Multiple drug resistance gene atrc of Aspergillus nidulans

Abstract

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

Description

TECHNICAL FIELD OF THE INVENTION

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 nidulans.

BACKGROUND OF THE INVENTION

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, Plasmodium falciparum, the yeast Saccharomyces cerevisiae, Caenorhabditis elegans, Leishmania donovanii, marine sponges, the plant Arabidopsis 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 nidulans. However, it is interesting to note that at higher concentrations some anti-Candida compounds are able to kill other fungal species such as A. fumigatus and A. nidulans. This type of observation suggests that the antifungal target(s) of these anti-Candida compounds is present in A. fumigatus and A. nidulans as well. Such results indicate that A. nidulans 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 A. nidulans has remained undescribed.

SUMMARY OF THE INVENTION

The invention provides, inter alia, isolated nucleic acid molecules that comprise nucleic acid encoding a multiple drug resistance protein from Aspergillus nidulans, herein referred to as atrC, vectors encoding atrC, 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) placing a culture of fungal cells, transformed with a vector capable of expressing atrC, in the presence of: (i) an antifungal agent to which said fungal cell is resistant, but to which said fungal 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 fungal cell.

In still another embodiment the present invention relates to strains of A. nidulans in which the atrC gene is disrupted or otherwise mutated such that the atrC protein is not produced in said strains.

In yet another embodiment, the present invention relates to a method for identifiying new antifungal compounds.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides isolated nucleic acid molecules that comprise a nucleic acid sequence encoding atrC. The cDNA (complementary deoxyribonucleic acid) sequence encoding atrC is provided in the Sequence Listing as SEQ ID NO: 1. The amino acid sequence of the protein encoded by atrC is provided in the Sequence Listing as SEQ ID NO: 2.

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 atrC-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. .sctn.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 atrC, or to amplify DNA or RNA that hybridizes with DNA or RNA encoding atrC.

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 atrC. Expression vectors of the present invention are replicable DNA constructs in which a DNA sequence encoding atrC is operably linked to suitable control sequences capable of effecting the expression of atrC 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, atrC may be synthesized by host cells transformed with vectors that provide for the expression of DNA encoding atrC. The DNA encoding atrC may be the natural sequence or a synthetic sequence or a combination of both ("semi-synthetic sequence"). The in vitro or in vivo transcription and translation of these sequences results in the production of atrC. Synthetic and semi-synthetic sequences encoding atrC 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. atrC-encoding DNA, or portions thereof, may be generated using a conventional DNA synthesizing apparatus such as the Applied Biosystems Model 380A,380B, 394 or 3948 DNA synthesizers (commercially available from Applied Biosystems, Inc., 850 Lincoln Center Drive, Foster City, Calif. 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 atrC. 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, et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989), or Current Protocols in Molecular Biology (F. M. Ausubel et al., 1989 and supplements). The contents of both of these references are incorporated herein by reference.

In another aspect, this invention provides the genomic DNA encoding atrC, which may be obtained by synthesizing the desired portion of SEQ ID No. 1 or by following the procedure carried out by Applicants. This procedure involved construction of a cosmid genomic DNA library from Aspergillus nidulans strain OC-1, a mutant derived from A42355. This library was screened for genes related to MDRs using a homologous probe generated by PCR. Degenerate PCR primers directed towards amplification of DNA sequences encoding highly conserved regions found in the ATP-binding domain of several MDR genes were synthesized. PCR using these primers and Aspergillus nidulans genomic DNA as template produced an approximately 400 base pair DNA fragment. The DNA sequence of this fragment was highly homologous to the ATP-binding region of several MDRs as predicted. This fragment was used as a hybridization probe to identify cosmid clones containing the entire atrC gene. A subclone from one such cosmid containing the entire atrC gene was sequenced to ascertain the entire sequence of atrC.

To effect the translation of atrC-encoding mRNA, one inserts the natural, synthetic, or semi-synthetic atrC-encoding DNA sequence into any of a large number of appropriate expression vectors through the use of appropriate restriction endonucleases and DNA ligases. Synthetic and semi-synthetic atrC-encoding DNA sequences can be designed, and natural atrC-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 atrC-encoding DNA with the control sequences to achieve proper in-frame transcription and translation of the atrC molecule. The atrC-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 atrC is to be expressed.

Expression of atrC in fungal cells, such as Saccharomyces 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. Pat. No. 4,935,350, Jun. 19, 1990) or other glycolytic enzymes such as enolase (found on plasmid pAC1 (ATCC 39532)), glyceraldehyde-3-phosphate dehydrogenase (derived from plasmid pHcGAPC1 (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), U.S. Pat. No. 4,840,896), glyceraldehyde 3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization (GAL1 found on plasmid pRY121 (ATCC 37658) and on plasmid pPST5, described below). Suitable vectors and promoters for use in yeast expression are further described by R. Hitzeman et al., in European Patent Publication No. 73,657A. Yeast enhancers such as the UAS Gal enhancer from Saccharomyces cerevisiae (found in conjunction with the CYC1 promoter on plasmid YEpsec--hI1beta, 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 et al., 1979, Nature 282:39; Kingsman et al., 1979, Gene 7:141 ; Tschemper et al., 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 44076 or PEP4-1 (Jones, 1977, Genetics 85:12).

Expression vectors useful in the expression of atrC can be constructed by a number of methods. For example, the cDNA sequence encoding atrC 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 atrC can be synthesized to contain NotI cohesive ends. Such a synthetic DNA fragment can be ligated into a NotI-digested expression vector such as pYES-2 (Invitrogen Corp., San Diego Calif. 92121).

An expression vector can also be constructed in the following manner. Logarithmic phase Aspergillus nidulans cells are disrupted by grinding under liquid nitrogen according to the procedure of Minuth et al., 1982 (Current Genetics 5:227-231). Aspergillus nidulans mRNA is preferably isolated from the disrupted cells using the QuickPrep.RTM. mRNA Purification Kit (Pharmacia Biotech) according to the instructions of the manufacturer. cDNA is produced from the isolated mRNA using the TimeSaver.RTM. cDNA Synthesis Kit (Pharmacia Biotech) using oligo (dT) according to the procedure described by the manufacturer. In this process an EcoRI/NotI adapter (Stratagene, Inc.) is ligated to each end of the double stranded cDNA. The adapter modified cDNA is ligated into the vector Lambda Zap.sup.R II.RTM. using the Predigested Lambda Zap.sup.R II.RTM./EcoRI/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 atrC using a .sup.32 P-radiolabeled fragment of the atrC gene. In this manner, a full-length cDNA clone is recovered from the Aspergillus nidulans cDNA library. A full-length cDNA clone recovered from the library is removed from the Lambda Zap.sup.R II.RTM. vector by digestion with the restriction endonuclease NotI which produces a DNA fragment encoding atrC. This plasmid further comprises the ColE1 origin of replication which allows replication in E. coli, and the ampicillin resistance gene for selection of E. coli transformants. The expression plasmid further comprises the yeast 2.mu. origin of replication (2.mu. 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 origin of replication from the f1 filamentous phage.

In a preferred embodiment of the invention Saccharomyces cerevisiae INVSc1 or INVSc2 cells (Invitrogen Corp., Sorrento Valley Blvd., San Diego Calif. 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, N. Y. 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 atrC, or a portion thereof, is also useful in producing nucleic acid molecules useful in diagnostic assays for the detection of atrC mRNA, atrC cDNA, or atrC genomic DNA. Further, nucleic acid, either RNA or DNA, which does not encode atrC, but which nonetheless is capable of hybridizing with atrC-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 atrC vectors and transformants and in in vitro diagnosis to detect atrC-like mRNA, cDNA, or genomic DNA from other organisms.

U.S. patent application Ser. No. 08/111,680, the entire contents of which are hereby incorporated herein by reference, describes the use of combination therapy involving an antifungal agent possessing a proven spectrum of activity, with a fungal MDR inhibitor to treat fungal infections. This combination therapy approach enables an extension of the spectrum of antifungal activity for a given antifungal compound which previously had only demonstrated limited clinically relevant antifungal activity. Similarly, 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 nidulans MDR inhibition activity. Host cells that express atrC provide an excellent means for the identification of compounds useful as inhibitors of Aspergillus nidulans MDR activity. Generally, the assay utilizes a culture of a yeast cell transformed with a vector which provides expression of atrC. The expression of atrC 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 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 nidulans MDR inhibitor blocks the ability of atrC 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.

EXAMPLE 1

Source of the atrC-Encoding Genomic DNA and cDNA of Aspergillus nidulans

Complementary DNA encoding atrC (sequence presented in SEQ ID NO: 1) may be from a natural sequence, a synthetic source or a combination of both ("semi-synthetic sequence"). The in vitro or in vivo transcription and translation of these sequences results in the production of atrC. Synthetic and semi-synthetic sequences encoding atrC 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. atrC-encoding DNA, or portions thereof, may be generated using a conventional DNA synthesizing apparatus such as the Applied Biosystems Model 380A, 380B, 384 or 3848 DNA synthesizers (commercially available from Applied Biosystems, Inc., 850 Lincoln Center Drive, Foster City, Calif. 94404). The polymerase chain reaction is especially useful in generating these DNA sequences. PCR primers are constructed which include the translational start (ATG) and translational stop codon (TAG) of atrC. Restriction enzyme sites may be included on these PCR primers outside of the atrC coding region to facilitate rapid cloning into expression vectors. Aspergillus nidulans genomic DNA is used as the PCR template for synthesis of atrC including introns which is useful for expression studies in closely related fungi. In contrast, cDNA is used as the PCR template for synthesis of atrC devoid of introns which is useful for expression in foreign hosts such as Saccharomyces cerevisiae or bacterial hosts such as Escherichia coli.

EXAMPLE 2

Expression of the atrC Protein

Saccharomyces cerevisiae INVSc1 cells (Invitrogen Corp., San Diego Calif. 92191) are transformed with the plasmid containing atrC 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, Mich.) supplemented with CSM-URA (Bio 101, Inc.)] supplemented with 4% raffinose) at 28.degree. C. in a shaker incubator until the culture is saturated. To induce expression of atrC, a portion of the culture is used to inoculate a flask containing YNB/CSM-Ura medium supplemented with 2% galactose (YNB/CSM-Ura/gal) rather than raffinose as the sole carbon source. The inoculated flask is incubated at 28.degree. C. for about 16 hours.

EXAMPLE 3

Antifungal Potentiator Assay

Approximately 1.times.10.sup.6 cells of a Saccharomyces cerevisiae INVSc1 culture expressing atrC are delivered to each of several agar plates containing YNB/CSM-Ura/gal. The agar surface is allowed to dry in a biohazard hood.

An antifungal compound that the untransformed yeast cell is typically sensitive to is dissolved in an appropriate solvent at a concentration that is biologically effective. Twenty .mu.l of the solution is delivered to an antibiotic susceptibility test disc (Difco Laboratories, Detroit, Mich.). 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 transformed Saccharomyces cerevisiae INVSc1 cells.

Compounds to be tested for the ability to inhibit atrC 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 .mu.l of the suspensions containing a compound to be tested are delivered to an antibiotic susceptibility test disc (Difco Laboratories, Detroit, Mich.). The disc is then placed on the surface of the dried petri plates containing the transformed Saccharomyces cerevisiae INVSc1 cells approximately 2 cm from the antifungal-containing disc. Petri plates containing the two discs are incubated at 28.degree. 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 atrC 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, atrC is allowed to act upon the antifungal compound to prevent its activity upon the host cell.

EXAMPLE 4

Screen For Novel Antifungal Compounds

A plasmid molecule is constructed which contains DNA sequence information required for replication and genetic transformation in E. coli (e.g. ampicillin resistance). The plasmid also comprises DNA sequences encoding a marker for selection in fungal cells (e.g. hygromycin B phosphotransferase, phleomycin resistance, G418 resistance) under the control of an A. nidulans promoter. Additionally, the plasmid contains an internal portion of the atrC gene (e.g. about 3000 base pairs which lack 500 base pairs at the N-terminal end, and about 500 base pairs at the C-terminal end of the coding region specified by SEQ ID NO:1). The atrC gene fragment enables a single crossover gene disruption when transformed or otherwise introduced into A. nidulans.

Alternatively, a 5 kilobase pair to 6 kilobase pair region of A. nidulans genomic DNA containing the atrC gene is subcloned into the aforementioned plasmid. Then, a central portion of the atrC gene is removed and replaced with a selectable marker, such as hyromycin B phosphotransferase, for a double crossover gene replacement.

Gene disruption and gene replacement procedures for A. nidulans are well known in the art (See e.g. May et al, J. Cell Biol. 101, 712, 1985; Jones and Sealy-Lewis, Curr. Genet. 17, 81, 1990). Transformants are recovered on an appropriate selection medium, for example, hygromycin (if hygromycin B gene is used in the construction of disruption cassette). Gene replacement, or gene disruption, is verified by any suitable method, for example, by Southern blot hybridization.

Gene disruption or gene replacement strains are rendered hypersensitive to antifungal compounds, and are useful in screens for new antifungal compounds in whole cell growth inhibition studies.

__________________________________________________________________________# SEQUENCE LISTING - - - - (1) GENERAL INFORMATION: - - (iii) NUMBER OF SEQUENCES: 3 - - - - (2) INFORMATION FOR SEQ ID NO:1: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 3927 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: cDNA - - (iii) HYPOTHETICAL: NO - - (iv) ANTI-SENSE: NO - - (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..3924 - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: - - ATG CGG AGG CTC GGA CCC TCA GTT TAC CGG CG - #T TCG GAC GTG TCT ACT 48 Met Arg Arg Leu Gly Pro Ser Val Tyr Arg Ar - #g Ser Asp Val Ser Thr 1 5 - # 10 - # 15 - - TTA AAA AAA AAG AAG CTC TCG TTG TCA CCA TC - #G TCA TGC TCG ACC GCG 96 Leu Lys Lys Lys Lys Leu Ser Leu Ser Pro Se - #r Ser Cys Ser Thr Ala 20 - # 25 - # 30 - - GCT GTA CCA GAC TCC GTC TCA GGA CGA GTC GA - #C CAC CAG TGT ACC ATG 144 Ala Val Pro Asp Ser Val Ser Gly Arg Val As - #p His Gln Cys Thr Met 35 - # 40 - # 45 - - CAC GGA GGC GCC TCT GGT CGA GGA AGG GGA GG - #A AGC AAG CTT TGG CGC 192 His Gly Gly Ala Ser Gly Arg Gly Arg Gly Gl - #y Ser Lys Leu Trp Arg 50 - # 55 - # 60 - - ATA CAA GGT GCC AAG CTG ATA TGC TCG CGC AA - #A AGA GGA TCT TTA CAT 240 Ile Gln Gly Ala Lys Leu Ile Cys Ser Arg Ly - #s Arg Gly Ser Leu His 65 - # 70 - # 75 - # 80 - - TCG CCG GCA GGA CAG AAC TTA TCC TTC AGG CC - #G TTG CTA TCC TTG CTG 288 Ser Pro Ala Gly Gln Asn Leu Ser Phe Arg Pr - #o Leu Leu Ser Leu Leu 85 - # 90 - # 95 - - CAT GCG CCT CTG GAG CAG GAA TTG CGC TTC AA - #A ACC TCA TCT TCG GCC 336 His Ala Pro Leu Glu Gln Glu Leu Arg Phe Ly - #s Thr Ser Ser Ser Ala 100 - # 105 - # 110 - - AGT TCG TCA CCG TCA TCA CCG ATT TCA CCA AC - #G GAA TCT CAA CGC CGG 384 Ser Ser Ser Pro Ser Ser Pro Ile Ser Pro Th - #r Glu Ser Gln Arg Arg 115 - # 120 - # 125 - - CAG ACT TTC GTG ACA ATG CCG CCG AGT TGG CG - #T ATC CTC TAC TTT GTA 432 Gln Thr Phe Val Thr Met Pro Pro Ser Trp Ar - #g Ile Leu Tyr Phe Val 130 - # 135 - # 140 - - TAC CTG GGC ATC GCG CGG CTC GTC CTC TCC TA - #C ACC TAC AAC ACC CTC 480 Tyr Leu Gly Ile Ala Arg Leu Val Leu Ser Ty - #r Thr Tyr Asn Thr Leu 145 1 - #50 1 - #55 1 -#60 - - CTA ACC TAC GCG GCC TAC CGC ATC GTC CGC AA - #T ATC CGA CAC GCCTAT 528 Leu Thr Tyr Ala Ala Tyr Arg Ile Val Arg As - #n Ile Arg His Ala Tyr 165 - # 170 - # 175 - - CTC AAA GCG GCG CTG AGC CAA GAA GTG GCA TA - #C TAC GAT TTC GGT AGC 576 Leu Lys Ala Ala Leu Ser Gln Glu Val Ala Ty - #r Tyr Asp Phe Gly Ser 180 - # 185 - # 190 - - GGG GGC TCC ATC GCC GCG CAG GCA ACT TCG AA - #C GGC AAA CTG ATC CAG 624 Gly Gly Ser Ile Ala Ala Gln Ala Thr Ser As - #n Gly Lys Leu Ile Gln 195 - # 200 - # 205 - - GCC GGC GCC TCG GAT AAG ATC GGT CTT CTC TT - #C CAG GGC CTC GCA GCA 672 Ala Gly Ala Ser Asp Lys Ile Gly Leu Leu Ph - #e Gln Gly Leu Ala Ala 210 - # 215 - # 220 - - TTC GTG ACG CTT TCA TTA TCG CGT TTG TGG TG - #C AAG TGG AAA CTC ACT 720 Phe Val Thr Leu Ser Leu Ser Arg Leu Trp Cy - #s Lys Trp Lys Leu Thr 225 2 - #30 2 - #35 2 -#40 - - CTG ATC TGC ATC TGC ATC CCC GTA GCC ACG AT - #C GGC ACG ACG GGGGTG 768 Leu Ile Cys Ile Cys Ile Pro Val Ala Thr Il - #e Gly Thr Thr Gly Val 245 - # 250 - # 255 - - GTA GCT GCG GTC GAG GCT GGG CAC GAG ACG AG - #G ATC TTG CAG ATA CAT 816 Val Ala Ala Val Glu Ala Gly His Glu Thr Ar - #g Ile Leu Gln Ile His 260 - # 265 - # 270 - - GCG CAG GCG AAT TCG TTT GCC GAG GGT ATT CT - #G GCG GGT GTG AAG GCT 864 Ala Gln Ala Asn Ser Phe Ala Glu Gly Ile Le - #u Ala Gly Val Lys Ala 275 - # 280 - # 285 - - GTT CAT GCT TTT GGG ATG CGG GAT AGT CTG GT - #C AGG AAG TTT GAT GAA 912 Val His Ala Phe Gly Met Arg Asp Ser Leu Va - #l Arg Lys Phe Asp Glu 290 - # 295 - # 300 - - TAT CTG GTG GAG GCG CAT AAG GTC GGT AAG AA - #G ATC TCG CCG CTG CTT 960 Tyr Leu Val Glu Ala His Lys Val Gly Lys Ly - #s Ile Ser Pro Leu Leu 305 3 - #10 3 - #15 3 -#20 - - GGT CTT CTC TTC TCG GCG GAG TAT ACG ATC AT - #C TAC CTT GGA TATGGG 1008 Gly Leu Leu Phe Ser Ala Glu Tyr Thr Ile Il - #e Tyr Leu Gly Tyr Gly 325 - # 330 - # 335 - - CTG GCG TTT TGG CAG GGG ATC CAT ATG TTC GG - #C AGG GGG GAG ATT GGG 1056 Leu Ala Phe Trp Gln Gly Ile His Met Phe Gl - #y Arg Gly Glu Ile Gly 340 - # 345 - # 350 - - ACT GCT GGG GAT ATC TTT ACG GTT TTG CTC TC - #T GTC GTC ATT GCG TCA 1104 Thr Ala Gly Asp Ile Phe Thr Val Leu Leu Se - #r Val Val Ile Ala Ser 355 - # 360 - # 365 - - ATC AAC CTG ACT TTA CTG GCG CCG TAT TCA AT - #T GAA TTT AGC AGG GCT 1152 Ile Asn Leu Thr Leu Leu Ala Pro Tyr Ser Il - #e Glu Phe Ser Arg Ala 370 - # 375 - # 380 - - GCT TCA GCG GCT GCG CAA CTG TTC CGA CTC AT - #A GAT CGA GAG TCT GAA 1200 Ala Ser Ala Ala Ala Gln Leu Phe Arg Leu Il - #e Asp Arg Glu Ser Glu 385 3 - #90 3 - #95 4 -#00 - - ATC AAC CCA TAC GGG AAG GAA GGC CTC GAG CC - #G GAA CGG GTA TTAGGC 1248 Ile Asn Pro Tyr Gly Lys Glu Gly Leu Glu Pr - #o Glu Arg Val Leu Gly 405 - # 410 - # 415 - - GAC GTC GAG CTC GAG AAT GTT ACG TTC TCG TA - #T CCC ACG AGG CCG GGG 1296 Asp Val Glu Leu Glu Asn Val Thr Phe Ser Ty - #r Pro Thr Arg Pro Gly 420 - # 425 - # 430 - - ATT ACC GTC CTC GAT AAC TTC AGT CTC AAG GT - #C CCA GCG GGA AAG GTG 1344 Ile Thr Val Leu Asp Asn Phe Ser Leu Lys Va - #l Pro Ala Gly Lys Val 435 - # 440 - # 445 - - ACT GCC CTG GTA GGG CAA TCT GGA TCG GGG AA - #G AGC ACG ATC GTG GGA 1392 Thr Ala Leu Val Gly Gln Ser Gly Ser Gly Ly - #s Ser Thr Ile Val Gly 450 - # 455 - # 460 - - TTG CTC GAG CGG TGG TAT AAC CCG ACC TCT GG - #G GCG ATC AGA CTC GAC 1440 Leu Leu Glu Arg Trp Tyr Asn Pro Thr Ser Gl - #y Ala Ile Arg Leu Asp 465 4 - #70 4 - #75 4 -#80 - - GGG AAC CTG ATC AGT GAG CTC AAT GTT GGC TG - #G CTG CGG AGG AATGTG 1488 Gly Asn Leu Ile Ser Glu Leu Asn Val Gly Tr - #p Leu Arg Arg Asn Val 485 - # 490 - # 495 - - CGG CTC GTA CAG CAG GAG CCG GTG CTC TTC CA - #G GGA AGC GTG TTC GAT 1536 Arg Leu Val Gln Gln Glu Pro Val Leu Phe Gl - #n Gly Ser Val Phe Asp 500 - # 505 - # 510 - - AAC ATC AGG TAC GGC CTC GTC GGG ACG CCG TG - #G GAG AAT GCC TCT CGG 1584 Asn Ile Arg Tyr Gly Leu Val Gly Thr Pro Tr - #p Glu Asn Ala Ser Arg 515 - # 520 - # 525 - - GAA GAG CAG ATG GAA CGG GTG CAG GAG GCC GC - #G AAG TTG GCA TAT GCG 1632 Glu Glu Gln Met Glu Arg Val Gln Glu Ala Al - #a Lys Leu Ala Tyr Ala 530 - # 535 - # 540 - - CAC GAA TTC ATC TCT GAG CTG ACC GAC GGA TA - #C GAT ACG CTG ATC GGC 1680 His Glu Phe Ile Ser Glu Leu Thr Asp Gly Ty - #r Asp Thr Leu Ile Gly 545 5 - #50 5 - #55 5 -#60 - - GAA CGG GGT GGT CTG CTT TCT GGA GGC CAG AA - #G CAG CGG GTT GCGATT 1728 Glu Arg Gly Gly Leu Leu Ser Gly Gly Gln Ly - #s Gln Arg Val Ala Ile 565 - # 570 - # 575 - - GCC CGC AGC GTC GTT TCT CAA CCG AAG GTC CT - #T CTG CTG GAT GAA GCA 1776 Ala Arg Ser Val Val Ser Gln Pro Lys Val Le - #u Leu Leu Asp Glu Ala 580 - # 585 - # 590 - - ACC AGT GCT CTT GAT CCG CAT GCA GAG ACG AT - #T GTT CAG AAG GCT CTG 1824 Thr Ser Ala Leu Asp Pro His Ala Glu Thr Il - #e Val Gln Lys Ala Leu 595 - # 600 - # 605 - - GAC AAA GCA GCT GAG GGG CGC ACG ACG ATT GT - #C ATT GCT CAC AAA CTT 1872 Asp Lys Ala Ala Glu Gly Arg Thr Thr Ile Va - #l Ile Ala His Lys Leu 610 - # 615 - # 620 - - GCT ACG ATC CGC AAG GCG GAC AAT ATC GTT GT - #C ATG AGC AAG GGT CAC 1920 Ala Thr Ile Arg Lys Ala Asp Asn Ile Val Va - #l Met Ser Lys Gly His 625 6 - #30 6 - #35 6 -#40 - - ATT GTC GAG CAA GGC ACA CAC GAG TCA CTG AT - #A GCC AAG GAC GGCGTC 1968 Ile Val Glu Gln Gly Thr His Glu Ser Leu Il - #e Ala Lys Asp Gly Val 645 - # 650 - # 655 - - TAT GCC GGT CTG GTC AAA ATC CAG AAC CTG GC - #A GTG AAT GCT TCA GCA 2016 Tyr Ala Gly Leu Val Lys Ile Gln Asn Leu Al - #a Val Asn Ala Ser Ala 660 - # 665 - # 670 - - CAT GAC AAT GTA AAT GAG GAG GGT GAA GGC GA - #A GAT GTC GCT CTC CTG 2064 His Asp Asn Val Asn Glu Glu Gly Glu Gly Gl - #u Asp Val Ala Leu Leu 675 - # 680 - # 685 - - GAG GTC ACC GAA ACA GCA GTA ACC CGC TAC CC - #A ACC TCC ATC CGC GGT 2112 Glu Val Thr Glu Thr Ala Val Thr Arg Tyr Pr - #o Thr Ser Ile Arg Gly 690 - # 695 - # 700 - - CGA ATG AAC TCC ATA AAG GAC CGC GAC GAT TA - #T GAG AAC CAC AAG CAC 2160 Arg Met Asn Ser Ile Lys Asp Arg Asp Asp Ty - #r Glu Asn His Lys His 705 7 - #10 7 - #15 7 -#20 - - ATG GAT ATG CTG GCC GCC TTA GCT TAT CTC GT - #C CGC GAA TGT CCAGAA 2208 Met Asp Met Leu Ala Ala Leu Ala Tyr Leu Va - #l Arg Glu Cys Pro Glu 725 - # 730 - # 735 - - CTG AAA TGG GCC TAT CTC GTC GTG CTA CTG GG - #G TGT CTT GGT GGT TGC 2256 Leu Lys Trp Ala Tyr Leu Val Val Leu Leu Gl - #y Cys Leu Gly Gly Cys 740 - # 745 - # 750 - - GCC ATG TAC CCC GGC CAA GCT ATC TTG ATG TC - #T CGC GTT GTC GAG GTC 2304 Ala Met Tyr Pro Gly Gln Ala Ile Leu Met Se - #r Arg Val Val Glu Val 755 - # 760 - # 765 - - TTC ACG CTC TCG GGA GAC GCT ATG CTA GAC AA - #A GGA GAC TTC TAT GCC 2352 Phe Thr Leu Ser Gly Asp Ala Met Leu Asp Ly - #s Gly Asp Phe Tyr Ala 770 - # 775 - # 780 - - AGT ATG CTG ATC GTT CTC GCG GCC GGG TGT CT - #G ATC TGT TAC TTA GCT 2400 Ser Met Leu Ile Val Leu Ala Ala Gly Cys Le - #u Ile Cys Tyr Leu Ala 785 7 - #90 7 - #95 8 -#00 - - GTC GGA TAT GCA ACC AAC ACT ATA GCC CAG CA - #T CTT AGT CAT TGGTTT 2448 Val Gly Tyr Ala Thr Asn Thr Ile Ala Gln Hi - #s Leu Ser His Trp Phe 805 - # 810 - # 815 - - CGA CGC CTC ATT CTG CAC GAC ATG CTG CGA CA - #G GAT ATC CAG TTC TTT 2496 Arg Arg Leu Ile Leu His Asp Met Leu Arg Gl - #n Asp Ile Gln Phe Phe 820 - # 825 - # 830 - - GAC CGT GAA GAG AAC ACT ACC GGT GCG CTG GT - #A AGC CGT ATC GAT TCG 2544 Asp Arg Glu Glu Asn Thr Thr Gly Ala Leu Va - #l Ser Arg Ile Asp Ser 835 - # 840 - # 845 - - TAC CCG CAT GCA ATT CTC GAA CTG ATG GGC TA - #C AAC ATC GCC CTG GTC 2592 Tyr Pro His Ala Ile Leu Glu Leu Met Gly Ty - #r Asn Ile Ala Leu Val 850 - # 855 - # 860 - - GTG ATT GCT GTC CTG CAG GTG GTA ACC TGT GG - #C ATC CTG GCC ATT GCA 2640 Val Ile Ala Val Leu Gln Val Val Thr Cys Gl - #y Ile Leu Ala Ile Ala 865 8 - #70 8 - #75 8 -#80 - - TTC TCC TGG AAA CTA GGG CTG GTC GTT GTC TT - #T GGC GGT ATT CCACCC 2688 Phe Ser Trp Lys Leu Gly Leu Val Val Val Ph - #e Gly Gly Ile Pro Pro 885 - # 890 - # 895 - - CTT GTC GGT GCT GGG ATG GTA CGA ATC CGC GT - #C GAC TCC CGC CTC GAT 2736 Leu Val Gly Ala Gly Met Val Arg Ile Arg Va - #l Asp Ser Arg Leu Asp 900 - # 905 - # 910 - - CGC CAG ACA TCG AAG AAA TAT GGC ACC AGC TC - #G TCC ATT GCC TCT GAA 2784 Arg Gln Thr Ser Lys Lys Tyr Gly Thr Ser Se - #r Ser Ile Ala Ser Glu 915 - # 920 - # 925 - - GCT GTA AAC GCT ATC CGG ACC GTT TCG TCC CT - #T GCA ATC GAA GAG ACG 2832 Ala Val Asn Ala Ile Arg Thr Val Ser Ser Le - #u Ala Ile Glu Glu Thr 930 - # 935 - # 940 - - GTG CTA CGT CGA TAC ACG GAG GAA CTA GAC CA - #C GCT GTC TCG TCT TCG 2880 Val Leu Arg Arg Tyr Thr Glu Glu Leu Asp Hi - #s Ala Val Ser Ser Ser 945 9 - #50 9 - #55 9 -#60 - - GTG AAA CCC ATG GCT GCC ACG ATG ATT TGT TT - #C GGG CTG ACG CAGTGC 2928 Val Lys Pro Met Ala Ala Thr Met Ile Cys Ph - #e Gly Leu Thr Gln Cys 965 - # 970 - # 975 - - ATT GAG TAC TGG TTT CAG GCG CTG GGA TTC TG - #G TAT GGG TGT CGT CTT 2976 Ile Glu Tyr Trp Phe Gln Ala Leu Gly Phe Tr - #p Tyr Gly Cys Arg Leu 980 - # 985 - # 990 - - GTG TCG CTG GGG GAG ACT AGC ATG TAT AGT TT - #C TTT GTC GCA TTC CTC 3024 Val Ser Leu Gly Glu Thr Ser Met Tyr Ser Ph - #e Phe Val Ala Phe Leu 995 - # 1000 - # 1005 - - AGT GTG TTC TTT GCG GGT CAG GCG TCA GCG CA - #G CTG TTC CAG TGG TCG 3072 Ser Val Phe Phe Ala Gly Gln Ala Ser Ala Gl - #n Leu Phe Gln Trp Ser 1010 - # 1015 - # 1020 - - ACC AGT ATT ACA AAG GGA ATC AAT GCG ACG AA - #C TAC ATC GCT TGG TTG 3120 Thr Ser Ile Thr Lys Gly Ile Asn Ala Thr As - #n Tyr Ile Ala Trp Leu 1025 1030 - # 1035 - # 1040 - - CAC CAG CTC CAA CCA ACA GTG CGC GAG ACG CC - #G GAG AAC CAC GAT AAA 3168 His Gln Leu Gln Pro Thr Val Arg Glu Thr Pr - #o Glu Asn His Asp Lys 1045 - # 1050 - # 1055 - - GGC CCT GGA TCT GGG GCG CCG ATT GCT ATG GA - #C AAT GTG CGC TTC TCG 3216 Gly Pro Gly Ser Gly Ala Pro Ile Ala Met As - #p Asn Val Arg Phe Ser 1060 - # 1065 - # 1070 - - TAC CCT CTA CGG CCA GAC GCC CCT ATC CTG AA - #A GGG GTG AAT CTG AAG 3264 Tyr Pro Leu Arg Pro Asp Ala Pro Ile Leu Ly - #s Gly Val Asn Leu Lys 1075 - # 1080 - # 1085 - - ATA AAC AAA GGC CAA TTC ATC GCT TTC GTC GG - #C TCC TCC GGC TGC GGC 3312 Ile Asn Lys Gly Gln Phe Ile Ala Phe Val Gl - #y Ser Ser Gly Cys Gly 1090 - # 1095 - # 1100 - - AAA TCC ACC ATG ATT GCC ATG CTC GAG CGC TT - #C TAC GAT CCA ACA ACA 3360 Lys Ser Thr Met Ile Ala Met Leu Glu Arg Ph - #e Tyr Asp Pro Thr Thr 1105 1110 - # 1115 - # 1120 - - GGG AGC ATC ACA ATC GAC GCT TCC ACC CTC AC - #C GAC ATA AAC CCC ATA 3408 Gly Ser Ile Thr Ile Asp Ala Ser Thr Leu Th - #r Asp Ile Asn Pro Ile 1125 - # 1130 - # 1135 - - TCC TAC CGA AAT ATT GTG GCA CTG GTG CAG CA - #A GAG CCA ACC CTT TTC 3456 Ser Tyr Arg Asn Ile Val Ala Leu Val Gln Gl - #n Glu Pro Thr Leu Phe 1140 - # 1145 - # 1150 - - CAA GGG ACA ATA CGG GAC AAC ATC TCG CTT GG - #C GAT GCA GTG AAG TCC 3504 Gln Gly Thr Ile Arg Asp Asn Ile Ser Leu Gl - #y Asp Ala Val Lys Ser 1155 - # 1160 - # 1165 - - GTG TCT GAT GAG CAG ATT GAG TCG GCC CTC CG - #C GCA GCT AAT GCC TGG 3552 Val Ser Asp Glu Gln Ile Glu Ser Ala Leu Ar - #g Ala Ala Asn Ala Trp 1170 - # 1175 - # 1180 - - GAC TTT GTC TCC TCA TTG CCG CAG GGG ATC TA - #C ACG CCC GCT GGC TCA 3600 Asp Phe Val Ser Ser Leu Pro Gln Gly Ile Ty - #r Thr Pro Ala Gly Ser 1185 1190 - # 1195 - # 1200 - - GGC GGG TCC CAA CTC TCT GGG GGG CAG CGG CA - #A CGC ATT GCC ATT GCC 3648 Gly Gly Ser Gln Leu Ser Gly Gly Gln Arg Gl - #n Arg Ile Ala Ile Ala 1205 - # 1210 - # 1215 - - CGC GCG CTC ATC CGA GAT CCA AAG ATC TTA CT - #C CTT GAC GAG GCT ACG 3696 Arg Ala Leu Ile Arg Asp Pro Lys Ile Leu Le - #u Leu Asp Glu Ala Thr 1220 - # 1225 - # 1230 - - AGT GCC CTG GAT ACA GAG AGT GAG AAG ATC GT - #G CAG AAG GCT CTC GAG 3744 Ser Ala Leu Asp Thr Glu Ser Glu Lys Ile Va - #l Gln Lys Ala Leu Glu 1235 - # 1240 - # 1245 - - GGG GCG GCC AGG GAC GGG GAC CGG CTT ACG GT - #T GCT GTT GCG CAT CGA 3792 Gly Ala Ala Arg Asp Gly Asp Arg Leu Thr Va - #l Ala Val Ala His Arg 1250 - # 1255 - # 1260 - - TTA AGC ACG ATT AAG GAT GCT AAT GTT ATC TG - #T GTA TTC TTT GGA GGA 3840 Leu Ser Thr Ile Lys Asp Ala Asn Val Ile Cy - #s Val Phe Phe Gly Gly 1265 1270 - # 1275 - # 1280 - - AAG ATT GCG GAG ATG GGA ACG CAT CAA GAG TT - #A ATA GTT AGG GGG GGG 3888 Lys Ile Ala Glu Met Gly Thr His Gln Glu Le - #u Ile Val Arg Gly Gly 1285 - # 1290 - # 1295 - - CTG TAT AGA CGG ATG TGT GAG GCG CAG GCC TT - #G GAC TAA - # 3927 Leu Tyr Arg Arg Met Cys Glu Ala Gln Ala Le - #u Asp 1300 - # 1305 - - - - (2) INFORMATION FOR SEQ ID NO:2: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1308 amino - #acids (B) TYPE: amino acid (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: protein - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: - - Met Arg Arg Leu Gly Pro Ser Val Tyr Arg Ar - #g Ser Asp Val Ser Thr 1 5 - # 10 - # 15 - - Leu Lys Lys Lys Lys Leu Ser Leu Ser Pro Se - #r Ser Cys Ser Thr Ala 20 - # 25 - # 30 - - Ala Val Pro Asp Ser Val Ser Gly Arg Val As - #p His Gln Cys Thr Met 35 - # 40 - # 45 - - His Gly Gly Ala Ser Gly Arg Gly Arg Gly Gl - #y Ser Lys Leu Trp Arg 50 - # 55 - # 60 - - Ile Gln Gly Ala Lys Leu Ile Cys Ser Arg Ly - #s Arg Gly Ser Leu His 65 - # 70 - # 75 - # 80 - - Ser Pro Ala Gly Gln Asn Leu Ser Phe Arg Pr - #o Leu Leu Ser Leu Leu 85 - # 90 - # 95 - - His Ala Pro Leu Glu Gln Glu Leu Arg Phe Ly - #s Thr Ser Ser Ser Ala 100 - # 105 - # 110 - - Ser Ser Ser Pro Ser Ser Pro Ile Ser Pro Th - #r Glu Ser Gln Arg Arg 115 - # 120 - # 125 - - Gln Thr Phe Val Thr Met Pro Pro Ser Trp Ar - #g Ile Leu Tyr Phe Val 130 - # 135 - # 140 - - Tyr Leu Gly Ile Ala Arg Leu Val Leu Ser Ty - #r Thr Tyr Asn Thr Leu 145 1 - #50 1 - #55 1 -#60 - - Leu Thr Tyr Ala Ala Tyr Arg Ile Val Arg As - #n Ile Arg His AlaTyr 165 - # 170 - # 175 - - Leu Lys Ala Ala Leu Ser Gln Glu Val Ala Ty - #r Tyr Asp Phe Gly Ser 180 - # 185 - # 190 - - Gly Gly Ser Ile Ala Ala Gln Ala Thr Ser As - #n Gly Lys Leu Ile Gln 195 - # 200 - # 205 - - Ala Gly Ala Ser Asp Lys Ile Gly Leu Leu Ph - #e Gln Gly Leu Ala Ala 210 - # 215 - # 220 - - Phe Val Thr Leu Ser Leu Ser Arg Leu Trp Cy - #s Lys Trp Lys Leu Thr 225 2 - #30 2 - #35 2 -#40 - - Leu Ile Cys Ile Cys Ile Pro Val Ala Thr Il - #e Gly Thr Thr GlyVal 245 - # 250 - # 255 - - Val Ala Ala Val Glu Ala Gly His Glu Thr Ar - #g Ile Leu Gln Ile His 260 - # 265 - # 270 - - Ala Gln Ala Asn Ser Phe Ala Glu Gly Ile Le - #u Ala Gly Val Lys Ala 275 - # 280 - # 285 - - Val His Ala Phe Gly Met Arg Asp Ser Leu Va - #l Arg Lys Phe Asp Glu 290 - # 295 - # 300 - - Tyr Leu Val Glu Ala His Lys Val Gly Lys Ly - #s Ile Ser Pro Leu Leu 305 3 - #10 3 - #15 3 -#20 - - Gly Leu Leu Phe Ser Ala Glu Tyr Thr Ile Il - #e Tyr Leu Gly TyrGly 325 - # 330 - # 335 - - Leu Ala Phe Trp Gln Gly Ile His Met Phe Gl - #y Arg Gly Glu Ile Gly 340 - # 345 - # 350 - - Thr Ala Gly Asp Ile Phe Thr Val Leu Leu Se - #r Val Val Ile Ala Ser 355 - # 360 - # 365 - - Ile Asn Leu Thr Leu Leu Ala Pro Tyr Ser Il - #e Glu Phe Ser Arg Ala 370 - # 375 - # 380 - - Ala Ser Ala Ala Ala Gln Leu Phe Arg Leu Il - #e Asp Arg Glu Ser Glu 385 3 - #90 3 - #95 4 -#00 - - Ile Asn Pro Tyr Gly Lys Glu Gly Leu Glu Pr - #o Glu Arg Val LeuGly 405 - # 410 - # 415 - - Asp Val Glu Leu Glu Asn Val Thr Phe Ser Ty - #r Pro Thr Arg Pro Gly 420 - # 425 - # 430 - - Ile Thr Val Leu Asp Asn Phe Ser Leu Lys Va - #l Pro Ala Gly Lys Val 435 - # 440 - # 445 - - Thr Ala Leu Val Gly Gln Ser Gly Ser Gly Ly - #s Ser Thr Ile Val Gly 450 - # 455 - # 460 - - Leu Leu Glu Arg Trp Tyr Asn Pro Thr Ser Gl - #y Ala Ile Arg Leu Asp 465 4 - #70 4 - #75 4 -#80 - - Gly Asn Leu Ile Ser Glu Leu Asn Val Gly Tr - #p Leu Arg Arg AsnVal 485 - # 490 - # 495 - - Arg Leu Val Gln Gln Glu Pro Val Leu Phe Gl - #n Gly Ser Val Phe Asp 500 - # 505 - # 510 - - Asn Ile Arg Tyr Gly Leu Val Gly Thr Pro Tr - #p Glu Asn Ala Ser Arg 515 - # 520 - # 525 - - Glu Glu Gln Met Glu Arg Val Gln Glu Ala Al - #a Lys Leu Ala Tyr Ala 530 - # 535 - # 540 - - His Glu Phe Ile Ser Glu Leu Thr Asp Gly Ty - #r Asp Thr Leu Ile Gly 545 5 - #50 5 - #55 5 -#60 - - Glu Arg Gly Gly Leu Leu Ser Gly Gly Gln Ly - #s Gln Arg Val AlaIle 565 - # 570 - # 575 - - Ala Arg Ser Val Val Ser Gln Pro Lys Val Le - #u Leu Leu Asp Glu Ala 580 - # 585 - # 590 - - Thr Ser Ala Leu Asp Pro His Ala Glu Thr Il - #e Val Gln Lys Ala Leu 595 - # 600 - # 605 - - Asp Lys Ala Ala Glu Gly Arg Thr Thr Ile Va - #l Ile Ala His Lys Leu 610 - # 615 - # 620 - - Ala Thr Ile Arg Lys Ala Asp Asn Ile Val Va - #l Met Ser Lys Gly His 625 6 - #30 6 - #35 6 -#40 - - Ile Val Glu Gln Gly Thr His Glu Ser Leu Il - #e Ala Lys Asp GlyVal 645 - # 650 - # 655 - - Tyr Ala Gly Leu Val Lys Ile Gln Asn Leu Al - #a Val Asn Ala Ser Ala 660 - # 665 - # 670 - - His Asp Asn Val Asn Glu Glu Gly Glu Gly Gl - #u Asp Val Ala Leu Leu 675 - # 680 - # 685 - - Glu Val Thr Glu Thr Ala Val Thr Arg Tyr Pr - #o Thr Ser Ile Arg Gly 690 - # 695 - # 700 - - Arg Met Asn Ser Ile Lys Asp Arg Asp Asp Ty - #r Glu Asn His Lys His 705 7 - #10 7 - #15 7 -#20 - - Met Asp Met Leu Ala Ala Leu Ala Tyr Leu Va - #l Arg Glu Cys ProGlu 725 - # 730 - # 735 - - Leu Lys Trp Ala Tyr Leu Val Val Leu Leu Gl - #y Cys Leu Gly Gly Cys 740 - # 745 - # 750 - - Ala Met Tyr Pro Gly Gln Ala Ile Leu Met Se - #r Arg Val Val Glu Val 755 - # 760 - # 765 - - Phe Thr Leu Ser Gly Asp Ala Met Leu Asp Ly - #s Gly Asp Phe Tyr Ala 770 - # 775 - # 780 - - Ser Met Leu Ile Val Leu Ala Ala Gly Cys Le - #u Ile Cys Tyr Leu Ala 785 7 - #90 7 - #95 8 -#00 - - Val Gly Tyr Ala Thr Asn Thr Ile Ala Gln Hi - #s Leu Ser His TrpPhe 805 - # 810 - # 815 - - Arg Arg Leu Ile Leu His Asp Met Leu Arg Gl - #n Asp Ile Gln Phe Phe 820 - # 825 - # 830 - - Asp Arg Glu Glu Asn Thr Thr Gly Ala Leu Va - #l Ser Arg Ile Asp Ser 835 - # 840 - # 845 - - Tyr Pro His Ala Ile Leu Glu Leu Met Gly Ty - #r Asn Ile Ala Leu Val 850 - # 855 - # 860 - - Val Ile Ala Val Leu Gln Val Val Thr Cys Gl - #y Ile Leu Ala Ile Ala 865 8 - #70 8 - #75 8 -#80 - - Phe Ser Trp Lys Leu Gly Leu Val Val Val Ph - #e Gly Gly Ile ProPro 885 - # 890 - # 895 - - Leu Val Gly Ala Gly Met Val Arg Ile Arg Va - #l Asp Ser Arg Leu Asp 900 - # 905 - # 910 - - Arg Gln Thr Ser Lys Lys Tyr Gly Thr Ser Se - #r Ser Ile Ala Ser Glu 915 - # 920 - # 925 - - Ala Val Asn Ala Ile Arg Thr Val Ser Ser Le - #u Ala Ile Glu Glu Thr 930 - # 935 - # 940 - - Val Leu Arg Arg Tyr Thr Glu Glu Leu Asp Hi - #s Ala Val Ser Ser Ser 945 9 - #50 9 - #55 9 -#60 - - Val Lys Pro Met Ala Ala Thr Met Ile Cys Ph - #e Gly Leu Thr GlnCys 965 - # 970 - # 975 - - Ile Glu Tyr Trp Phe Gln Ala Leu Gly Phe Tr - #p Tyr Gly Cys Arg Leu 980 - # 985 - # 990 - - Val Ser Leu Gly Glu Thr Ser Met Tyr Ser Ph - #e Phe Val Ala Phe Leu 995 - # 1000 - # 1005 - - Ser Val Phe Phe Ala Gly Gln Ala Ser Ala Gl - #n Leu Phe Gln Trp Ser 1010 - # 1015 - # 1020 - - Thr Ser Ile Thr Lys Gly Ile Asn Ala Thr As - #n Tyr Ile Ala Trp Leu 1025 1030 - # 1035 - # 1040 - - His Gln Leu Gln Pro Thr Val Arg Glu Thr Pr - #o Glu Asn His Asp Lys 1045 - # 1050 - # 1055 - - Gly Pro Gly Ser Gly Ala Pro Ile Ala Met As - #p Asn Val Arg Phe Ser 1060 - # 1065 - # 1070 - - Tyr Pro Leu Arg Pro Asp Ala Pro Ile Leu Ly - #s Gly Val Asn Leu Lys 1075 - # 1080 - # 1085 - - Ile Asn Lys Gly Gln Phe Ile Ala Phe Val Gl - #y Ser Ser Gly Cys Gly 1090 - # 1095 - # 1100 - - Lys Ser Thr Met Ile Ala Met Leu Glu Arg Ph - #e Tyr Asp Pro Thr Thr 1105 1110 - # 1115 - # 1120 - - Gly Ser Ile Thr Ile Asp Ala Ser Thr Leu Th - #r Asp Ile Asn Pro Ile 1125 - # 1130 - # 1135 - - Ser Tyr Arg Asn Ile Val Ala Leu Val Gln Gl - #n Glu Pro Thr Leu Phe 1140 - # 1145 - # 1150 - - Gln Gly Thr Ile Arg Asp Asn Ile Ser Leu Gl - #y Asp Ala Val Lys Ser 1155 - # 1160 - # 1165 - - Val Ser Asp Glu Gln Ile Glu Ser Ala Leu Ar - #g Ala Ala Asn Ala Trp 1170 - # 1175 - # 1180 - - Asp Phe Val Ser Ser Leu Pro Gln Gly Ile Ty - #r Thr Pro Ala Gly Ser 1185 1190 - # 1195 - # 1200 - - Gly Gly Ser Gln Leu Ser Gly Gly Gln Arg Gl - #n Arg Ile Ala Ile Ala 1205 - # 1210 - # 1215 - - Arg Ala Leu Ile Arg Asp Pro Lys Ile Leu Le - #u Leu Asp Glu Ala Thr 1220 - # 1225 - # 1230 - - Ser Ala Leu Asp Thr Glu Ser Glu Lys Ile Va - #l Gln Lys Ala Leu Glu 1235 - # 1240 - # 1245 - - Gly Ala Ala Arg Asp Gly Asp Arg Leu Thr Va - #l Ala Val Ala His Arg 1250 - # 1255 - # 1260 - - Leu Ser Thr Ile Lys Asp Ala Asn Val Ile Cy - #s Val Phe Phe Gly Gly 1265 1270 - # 1275 - # 1280 - - Lys Ile Ala Glu Met Gly Thr His Gln Glu Le - #u Ile Val Arg Gly Gly 1285 - # 1290 - # 1295 - - Leu Tyr Arg Arg Met Cys Glu Ala Gln Ala Le - #u Asp 1300 - # 1305 - - - - (2) INFORMATION FOR SEQ ID NO:3: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 3924 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: mRNA - - (iii) HYPOTHETICAL: NO - - (iv) ANTI-SENSE: NO - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: - - AUGCGGAGGC UCGGACCCUC AGUUUACCGG CGUUCGGACG UGUCUACUUU AA -#AAAAAAAG 60 - - AAGCUCUCGU UGUCACCAUC GUCAUGCUCG ACCGCGGCUG UACCAGACUC CG -#UCUCAGGA 120 - - CGAGUCGACC ACCAGUGUAC CAUGCACGGA GGCGCCUCUG GUCGAGGAAG GG -#GAGGAAGC 180 - - AAGCUUUGGC GCAUACAAGG UGCCAAGCUG AUAUGCUCGC GCAAAAGAGG AU -#CUUUACAU 240 - - UCGCCGGCAG GACAGAACUU AUCCUUCAGG CCGUUGCUAU CCUUGCUGCA UG -#CGCCUCUG 300 - - GAGCAGGAAU UGCGCUUCAA AACCUCAUCU UCGGCCAGUU CGUCACCGUC AU -#CACCGAUU 360 - - UCACCAACGG AAUCUCAACG CCGGCAGACU UUCGUGACAA UGCCGCCGAG UU -#GGCGUAUC 420 - - CUCUACUUUG UAUACCUGGG CAUCGCGCGG CUCGUCCUCU CCUACACCUA CA -#ACACCCUC 480 - - CUAACCUACG CGGCCUACCG CAUCGUCCGC AAUAUCCGAC ACGCCUAUCU CA -#AAGCGGCG 540 - - CUGAGCCAAG AAGUGGCAUA CUACGAUUUC GGUAGCGGGG GCUCCAUCGC CG -#CGCAGGCA 600 - - ACUUCGAACG GCAAACUGAU CCAGGCCGGC GCCUCGGAUA AGAUCGGUCU UC -#UCUUCCAG 660 - - GGCCUCGCAG CAUUCGUGAC GCUUUCAUUA UCGCGUUUGU GGUGCAAGUG GA -#AACUCACU 720 - - CUGAUCUGCA UCUGCAUCCC CGUAGCCACG AUCGGCACGA CGGGGGUGGU AG -#CUGCGGUC 780 - - GAGGCUGGGC ACGAGACGAG GAUCUUGCAG AUACAUGCGC AGGCGAAUUC GU -#UUGCCGAG 840 - - GGUAUUCUGG CGGGUGUGAA GGCUGUUCAU GCUUUUGGGA UGCGGGAUAG UC -#UGGUCAGG 900 - - AAGUUUGAUG AAUAUCUGGU GGAGGCGCAU AAGGUCGGUA AGAAGAUCUC GC -#CGCUGCUU 960 - - GGUCUUCUCU UCUCGGCGGA GUAUACGAUC AUCUACCUUG GAUAUGGGCU GG -#CGUUUUGG 1020 - - CAGGGGAUCC AUAUGUUCGG CAGGGGGGAG AUUGGGACUG CUGGGGAUAU CU -#UUACGGUU 1080 - - UUGCUCUCUG UCGUCAUUGC GUCAAUCAAC CUGACUUUAC UGGCGCCGUA UU -#CAAUUGAA 1140 - - UUUAGCAGGG CUGCUUCAGC GGCUGCGCAA CUGUUCCGAC UCAUAGAUCG AG -#AGUCUGAA 1200 - - AUCAACCCAU ACGGGAAGGA AGGCCUCGAG CCGGAACGGG UAUUAGGCGA CG -#UCGAGCUC 1260 - - GAGAAUGUUA CGUUCUCGUA UCCCACGAGG CCGGGGAUUA CCGUCCUCGA UA -#ACUUCAGU 1320 - - CUCAAGGUCC CAGCGGGAAA GGUGACUGCC CUGGUAGGGC AAUCUGGAUC GG -#GGAAGAGC 1380 - - ACGAUCGUGG GAUUGCUCGA GCGGUGGUAU AACCCGACCU CUGGGGCGAU CA -#GACUCGAC 1440 - - GGGAACCUGA UCAGUGAGCU CAAUGUUGGC UGGCUGCGGA GGAAUGUGCG GC -#UCGUACAG 1500 - - CAGGAGCCGG UGCUCUUCCA GGGAAGCGUG UUCGAUAACA UCAGGUACGG CC -#UCGUCGGG 1560 - - ACGCCGUGGG AGAAUGCCUC UCGGGAAGAG CAGAUGGAAC GGGUGCAGGA GG -#CCGCGAAG 1620 - - UUGGCAUAUG CGCACGAAUU CAUCUCUGAG CUGACCGACG GAUACGAUAC GC -#UGAUCGGC 1680 - - GAACGGGGUG GUCUGCUUUC UGGAGGCCAG AAGCAGCGGG UUGCGAUUGC CC -#GCAGCGUC 1740 - - GUUUCUCAAC CGAAGGUCCU UCUGCUGGAU GAAGCAACCA GUGCUCUUGA UC -#CGCAUGCA 1800 - - GAGACGAUUG UUCAGAAGGC UCUGGACAAA GCAGCUGAGG GGCGCACGAC GA -#UUGUCAUU 1860 - - GCUCACAAAC UUGCUACGAU CCGCAAGGCG GACAAUAUCG UUGUCAUGAG CA -#AGGGUCAC 1920 - - AUUGUCGAGC AAGGCACACA CGAGUCACUG AUAGCCAAGG ACGGCGUCUA UG -#CCGGUCUG 1980 - - GUCAAAAUCC AGAACCUGGC AGUGAAUGCU UCAGCACAUG ACAAUGUAAA UG -#AGGAGGGU 2040 - - GAAGGCGAAG AUGUCGCUCU CCUGGAGGUC ACCGAAACAG CAGUAACCCG CU -#ACCCAACC 2100 - - UCCAUCCGCG GUCGAAUGAA CUCCAUAAAG GACCGCGACG AUUAUGAGAA CC -#ACAAGCAC 2160 - - AUGGAUAUGC UGGCCGCCUU AGCUUAUCUC GUCCGCGAAU GUCCAGAACU GA -#AAUGGGCC 2220 - - UAUCUCGUCG UGCUACUGGG GUGUCUUGGU GGUUGCGCCA UGUACCCCGG CC -#AAGCUAUC 2280 - - UUGAUGUCUC GCGUUGUCGA GGUCUUCACG CUCUCGGGAG ACGCUAUGCU AG -#ACAAAGGA 2340 - - GACUUCUAUG CCAGUAUGCU GAUCGUUCUC GCGGCCGGGU GUCUGAUCUG UU -#ACUUAGCU 2400 - - GUCGGAUAUG CAACCAACAC UAUAGCCCAG CAUCUUAGUC AUUGGUUUCG AC -#GCCUCAUU 2460 - - CUGCACGACA UGCUGCGACA GGAUAUCCAG UUCUUUGACC GUGAAGAGAA CA -#CUACCGGU 2520 - - GCGCUGGUAA GCCGUAUCGA UUCGUACCCG CAUGCAAUUC UCGAACUGAU GG -#GCUACAAC 2580 - - AUCGCCCUGG UCGUGAUUGC UGUCCUGCAG GUGGUAACCU GUGGCAUCCU GG -#CCAUUGCA 2640 - - UUCUCCUGGA AACUAGGGCU GGUCGUUGUC UUUGGCGGUA UUCCACCCCU UG -#UCGGUGCU 2700 - - GGGAUGGUAC GAAUCCGCGU CGACUCCCGC CUCGAUCGCC AGACAUCGAA GA -#AAUAUGGC 2760 - - ACCAGCUCGU CCAUUGCCUC UGAAGCUGUA AACGCUAUCC GGACCGUUUC GU -#CCCUUGCA 2820 - - AUCGAAGAGA CGGUGCUACG UCGAUACACG GAGGAACUAG ACCACGCUGU CU -#CGUCUUCG 2880 - - GUGAAACCCA UGGCUGCCAC GAUGAUUUGU UUCGGGCUGA CGCAGUGCAU UG -#AGUACUGG 2940 - - UUUCAGGCGC UGGGAUUCUG GUAUGGGUGU CGUCUUGUGU CGCUGGGGGA GA -#CUAGCAUG 3000 - - UAUAGUUUCU UUGUCGCAUU CCUCAGUGUG UUCUUUGCGG GUCAGGCGUC AG -#CGCAGCUG 3060 - - UUCCAGUGGU CGACCAGUAU UACAAAGGGA AUCAAUGCGA CGAACUACAU CG -#CUUGGUUG 3120 - - CACCAGCUCC AACCAACAGU GCGCGAGACG CCGGAGAACC ACGAUAAAGG CC -#CUGGAUCU 3180 - - GGGGCGCCGA UUGCUAUGGA CAAUGUGCGC UUCUCGUACC CUCUACGGCC AG -#ACGCCCCU 3240 - - AUCCUGAAAG GGGUGAAUCU GAAGAUAAAC AAAGGCCAAU UCAUCGCUUU CG -#UCGGCUCC 3300 - - UCCGGCUGCG GCAAAUCCAC CAUGAUUGCC AUGCUCGAGC GCUUCUACGA UC -#CAACAACA 3360 - - GGGAGCAUCA CAAUCGACGC UUCCACCCUC ACCGACAUAA ACCCCAUAUC CU -#ACCGAAAU 3420 - - AUUGUGGCAC UGGUGCAGCA AGAGCCAACC CUUUUCCAAG GGACAAUACG GG -#ACAACAUC 3480 - - UCGCUUGGCG AUGCAGUGAA GUCCGUGUCU GAUGAGCAGA UUGAGUCGGC CC -#UCCGCGCA 3540 - - GCUAAUGCCU GGGACUUUGU CUCCUCAUUG CCGCAGGGGA UCUACACGCC CG -#CUGGCUCA 3600 - - GGCGGGUCCC AACUCUCUGG GGGGCAGCGG CAACGCAUUG CCAUUGCCCG CG -#CGCUCAUC 3660 - - CGAGAUCCAA AGAUCUUACU CCUUGACGAG GCUACGAGUG CCCUGGAUAC AG -#AGAGUGAG 3720 - - AAGAUCGUGC AGAAGGCUCU CGAGGGGGCG GCCAGGGACG GGGACCGGCU UA -#CGGUUGCU 3780 - - GUUGCGCAUC GAUUAAGCAC GAUUAAGGAU GCUAAUGUUA UCUGUGUAUU CU -#UUGGAGGA 3840 - - AAGAUUGCGG AGAUGGGAAC GCAUCAAGAG UUAAUAGUUA GGGGGGGGCU GU -#AUAGACGG 3900 - - AUGUGUGAGG CGCAGGCCUU GGAC - # - # 3924__________________________________________________________________________

Claims

1. A method for determining the fungal MDR inhibition activity of a compound which comprises:

a) placing a culture of fungal cells, transformed with a vector capable of expressing atrC, in the presence of:

(i) an antifungal agent to which said fungal cell is resistant, but to which said fungal cell is sensitive in its untransformed state;

(ii) a compound suspected of possessing Aspergillus nidulans 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 fungal cell.

2. A method of claim 1 wherein the fungal cell is Saccharomyces cerevisiae.

3. The protein of SEQ ID No. 2 in purified form.

4. A strain of A. nidulans wherein said strain carries a gene disruption or gene replacement at the atrC locus such that said strain does not produce the atrC protein product.

5. A method for identifying an antifungal compound comprising the steps of:

(a) culturing in the presence of a test compound a strain of claim 4;

(b) culturing said strain in the absence of said test compound; and

(c) comparing the growth of said strain in step (a) with the growth in step (b).