics, antihypertensives, antibiotics, antihistamines, immuno-suppressants, steroid hormones, modified steroids, lipophilic cations, diterpenes, detergents, antidepressants, and antipsychotics (Gottesman and Pastan, 1993, Annual Review of Biochemistry 62:385- 427) . Clinical application of human MDR inhibitors to cancer chemotherapy has become an area of intensive focus for research. On another front, the discovery and development of antifungal compounds for specific fungal species has also met with some degree of success. Candida species represent the majority of fungal infections, and screens for new antifungal compounds have been designed to discover anti- Candida compounds. During development of antifungal agents, activity has generally been optimized based on activity against Candida albicans . As a consequence, these anti -Candida compounds frequently do not possess clinically significant activity against other fungal species such as Aspergillus fumigatus . However, it is interesting to note that at higher concentrations some anti-Candida compounds are able to kill Aspergillus fungal species. This suggests that the antifungal target (s) of these anti-Candida compounds is present in Aspergillus as well. Such results indicate that Aspergillus may possess a natural mechanism of resistance that permits them to survive in clinically relevant concentrations of antifungal compounds. Until the present invention, such a general mechanism of resistance to antifungal compounds in Aspergillus fumigatus has remained undescribed. The invention provides, inter alia, isolated nucleic acid molecules that comprise nucleic acid encoding a multiple drug resistance protein from Aspergillus fumigatus , known as Afu-MDRl, vectors encoding Afu-MDRl, and host cells transformed with these vectors. In another embodiment, the invention provides a method for determining the fungal MDR inhibition activity of a compound which comprises: a) growing a culture of yeast cells, transformed with a vector which provides expression of Afu-MDRl, in the presence of: (i) an antifungal agent to which said yeast cell is resistant, but to which said yeast cell is sensitive in its untransformed state; (ii) a compound suspected of possessing fungal MDR inhibition activity; and b) determining the fungal MDR inhibition activity of said compound by measuring the ability of the antifungal agent to inhibit the growth of said yeast cell. The restriction enzyme site and function maps presented in the accompanying drawings are approximate representations of cosmid pPSR-7 and plasmid pPSR-9, discussed herein. The restriction enzyme site information is not exhaustive. There may be more restriction enzyme sites of a given type on the vectors than actually shown on the maps. Figure 1 - A restriction enzyme site and function map of cosmid pPSR-7. Figure 2 - A restriction enzyme site and function map of plasmid pPSR-9. The present invention provides isolated nucleic acid molecules that comprise a nucleic acid sequence encoding Afu-MDRl. The amino acid sequence of Afu-MDRl is provided in the Sequence Listing as SEQ ID NO: 2. The cDNA (complementary deoxyribonucleic acid) sequence encoding Afu- MDRl is provided in the Sequence Listing as SEQ ID NO: 1. Those skilled in the art will recognize that the degenerate nature of the genetic code enables one to construct many different nucleic acid sequences that encode the amino acid sequence of SEQ ID NO: 2. The cDNA sequence depicted by SEQ ID NO: 1 is only one of many possible Afu- MDRl encoding sequences. Consequently, the constructions described below and in the accompanying examples for the preferred nucleic acid molecules, vectors, and transformants of the invention are illustrative and are not intended to limit the scope of the invention. All nucleotide and amino acid abbreviations used in this disclosure are those accepted by the United States Patent and Trademark Office as set forth in 37 C.F.R. §1.822 (b) (1994) . The term "vector" refers to any autonomously replicating or integrating agent, including but not limited to plasmids, cosmids, and viruses (including phage) , comprising a nucleic acid molecule to which one or more additional nucleic acid molecules can be added. Included in the definition of "vector" is the term "expression vector". Vectors are used either to amplify and/or to express deoxyribonucleic acid (DNA) , either genomic or cDNA, or RNA (ribonucleic acid) which encodes Afu-MDRl, or to amplify DNA or RNA that hybridizes with DNA or RNA encoding Afu-MDRl. The term "expression vector" refers to vectors which comprise a transcriptional promoter (hereinafter "promoter") and other regulatory sequences positioned to drive expression of a DNA segment that encodes Afu-MDRl. Expression vectors of the present invention are replicable DNA constructs in which a DNA sequence encoding Afu-MDRl is operably linked to suitable control sequences capable of effecting the expression of Afu-MDRl in a suitable host. Such control sequences include a promoter, an optional operator sequence to control transcription, a sequence encoding suitable mRNA ribosomal binding sites, and sequences which control termination of transcription and translation. DNA regions are operably linked when they are functionally related to each other. For example, a promoter is operably linked to a DNA coding sequence if it controls the transcription of the sequence, or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to permit translation. The term "MDR inhibition activity" refers to the ability of a compound to inhibit the MDR activity of a host cell, thereby increasing the antifungal activity of an antifungal compound against said host cell. In the present invention, Afu-MDRl may be synthesized by host cells transformed with vectors that provide for the expression of DNA encoding Afu-MDRl. The DNA encoding Afu- MDRl may be the natural sequence or a synthetic sequence or a combination of both ( "semi-synthetic sequence") . The in vi tro or in vivo transcription and translation of these sequences results in the production of Afu-MDRl. Synthetic and semi-synthetic sequences encoding Afu-MDRl may be constructed by techniques well known in the art. See Brown et al. (1979) Methods in Enzymology, Academic Press, N.Y., 68:109-151. Afu-MDRl-encoding DNA, or portions thereof, may be generated using a conventional DNA synthesizing apparatus such as the Applied Biosystems Model 380A or 380B DNA synthesizers (commercially available from Applied Biosystems, Inc., 850 Lincoln Center Drive, Foster City, CA 94404) . Owing to the natural degeneracy of the genetic code, the skilled artisan will recognize that a sizable yet definite number of nucleic acid sequences may be constructed which encode Afu-MDRl. All such nucleic acid sequences are provided by the present invention. These sequences can be prepared by a variety of methods and, therefore, the invention is not limited to any particular preparation means. The nucleic acid sequences of the invention can be produced by a number of procedures, including DNA synthesis, cDNA cloning, genomic cloning, polymerase chain reaction (PCR) technology, or a combination of these approaches. These and other techniques are described by Maniatis, e_t j__,. Molecular Cloninσ: A Laboratory Manual. Cold Spring Harbor Press, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1989), or Current Protocols in Molecular Bioloσv (F. M. Ausbel et al.. 1989). The contents of both of these references are incorporated herein by reference. In another aspect, this invention provides the genomic DNA encoding the Afu-MDRl. This DNA sequence is preferably obtained from cosmid pPSR-7. Cosmid pPSR-7 can be obtained from the host cell Escherichia coli XLl-Blue/pPSR-7 which was deposited in the permanent culture collection of the Northern Regional Research Laboratory (NRRL) , United States Department of Agriculture Service, 1815 North University Street, Peoria, IL 61604, on February 10, 1995, and is available under accession number NRRL B-21407. A restriction site and function map of cosmid pPSR-7 is provided as Figure 1 of the drawings. Cosmid pPSR-7 comprises the ColEl origin of replication (ColEl) which allows replication in E. coli host cells, and the ampicillin resistance gene (Amp) for selection of E. coli cells transformed with the cosmid grown in the presence of ampicillin. The cosmid further comprises the cos site (cos) from bacteriophage lambda, the neomycin resistance gene and the SV40 origin of replication. The genomic DNA encoding Afu-MDRl can be obtained from cosmid pPSR-7 on adjacent approximately 5.5 kilobase pair Xbal and 3.5 kilobase pair Xbal restriction enzyme fragments. Examination of the genomic DNA sequence encoding Afu- MDRl suggested the presence of introns. The precise location of suspected introns was determined by comparing the cDNA sequence encoding Afu-MDRl to the sequence of the genomic DNA encoding Afu-MDRl. The cDNA sequence was obtained in the following manner. Messenger RNA (mRNA) isolated from Aspergillus fumigatus was converted to cDNA by conventional methodologies. Polymeraεe Chain Reaction (PCR) primers were designed to amplify regions of the gene encoding Afu-MDRl suspected to contain introns. These regions were amplified from Afu-MDRl encoding cDNA using standard PCR methodologies. The PCR fragments generated from Afu-MDRl encoding cDNA were subcloned into the vector pCRScript™ (Stratagene, Inc.) . The DNA sequence of the subcloned DNA was determined using the dideoxy sequencing method. The cDNA sequence, which lacks introns, was then compared to the genomic DNA sequence to locate the precise position of the introns in the genomic DNA. To effect the translation of Afu-MDRl encoding DNA, one inserts the natural, synthetic, or semi-synthetic Afu- MDRl-encoding DNA sequence into any of a large number of appropriate expression vectors through the use of appropriate restriction endonucleases and DNA ligaseε. Synthetic and semi-synthetic Afu-MDRl encoding DNA sequences can be designed, and natural Afu-MDRl encoding nucleic acid can be modified, to possess restriction endonuclease cleavage sites to facilitate isolation from and integration into these vectors. Particular restriction endonucleases employed will be dictated by the restriction endonuclease cleavage pattern of the expression vector utilized. Restriction enzyme sites are chosen so as to properly orient the Afu-MDRl encoding DNA with the control sequences to achieve proper in-frame transcription and translation of the Afu-MDRl molecule. The Afu-MDRl encoding DNA must be positioned so as to be in proper reading frame with the promoter and ribosome binding site of the expression vector, both of which are functional in the host cell in which Afu-MDRl is to be expressed. Expression of Afu-MDRl in yeast cells, such as Saccharomyceε cerevisiae is preferred. Suitable promoter sequences for use with yeast hosts include the promoters for 3-phosphoglycerate kinase (found on plasmid pAP12BD ATCC 53231 and described in U.S. Patent No. 4,935,350, June 19, 1990) or other glycolytic enzymes such as enolase (found on plasmid pACl ATCC 39532), glyceraldehyde-3- phosphate dehydrogenase (derived from plasmid pHcGAPCl ATCC 57090, 57091), hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3- phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. Inducible yeast promoters have the additional advantage of transcription controlled by growth conditions. Such promoters include the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphotase, degradative enzymes associated with nitrogen metabolism, metallothionein (contained on plasmid vector pCL28XhoLHBPV ATCC 39475, United States Patent No. 4,840,896) , glyceraldehyde 3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization (GAL1 found on plasmid pRYl21 ATCC 37658 and on plasmid pPSR-9, described below) . Suitable vectors and promoters for use in yeast expression are further described by R. Hitzeman e_£ _ .. , in European Patent Publication No. 73,657A. Yeast enhancers such as the UAS Gal enhancer from Saccharomyceε cerevisiae (found in conjunction with the CYC1 promoter on plasmid YEpsec--hIlbeta, ATCC 67024), also are advantageously used with yeast promoters. A variety of expression vectors useful in the present invention are well known in the art. For expression in Saccharomyces , the plasmid YRp7, for example, (ATCC-40053, Stinchcomb, e_£ __ % . , 1979, Nature 282:39; Kingsman __, al.. 1979, Gene 7:141; Tschemper e_£. _ . , 1980, Gene 10:157) is commonly used. This plasmid contains the trp gene which provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example ATCC no. 44076 or PEP4-1 (Jones, 1977, Genetics 85:12) . Expression vectors useful in the expression of Afu- MDRl can be constructed by a number of methods. For example, the cDNA sequence encoding Afu-MDRl can be synthesized using DNA synthesis techniques such as those described above. Such synthetic DNA can be synthesized to contain cohesive ends that allow facile cloning into an appropriately digested expression vector. For example, the cDNA encoding Afu-MDRl can be synthesized to contain iVotl cohesive ends. Such a synthetic DNA fragment can be ligated into a Notl-digested expression vector such as pYES-2 (Invitrogen Corp., San Diego CA 92121). The resultant plasmid is designated herein as pPSR-9. Plasmid pPSR-9 is useful for the expression of the Afu-MDRl in Saccharomyces cerevisiae. Plasmid pPSR-9 can also be constructed in the following manner. Logarithmic phase Aspergillus fumigatus mycelia are disrupted by grinding under liquid nitrogen according to the procedure of Minuth et al . , 1982 (Current Genetics 5:227-231) . Aspergillus fumigatus mRNA is preferably isolated from the disrupted mycelia using the TM QuickPrep mRNA Purification Kit (Pharmacia Biotech) according to the instructions of the manufacturer. cDNA is produced from the isolated mRNA using the TimeSaver® cDNA Synthesis Kit (Pharmacia Biotech) using oligo (dT) according to the procedure described by the manufacturer. In this process an EcoRI/Notl adapter (Stratagene, Inc.) is ligated to each end of the double stranded cDNA. The adapter modified cDNA is ligated into the vector Lambda Zap R II using the Predigested Lambda Zap R II/£coRI/CIAP Cloning Kit (Stratagene, Inc.) according to the instructions of the manufacturer to create a cDNA library. The library is screened for full-length cDNA encoding Afu-MDRl using a 32p-radiolabeled fragment of the Afu-MDRl gene. This radiolabeled fragment is produced from the Afu- MDRl genomic DNA present in cosmid pPSR-7. Utilizing the polymerase chain reaction (Gene Amp® Kit, Perkin Elmer Cetus, Norwalk, CT) and the following primers: 5'- AGCGATTCGAAACCTGAGTCG-3 " (SEQ. I D. NO: 3); and 5'- TCGTTGATTTTCCACCTCCTG-3 ' (SEQ. ID. No: 4), a Afu-MDRl radiolabeled fragment is obtained. This radiolabeled fragment is used to probe the Aspergillus fumigatus cDNA library using standard hybridization techniques (Maniatis et al . 1992). In this manner, a full-length cDNA clone is recovered from the Aspergillus fumigatus cDNA library. A full-length cDNA clone recovered from the library is removed from the Lambda Zap R II vector by digestion with the restriction endonuclease Notl which produces an approximately 2600 base pair D A fragment encoding Afu- MDRl. The vector DΝA fragment and the desired approximately 2600 base pair Notl DΝA restriction fragment are separated by agarose gel electrophoresis. The desired approximately 2600 base pair Notl DΝA fragment encoding Afu-MDRl is isolated by cutting out that portion of the agarose gel containing the fragment and electroeluting the DΝA using a Centrilutor Micro-electroeluter (Amicon, Inc.) according to the manufacturer's instructions. The isolated approximately 2600 base pair Notl restriction fragment encoding Afu-MDRl is ligated to Notl digested pYES2 vector DΝA. The correct orientation of the insert DΝA is screened for by restriction endonuclease digestion using asymmetric sites located with the coding region and the flanking vector. The resultant plasmid, pPSR-9, is useful for the expression of the Afu-MDRl in Saccharomyces cerevisiae. A representation of plasmid pPSR-9 is provided as Figure 2. As noted above, this plasmid contains the Afu- MDRl encoding DΝA operably linked to the Saccharomyces cerevisiae GALl promoter (P Gall) . Plasmid pPSR-9 also comprises the yeast transcription terminator cycl (T cycl) located in a position 3' to the Afu-MDRl encoding DΝA. Plasmid pPΞR-9 further comprises the ColEl origin of replication (ColEl) which allows replication in Escheri chia coli host cells, and the ampicillin resistance gene (Amp) for selection of E. coli cells transformed with the plasmid and grown in the presence of ampicillin. Plasmid pPSR-9 further comprises the yeast 2μ origin of replication (2μ ori) allowing replication in yeast host cells, the yeast URA3 gene for selection of S. cerevisiae cells transformed with the plasmid grown in a medium lacking uracil, and the fl origin of replication (fl ori) from filamentous phage fl. In a preferred embodiment of the invention Saccharomyces cerevisiae IΝVScl or IΝVSc2 cells (Invitrogen Corp., Sorrento Valley Blvd., San Diego CA 92121) are employed as host cells, but numerous other cell lines are available for this use. The transformed host cells are plated on an appropriate medium under selective pressure (minimal medium lacking uracil) . The cultures are then incubated for a time and temperature appropriate to the host cell line employed. The techniques involved in the transformation of yeast cells such as Saccharomyces cerevisiae cells are well known in the art and may be found in such general references as Ausubel et al . , Current Protocols in Molecular Biology (1989), John Wiley & Sons, New York, NY and supplements. The precise conditions under which the transformed yeast cells are cultured is dependent upon the nature of the yeast host cell line and the vectors employed. Nucleic acid, either RNA or DNA, which encodes Afu- MDRl, or a portion thereof, is also useful in producing nucleic acid molecules useful in diagnostic assays for the detection of Afu-MDRl mRNA, Afu-MDRl cDNA, or Afu-MDRl genomic DNA. Further, nucleic acid, either RNA or DNA, which does not encode Afu-MDRl, but which nonetheless is capable of hybridizing with Afu-MDRl-encoding DNA or RNA is also useful in such diagnostic assays. These nucleic acid molecules may be covalently labeled by known methods with a detectable moiety such as a fluorescent group, a radioactive atom or a chemiluminescent group. The labeled nucleic acid is then used in conventional hybridization assays, such as Southern or Northern hybridization assays, or polymerase chain reaction assays (PCR) , to identify hybridizing DNA, cDNA, or RNA molecules. PCR assays may also be performed using unlabeled nucleic acid molecules. Such assays may be employed to identify Afu-MDRl vectors and transformants and in _j_ vitro diagnosis to detect Afu- MDRl-like mRNA, cDNA, or genomic DNA from other organisms. Combination therapy involving an antifungal agent possessing a proven spectrum of activity in combination with a fungal MDR inhibitor to treat fungal infections enables an extension of the spectrum of antifungal activity for a given antifungal compound which previously had only demonstrated limited clinically relevant antifungal activity. Compounds with demonstrated antifungal activity can also be potentiated by a fungal MDR inhibitor such that the antifungal activity of these compounds is extended to previously resistant species. To identify compounds useful in such combination therapy the present invention provides an assay method for identifying compounds with Aspergillus fumigatus MDR inhibition activity. Host cells that express Afu-MDRl provide an excellent means for the identification of compounds useful as inhibitors of Aspergillus fumigatus MDR activity. Generally, the assay utilizes a culture of a yeast cell transformed with a vector which provides expression of Afu-MDRl. The expression of the Afu-MDRl by the host cell enables the host cell to grow in the presence of an antifungal compound to which the yeast cell is sensitive to in the untransformed state. Thus, the transformed yeast cell culture is grown in the presence of i) an antifungal agent to which the untransformed yeast cell is sensitive, but to which the transformed host cell is resistant, and ii) a compound that is suspected of being an Aspergillus fumigatus MDR inhibitor. The effect of the suspected MDR inhibitor is measured by testing for the ability of the antifungal compound to inhibit the growth of the transformed yeast cell. Such inhibition will occur if the suspected Aspergillus fumigatus MDR inhibitor blocks the ability of the Afu-MDRl to prevent the antifungal compound from acting on the yeast cell. An illustrative example of such an assay is provided in Example 3. In order to illustrate more fully the operation of this invention, the following examples are provided, but are not to be construed as a limitation on the scope of the invention. Source of the MDR encoding Genomic DNA of AsOerσi llus fumiαatus Isolation of Cosmid DPSR-7 A lyophil of Escherichia coli XLl-Blue/pPSR-7 can be obtained from the Northern Regional Research Laboratories (NRRL), Peoria, Illinois 61604, under the accession number NRRL B-21407. Cosmid pPSR-7 may be isolated from NRRL B- 21407 using techniques that are well-known to those skilled in the art. See Sambrook et al . , Molecular Cloning: A Laboratory Manual (1988), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY or Ausubel et al., Current Protocols in Molecular Biology (1989), John Wiley & Sons, New York, NY and supplements. Example 2. Expression of the Afu-MDRl Protein Saccharomyces cerevisiae INVScl cells (Invitrogen Corp., San Diego CA 92191) are transformed with the plasmid pPSR-9 by the technique described by J. D. Beggs, 1988, Nature 275:104-109). The transformed yeast cells are grown in a broth medium containing YNB/CSM-Ura/raf (YNB/CSM-Ura [Yeast Nitrogen Base (Difco Laboratories, Detroit, MI) supplemented with CSM-URA (Bio 101, Inc.)] supplemented with 4% raffinose) at 28°C in a shaker incubator until the culture is saturated. To induce expression of the Afu- MDRl, a portion of the culture is used to inoculate a flask containing YNB/CSM-Ura medium supplemented with 2% galactose (YΝB/CSM-Ura/gal) rather than raffinose as the sole carbon source. The inoculated flask is incubated at 28°C for about 16 hours. Example Antifungal Potentiator Assay Approximately 1 x 10^ cells of a Saccharomyces cerevisiae IΝVScl/pPSR-9 culture are delivered to each of several agar plates containing YNB/CSM-Ura/gal. The agar surface is allowed to dry in a biohazard hood. Saccharomyces cerevisiae INVScl/pPSR-9 cells express the Afu-MDRl activity. An antifungal compound that the untransformed yeast cell is typically sensitive to, such as R106I (United States Patent No. 5,057,493, which is hereby incorporated herein by reference) , is dissolved in 100% ethanol at a concentration of either 1 or 7 mg/ml. Twenty μl of the 1 mg/ml solution is delivered to an antibiotic susceptibility test disc (Difco Laboratories, Detroit, MI) . After addition of the antifungal solution the disc is allowed to air dry in a biohazard hood. When dry, the disc is placed on the surface of the petri plates containing the Saccharomyces cerevisiae INVScl/pPSR-9 cells. Compounds to be tested for the ability to inhibit Afu- MDRl are dissolved in dimethylsulfoxide (DMSO) . The amount of compound added to the DMSO depends on the solubility of the individual compound to be tested. Twenty μl of the suspensions containing a compound to be tested are delivered to an antibiotic susceptibility test disc (Difco Laboratories, Detroit, MI) . The disc is then placed on the surface of the dried petri plates containing the Saccharomyces cerevisiae INVScl/pPSR-9 cells approximately 2 cm from the antifungal-containing disc. Petri plates containing the two discs are incubated at 28°C for about 16-48 hours. Following this incubation period, the petri plates are examined for zones of growth inhibition around the discs. A zone of growth inhibition near the antifungal disc on the test plate indicates that the compound being tested for MDR inhibition activity blocks the activity of Afu-MDRl and allows the antifungal compound to inhibit the growth of the yeast host cell. Such compounds are said to possess MDR inhibition activity. Little or no zone of growth inhibition indicates that the test compound does not block MDR activity and, thus, the Afu-MDRl is allowed to act upon the antifungal compound to prevent its activity upon the host cell. SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: Peery, Robert B. Skatrud, Paul L. (ii) TITLE OF INVENTION: Multiple Drug Resistance Gene Of Aspergillus Fumigatus (iii) NUMBER OF SEQUENCES: 2 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Eli Lilly and Company/Patent Division (B) STREET: Lilly Corporate Center (C) CITY: Indianapolis (D) STATE: Indiana (E) COUNTRY: US (F) ZIP: 46285 (V) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentin Release #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE: (C) CLASSIFICATION: (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Plant G. , Thomas (B) REGISTRATION NUMBER: 35784 (C) REFERENCE/DOCKET NUMBER: X-9682 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: (317) 276-2459 (B) TELEFAX: (317) 277-1917 (2) INFORMATION FOR SEQ ID NO:l: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2376 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA ( ix ) FEATURE : (A) NAME/KEY: CDS (B) LOCATION: 1..2376 (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: ATG CGC GGG ATT AGG TCG CTT CCC TGC TGG GCT CCT GGG TTG AGC ACG 48 Met Arg Gly lie Arg Ser Leu Pro Cys Trp Ala Pro Gly Leu Ser Thr 1 5 10 15 AAG CGA ATT CCT CCA CGA GAA CTG TTT GCA GAC CTA TTT CCT AAT GCC 96 Lys Arg lie Pro Pro Arg Glu Leu Phe Ala Asp Leu Phe Pro Asn Ala 20 25 30 TGC GTA ATT TCT GCG AGA CAC AGC GCT CGC AAT GGT CTC ATC CGA CAG 144 Cys Val lie Ser Ala Arg His Ser Ala Arg Asn Gly Leu lie Arg Gin 35 40 45 TTT TCG GGT TGC TCC GGG TCA ATA AGC AAC AGT TGC AAC CCG CGT CCG 192 Phe Ser Gly Cys Ser Gly Ser lie Ser Asn Ser Cys Asn Pro Arg Pro 50 55 60 TAT CGA TCC GCA ATT ACG TCC TTG TTA TCC GCC AAT GTC TGT AGT AAA 240 Tyr Arg Ser Ala lie Thr Ser Leu Leu Ser Ala Asn Val Cys Ser Lys 65 70 75 80 GGG GTT TCG GCC GTT CAA CCG CGA TTT TTG AGT ACA GTC AGG CTG TTT 288 Gly Val Ser Ala Val Gin Pro Arg Phe Leu Ser Thr Val Arg Leu Phe 85 90 95 TCT ACC TCC CAG CGA TCA TTG GAG CCA AAG TCG AAC GTC AAG TCG ACA 336 Ser Thr Ser Gin Arg Ser Leu Glu Pro Lys Ser Asn Val Lys Ser Thr 100 105 110 GGC GGT CAG GTA GTG AGG CCA GAG TTA CAC CAG GAT CAG GAG CAT GAG 384 Gly Gly Gin Val Val Arg Pro Glu Leu His Gin Asp Gin Glu His Glu 115 120 125 GAC ATC GAG AAA GGC TTT GAG CTA TCG GAG AGA GCC GCT CAG GCA GCA 432 Asp lie Glu Lys Gly Phe Glu Leu Ser Glu Arg Ala Ala Gin Ala Ala 130 135 140 CAG GTC AAC CTC AGC GCG AAG CTC GCC AAA GAC GGT GCT GCT GGA AAG 480 Gin Val Asn Leu Ser Ala Lys Leu Ala Lys Asp Gly Ala Ala Gly Lys 145 150 155 160 AAG GCC GGG TTC AAG GAA ATA TGG AGG CTC TTA CTG ATT GCT CGA CCG 528 Lys Ala Gly Phe Lys Glu lie Trp Arg Leu Leu Leu lie Ala Arg Pro 165 170 175 GAG GCG AAG AAG CTC GCC TTA GCC TTC CTT TTC TTG CTG GTA TCA TCT 576 Glu Ala Lys Lys Leu Ala Leu Ala Phe Leu Phe Leu Leu Val Ser Ser 180 185 190 GGT ATT ACA ATG TCC ATC CCA TTT TCG ATT GGA AAG ATC ATG GAC ACC 624 Gly lie Thr Met Ser lie Pro Phe Ser lie Gly Lys lie Met Asp Thr 195 200 205 TCG ACA AAA GCG ACT ACA GAA GGT GGT AAT GAG CTC TTT GGT CTG AGC 672 Ser Thr Lys Ala Thr Thr Glu Gly Gly Asn Glu Leu Phe Gly Leu Ser 210 215 220 CTT CCC ATG TTC TAT GGT GCG TTG GCT GGA ATT CTC ACT CTG GGC GCT 720 Leu Pro Met Phe Tyr Gly Ala Leu Ala Gly lie Leu Thr Leu Gly Ala 225 230 235 240 GCG GCG AAT TAT GGT CGC ATT ATC ATC CTG CGT ATC GTT GGT GAG CGT 768 Ala Ala Asn Tyr Gly Arg lie lie lie Leu Arg lie Val Gly Glu Arg 245 250 255 ATT GTT GCC AGA CTT CGC TCG AAG CTC TTC CGC CAG ACA TTT GTG CAA 816 lie Val Ala Arg Leu Arg Ser Lys Leu Phe Arg Gin Thr Phe Val Gin 260 265 270 GAT GCA GAG TTC TTC GAT GCG AAT CGG GTC GGT GAC TTG ATT TCT CGC 864 Asp Ala Glu Phe Phe Asp Ala Asn Arg Val Gly Asp Leu lie Ser Arg 275 280 285 CTT AGC TCT GAT ACC ATT ATT GTC GGC AAG AGT ATC ACA CAG AAC CTG 912 Leu Ser Ser Asp Thr lie lie Val Gly Lys Ser lie Thr Gin Asn Leu 290 295 300 TCC GAT GGA CTG CGC GCT GCA GTT AGC GGT GCA GCA GGG TTC GGC TTA 960 Ser Asp Gly Leu Arg Ala Ala Val Ser Gly Ala Ala Gly Phe Gly Leu 305 310 315 320 ATG GCC TAC GTC AGT CTC AAG CTC TCC AGT ATA CTG GCT CTC CTT CTC 1008 Met Ala Tyr Val Ser Leu Lys Leu Ser Ser He Leu Ala Leu Leu Leu 325 330 335 CCT CCT ATC GGC CTC GGG GCT TTT TTC TAC GGA CGA GCG ATT CGA AAC 1056 Pro Pro He Gly Leu Gly Ala Phe Phe Tyr Gly Arg Ala He Arg Asn 340 345 350 CTG AGT CGT CAA ATC CAG AGG AAT CTT GGG ACC TTG ACT AAG ATT GCG 1104 Leu Ser Arg Gin He Gin Arg Asn Leu Gly Thr Leu Thr Lys He Ala 355 360 365 GAG GAG CGC CTA GGC AAC GTC AAG ACT AGC CAG TCC TTT GCT GGC GAG 1152 Glu Glu Arg Leu Gly Asn Val Lys Thr Ser Gin Ser Phe Ala Gly Glu 370 375 380 GTT CTC GAG GTT CGT CGG TAT AAC AAT CAA GTG CGA AAG ATC TTT GAA 1200 Val Leu Glu Val Arg Arg Tyr Asn Asn Gin Val Arg Lys He Phe Glu 385 390 395 400 CTT GGC AAA AAA GAA TCC CTT ATC AGT GCC ACA TTC TTT AGC TCC ACC 1248 Leu Gly Lys Lys Glu Ser Leu He Ser Ala Thr Phe Phe Ser Ser Thr 405 410 415 GGG TTT GCT GGC AAC ATG ACC ATC CTG GCA TTG CTG TAC GTC GGA GGA 1296 Gly Phe Ala Gly Asn Met Thr He Leu Ala Leu Leu Tyr Val Gly Gly 420 425 430 GGC ATG GTC CAA TCT GGT GCC ATA ACA ATT GGA GAA TTA ACT TCA TTC 1344 Gly Met Val Gin Ser Gly Ala He Thr He Gly Glu Leu Thr Ser Phe 435 440 445 CTG ATG TAC ACA GCG TAC GCA GGG TCA AGC ATG TTC GGT CTT TCG AGC 1392 Leu Met Tyr Thr Ala Tyr Ala Gly Ser Ser Met Phe Gly Leu Ser Ser 450 455 460 TTT TAC TCT GAA CTG ATG AAA GGT GTC GGG GCA GCT AGT CGA CTG TTT 1440 Phe Tyr Ser Glu Leu Met Lys Gly Val Gly Ala Ala Ser Arg Leu Phe 465 470 475 480 GAA TTA CAG GAT CGT CAG CCA ACG ATA TCT CCA ACC AAG GGC GAG AAG 1488 Glu Leu Gin Asp Arg Gin Pro Thr He Ser Pro Thr Lys Gly Glu Lys 485 490 495 GTG GCT TCG GCG CGA GGA CCA ATT CGC TTC GAA AAC GTG ACT TTT AGT 1536 Val Ala Ser Ala Arg Gly Pro He Arg Phe Glu Asn Val Thr Phe Ser 500 505 510 TAC CCG ACT CGT CCT GCT GTT CCT ATT TTT AGG GAT CTC AAT TTC GAA 1584 Tyr Pro Thr Arg Pro Ala Val Pro He Phe Arg Asp Leu Asn Phe Glu 515 520 525 ATT CCC CAA GGG ACC AAT GTT GCT ATC GTT GGG CCC TCA GGA GGT GGA 1632 He Pro Gin Gly Thr Asn Val Ala He Val Gly Pro Ser Gly Gly Gly 530 535 540 AAA TCA ACG ATC GCT TCT ATT TTG CTC CGT TTC TAC TCT CCT ACC GAA 1680 Lys Ser Thr He Ala Ser He Leu Leu Arg Phe Tyr Ser Pro Thr Glu 545 550 555 560 GGA AGA GTT CTC ATC GGT GGC AAA GAC ATC ACC CAT ATG AAC GCC AAA 1728 Gly Arg Val Leu He Gly Gly Lys Asp He Thr His Met Asn Ala Lys 565 570 575 TCT CTT CGA CGA AAG ATT GGA ATC GTC TCC CAA GAG CCT GTC CTC TTT 1776 Ser Leu Arg Arg Lys He Gly He Val Ser Gin Glu Pro Val Leu Phe 580 585 590 TCG GGG ACT ATA GCG GAG AAC ATT GCG TAT GGT AAA CCT CAG GCC AAG 1824 Ser Gly Thr He Ala Glu Asn He Ala Tyr Gly Lys Pro Gin Ala Lys 595 600 605 CGG TCA GAG ATC GTT GCA GCT GCG CGC AAG GCG AAC TGC CAG TTC ATA 1872 Arg Ser Glu He Val Ala Ala Ala Arg Lys Ala Asn Cys Gin Phe He 610 615 620 AGT GAC TTT CCG GAT GGT CTT GAC ACC CAG GTC GGA CCC CGG GGC GCT 1920 Ser Asp Phe Pro Asp Gly Leu Asp Thr Gin Val Gly Pro Arg Gly Ala 625 630 635 640 CAG CTT TCT GGT GGC CAG AAG CAG CGC ATT GCG ATT GCC CGG GCT CTG 1968 Gin Leu Ser Gly Gly Gin Lys Gin Arg He Ala He Ala Arg Ala Leu 645 650 655 ATC AAG GAT CCT GAT ATT CTC ATA CTC GAT GAA GCA ACT TCT GCT CTC 2016 He Lys Asp Pro Asp He Leu He Leu Asp Glu Ala Thr Ser Ala Leu 660 665 670 GAT GCG GAG TCC GAA ACT CTT GTG AAC AGT GCT CTT ACC GCG CTC CTT 2064 Asp Ala Glu Ser Glu Thr Leu Val Asn Ser Ala Leu Thr Ala Leu Leu 675 680 685 CGT GGT AAC AAT ACT ACC ATT AGC ATC GCT CAT CGA CTC TCT ACT ATT 2112 Arg Gly Asn Asn Thr Thr He Ser He Ala His Arg Leu Ser Thr He 690 695 700 AAG AGA TCC GAT ACG ATT ATT GTC CTT GGT CCC GAC GGA AGA GTA GCC 2160 Lys Arg Ser Asp Thr He He Val Leu Gly Pro Asp Gly Arg Val Ala 705 710 715 720 GAA CAA GGC AGT TAT GAG GAG CTC AGT GCT CGC CCT GAT GGC GCC TTC 2208 Glu Gin Gly Ser Tyr Glu Glu Leu Ser Ala Arg Pro Asp Gly Ala Phe 725 730 735 ACC AAA TTG ATG GAG TGG CAG ATG AGC GGC GGT GAG GTG ATG GAC CAG 2256 Thr Lys Leu Met Glu Trp Gin Met Ser Gly Gly Glu Val Met Asp Gin 740 745 750 CTC GCC AAC ACT CCT GCC AAC CCG GTG GCG CAG GAA ACT TCT TGG GAC 2304 Leu Ala Asn Thr Pro Ala Asn Pro Val Ala Gin Glu Thr Ser Trp Asp 755 760 765 TTG CAG AGT GAT GAC GGG ACA GAG ATA TCC GAG GAT ACC AAC ATC CCC 2352 Leu Gin Ser Asp Asp Gly Thr Glu He Ser Glu Asp Thr Asn He Pro 770 775 780 TCA GAG CCG CGA AAA ATT GAT TA 2376 Ser Glu Pro Arg Lys He Asp 785 790 (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 791 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Met Arg Gly He Arg Ser Leu Pro Cys Trp Ala Pro Gly Leu Ser Thr 1 5 10 15 Lys Arg He Pro Pro Arg Glu Leu Phe Ala Asp Leu Phe Pro Asn Ala 20 25 30 Cys Val He Ser Ala Arg His Ser Ala Arg Asn Gly Leu He Arg Gin 35 40 45 Phe Ser Gly Cys Ser Gly Ser He Ser Asn Ser Cys Asn Pro Arg Pro 50 55 60 Tyr Arg Ser Ala He Thr Ser Leu Leu Ser Ala Asn Val Cys Ser Lys 65 70 75 80 Gly Val Ser Ala Val Gin Pro Arg Phe Leu Ser Thr Val Arg Leu Phe 85 90 95 Ser Thr Ser Gin Arg Ser Leu Glu Pro Lys Ser Asn Val Lys Ser Thr 100 105 110 Gly Gly Gin Val Val Arg Pro Glu Leu His Gin Asp Gin Glu His Glu 115 120 125 Asp He Glu Lys Gly Phe Glu Leu Ser Glu Arg Ala Ala Gin Ala Ala 130 135 140 Gin Val Asn Leu Ser Ala Lys Leu Ala Lys Asp Gly Ala Ala Gly Lys 145 150 155 160 Lys Ala Gly Phe Lys Glu He Trp Arg Leu Leu Leu He Ala Arg Pro 165 170 175 Glu Ala Lys Lys Leu Ala Leu Ala Phe Leu Phe Leu Leu Val Ser Ser 180 185 190 Gly He Thr Met Ser He Pro Phe Ser He Gly Lys He Met Asp Thr 195 200 205 Ser Thr Lys Ala Thr Thr Glu Gly Gly Asn Glu Leu Phe Gly Leu Ser 210 215 220 Leu Pro Met Phe Tyr Gly Ala Leu Ala Gly He Leu Thr Leu Gly Ala 225 230 235 240 Ala Ala Asn Tyr Gly Arg He He He Leu Arg He Val Gly Glu Arg 245 250 255 He Val Ala Arg Leu Arg Ser Lys Leu Phe Arg Gin Thr Phe Val Gin 260 265 270 Asp Ala Glu Phe Phe Asp Ala Asn Arg Val Gly Asp Leu He Ser Arg 275 280 285 Leu Ser Ser Asp Thr He He Val Gly Lys Ser He Thr Gin Asn Leu 290 295 300 Ser Asp Gly Leu Arg Ala Ala Val Ser Gly Ala Ala Gly Phe Gly Leu 305 310 315 320 Met Ala Tyr Val Ser Leu Lys Leu Ser Ser He Leu Ala Leu Leu Leu 325 330 335 Pro Pro He Gly Leu Gly Ala Phe Phe Tyr Gly Arg Ala He Arg Asn 340 345 350 Leu Ser Arg Gin He Gin Arg Asn Leu Gly Thr Leu Thr Lys He Ala 355 360 365 Glu Glu Arg Leu Gly Asn Val Lys Thr Ser Gin Ser Phe Ala Gly Glu 370 375 380 Val Leu Glu Val Arg Arg Tyr Asn Asn Gin Val Arg Lys He Phe Glu 385 390 395 400 Leu Gly Lys Lys Glu Ser Leu He Ser Ala Thr Phe Phe Ser Ser Thr 405 410 415 Gly Phe Ala Gly Asn Met Thr He Leu Ala Leu Leu Tyr Val Gly Gly 420 425 430 Gly Met Val Gin Ser Gly Ala He Thr He Gly Glu Leu Thr Ser Phe 435 440 445 Leu Met Tyr Thr Ala Tyr Ala Gly Ser Ser Met Phe Gly Leu Ser Ser 450 455 460 Phe Tyr Ser Glu Leu Met Lys Gly Val Gly Ala Ala Ser Arg Leu Phe 465 470 475 480 Glu Leu Gin Asp Arg Gin Pro Thr He Ser Pro Thr Lys Gly Glu Lys 485 490 495 Val Ala Ser Ala Arg Gly Pro He Arg Phe Glu Asn Val Thr Phe Ser 500 505 510 Tyr Pro Thr Arg Pro Ala Val Pro He Phe Arg Asp Leu Asn Phe Glu 515 520 525 He Pro Gin Gly Thr Asn Val Ala He Val Gly Pro Ser Gly Gly Gly 530 535 540 Lys Ser Thr He Ala Ser He Leu Leu Arg Phe Tyr Ser Pro Thr Glu 545 550 555 560 Gly Arg Val Leu He Gly Gly Lys Asp He Thr His Met Asn Ala Lys 565 570 575 Ser Leu Arg Arg Lys He Gly He Val Ser Gin Glu Pro Val Leu Phe 580 585 590 Ser Gly Thr He Ala Glu Asn He Ala Tyr Gly Lys Pro Gin Ala Lys 595 600 605 Arg Ser Glu He Val Ala Ala Ala Arg Lys Ala Asn Cys Gin Phe He 610 615 620 Ser Asp Phe Pro Asp Gly Leu Asp Thr Gin Val Gly Pro Arg Gly Ala 625 630 635 640 Gin Leu Ser Gly Gly Gin Lys Gin Arg He Ala He Ala Arg Ala Leu 645 650 655 He Lys Asp Pro Asp He Leu He Leu Asp Glu Ala Thr Ser Ala Leu 660 665 670 Asp Ala Glu Ser Glu Thr Leu Val Asn Ser Ala Leu Thr Ala Leu Leu 675 680 685 Arg Gly Asn Asn Thr Thr He Ser He Ala His Arg Leu Ser Thr He 690 695 700 Lys Arg Ser Asp Thr He He Val Leu Gly Pro Asp Gly Arg Val Ala 705 710 715 720 Glu Gin Gly Ser Tyr Glu Glu Leu Ser Ala Arg Pro Asp Gly Ala Phe 725 730 735 Thr Lys Leu Met Glu Trp Gin Met Ser Gly Gly Glu Val Met Asp Gin 740 745 750 Leu Ala Asn Thr Pro Ala Asn Pro Val Ala Gin Glu Thr Ser Trp Asp 755 760 765 Leu Gin Ser Asp Asp Gly Thr Glu He Ser Glu Asp Thr Asn He Pro 770 775 780 Ser Glu Pro Arg Lys He Asp 785 790

Claims: _____}__ 1. An isolated nucleic acid molecule that comprises nucleic acid encoding Aspergillus fumigatus MDR-1. 2. A nucleic acid molecule of Claim 1 which is DNA. 3. The nucleic acid molecule of Claim 2 that is SEQ. ID. No. 1. 4. The nucleic acid molecule of Claim 1 that is genomic DNA. 5. A nucleic acid molecule of Claim 1 or a portion thereof, which is labeled with a detectable moiety. 6. A nucleic acid molecule of Claim 4 wherein the detectable moiety is selected from the group consisting of a fluorescent label, a radioactive atom, and a chemiluminescent label. 7. A vector comprising the nucleic acid of Claim 1. 8. A vector comprising the nucleic acid of Claim 2. 9. A vector comprising the nucleic acid of Claim 3. 10. A vector comprising the nucleic acid of Claim 4. 11. A host cell containing the vector of Claim 7. 12. A host cell containing the vector of Claim 8. 13. A host cell containing the vector of Claim 9. 14. A host cell containing the vector of Claim 10. 15. The host cell of Claim 11 that is a yeast cell. 16. The host cell of Claim 12 that is a yeast cell. 17. The host cell of Claim 13 that is a yeast cell. 18. The host cell of Claim 14 that is a yeast cell. 19. A method for determining the fungal MDR inhibition activity of a compound which comprises: a) growing a culture of yeast cells, transformed with a vector which provides expression of the Afu-MDR-1, in the presence of: (i) an antifungal agent to which said yeast cell is resistant, but to which said yeast cell is sensitive in its untransformed state; (ii) a compound suspected of possessing Aspergillus fumigatus MDR inhibition activity; and b) determining the fungal MDR inhibition activity of said compound by measuring the ability of the antifungal agent to inhibit the growth of said yeast cell. 20. A method of Claim 19 wherein the yeast cell is Saccharomyces cerevisiae.

Inventors: PEERY ROBERT B, SKATRUD PAUL L, PEERY, ROBERT, B., SKATRUD, PAUL, L.
Applicants: LILLY CO ELI, PEERY ROBERT B, SKATRUD PAUL L, ELI LILLY AND COMPANY, PEERY, ROBERT, B., SKATRUD, PAUL, L.
Application year: 1996
Main classification: G01N33
IPC codes: C07K14, C12N1, C12N15, C12R1, G01N33