GS-X pumps are considered to constitute a terminal phase of xenobiotic detoxification in animals and plants.

The metabolism and detoxification of xenobiotics comprises three main phases (Ishikawa, 1992, supra). Phase I is a preparatory step in which toxins are oxidized, reduced or hydrolyzed to introduce or expose functional groups having an appropriate reactivity. Cytochrome P450 monooxygenases and mixed function oxidases are examples of phase I enzymes. In phase II, the activated derivative is

conjugated with GSH, glucuronic acid or glucose. In the case of the GSH-dependent pathway, S-conjugates of GSH are formed by cytosolic glutathione-S-transferases (GSTs). In the final phase, phase III, of the GSH-dependent pathway, GS-conjugates are eliminated from the cytosol by the GS-Xpump.

The GS-Xpump is unique in its exclusive use of MgATP, rather than preformed transmembrane ion gradients, as a direct energy source for organic solute transport. Although an understanding of the constituents of US-X pumps is relevant to an understanding of the mechanism by which cells combat, for example, chemotherapeutic agents and herbicides, there has until recently been a paucity of information on the molecular identity of US-Xpumps, particularly in plants.

A 190 kDa membrane glycoprotein encoded by the human multidrug resistance-associated protein gene (MRP1) has been implicated in the resistance of small cell lung cancer cell lines to a number of chemotherapeutic drugs (Cole et al., 1992, Science 258:1650-1654). This glycoprotein catalyzes the MgATP-dependent transport of leukotriene C4 and related glutathione-S-conjugates (Leier et al., 1994, J.

Biol. Chem. 269:27807-27810; Muller et al., 1994, Proc. Natl. Acad. Sci. USA 91:13033-13037; Zamam et al., 1995, Proc. Natl. Acad. Sci. USA 92:7690-7694).

MRP 1 is a member of the ATP binding cassette (ABC) superfamily of transporter proteins. Distributed throughout the major taxa, ABC transporters catalyze the MgATP-dependent transport of peptides, sugars, ions and lipophiles across membranes. ABC transporters comprise one or two copies each of two basic structural elements, a hydrophobic integral membrane sector containing approximately six transmembrane a helices and a cytoplasmically oriented ATP-binding domain known as a nucleotide binding fold (NBF) (Hyde et al., 1990, Nature 346:362-365; Higgins, 1995, Cell 82:693-696). The NBFs are a diagnostic feature of ABC transporters and are 30% identical between family members over a span of about 200 amino acid residues, having two regions known as a Walker A and a Walker B box (Walker et al., 1992, EMBO J. 1:945-951), and also having an ABC signature motif (Higgins, 1995, supra).

ABC family members in eukaryotes include mammalian P- glycoproteins (P-gps or MDRs), some of which are implicated in drug resistance and others in lipid translocation (Ruetz et al., 1994, Cell 77: - 1081), the pleiotropic drug resistance protein (PDR5) and STE6 peptide mating pheromone transporter of yeast, the cystic fibrosis transmembrane conductance regulator (CFTR) Cl channel, the malarial Plasmodiumfalciparum chloroquine transporter (PFMDRl) and the major histocompatibility (MHC) transporters responsible for peptide translocation and antigen presentation (Balzi et al., 1994, J. Bioenerg. Biomemb. 27:71-76; Higgins, 1995, supra).

Sequence comparisons between MRP 1 and other ABC transporters reveal two major subgroups among these proteins (Cole et al., 1992, supra; Szczypka et al., 1994, J. Biol. Chem. 269:22853-22857). One subgroup comprises MRP1, the Saccharomyces cerevisiae cadmium factor (YCFl) gene, the Leishmania P- glycoprotein-related molecule (Lei/PgpA) and the CFTRs. The other subgroup comprises the multiple drug resistance proteins (MDRs), MHC transporters and STE6.

The invention described herein relates to bioremediation (specifically phytoremediation), plant responses to herbicides, plant-pathogen interactions and plant pigmentation.

With respect to bioremediation, the massive global expansion in industrial and mining activities during the last two decades together with changes in agricultural practices, has markedly increased contamination of groundwaters and soils with heavy metals. Indeed, it is estimated that the annual toxicity of metal emissions exceeds that of organics and radionuclides combined (Nriagu et al., 1988, Nature 333:1340138). Since soil and water contamination results in the uptake of heavy metals and toxins by crop plants, and eventually humans, there remains a need for a means of manipulating the ability of a plant to sequester compounds from the soil in order to better manage soil detoxification through bioremediation using native species or genetically engineered organisms.

Regarding herbicides, these compounds are generally low molecular weight, lipophilic compounds that readily penetrate cells in a passive manner. Having entered cells, herbicides inhibit plant-specific processes such as photosynthetic electron transport (e.g., atrazine, chlortoluron) or the biosynthesis of essential amino acids (e.g., glyphosate, chlorsulfuron or phosphotricine), porphyrins (e.g., acidofluorfen), carotenoids (e.g., norfiurazon), fatty acids (e.g., diclofop) or cellulose (e.g., dichlobenil) (Boger et al., 1989, Target Sites ofHerbicide Action, CRC Press, Boca Raton, FL; Devine et al., 1993, Physiology ofHerbicide Action, Prentice Hall, Englewood Cliffs, NJ). Plants that are naturally tolerant of certain herbicides either contain a cellular target that does not interact with the herbicide, have efficient systems for inactivation of the herbicide, or have a high capacity for excluding or eliminating the herbicide from the target.

Herbicide metabolism comprises the three phases described above for general xenobiotic metabolism. The first two phases (the first being oxidation and hydrolysis and the second being conjugation with GSH or glucose) contribute to detoxification by decreasing the intrinsic biochemical activity of the herbicide and/or by increasing its hydrophilicity. These two phases render the herbicide less mobile in the plant. The third phase (compartmentation) is often critical for sustained detoxification since the conjugates themselves may interfere with metabolism. For example, the herbicide synergist tridiphane, is converted to its corresponding GS- conjugate in plants to generate a potent inhibitor of atrazine metabolism. (Lamoureux et al., 1986, Pestic. Biochem. Physiol. 26:323-342).

Likewise, and more generally, GS-conjugates of any given herbicide would be expected to act as end-product inhibitors of GSTs and thereby impair long- term detoxification unless they are removed from the intracellular compartment, usually the cytosol, in which they are formed. Since the vacuolar GS-Xpump of plants is known to transport several GS-herbicide conjugates, for example, those of the chloroacetanilide herbicides (metolachlor) and triazines (simetryn) (Martinoia et al., 1993, supra; Li et al., 1995, supra), there is a long felt need for a knowledge of the

molecular identity of this transporter or family of transporters. Such knowledge will enable the development of new strategies for increasing or decreasing the resistance of plants to herbicides.

With regard to plant-pathogen interactions, a key event in the disease resistance response of legumes is the rapid and localized accumulation of isoflavonoid phytoalexins. The majority of the research on plant-pathogen interactions has centered on the enzymology and molecular biology of the isoflavonoid biosynthetic pathway (Dixon et al., 1995, Physiol. Plant 93:385). However, the mechanism and sites of intracellular accumulation of these compounds is not understood. Since many isoflavonoid phytoalexins are as toxic to the host plant as they are to its pathogens, the discovery of the molecular mechanism by which these compounds are sequestered within a plant is crucial to the development of plants with increased pathogen resistance.

With regard to plant pigmentation, functional analyses of the maize gene, Bronze-2, which participates in anthocyanin pigment biosynthesis, suggest that one of the endogenous substrates for the plant vacuolar GS-Xpump are anthocyanin- GS conjugates (Marrs et al., 1995, Nature, 375:397-400). Anthocyanins share a common biosynthetic origin and core structure based on cyanidin-3-glucoside. It is through the species-specific decoration of cyanidin-3-glucoside by hydroxylation, methylation, glucosylation and acylation that the wide spectrum of red, blue and purple colors in the vacuoles of flowers, fruits and leaves is produced. The molecular nature of the plant GS-X pump which mediates transport of anthocyanin-GS conjugates was not known in the art until the present invention. There remains a need to determine the molecular nature of the GS-Xpump responsible for transport of anthocyanin-GS conjugates in order that plant coloration may be manipulated at the molecular level.

The present invention satisfies the aforementioned needs.

BRIEF SUMMARY OF THE INVENTION The invention includes an isolated DNA encoding a plant GS-Xpump polypeptide. In one aspect, the isolated DNA is selected from the group consisting of DNA comprising AtMRP1 and AtMRP2, and any mutants, derivatives, homologs and fragments thereof encoding GS-X pump activity.

The invention also includes an isolated preparation of a polypeptide comprising a plant US-Xpump. In one aspect of this aspect of the invention, the polypeptide is selected from the group consisting of AtMRP 1, AtMRP2, and any mutants, derivatives, homologs and fragments thereof having GS-Xpump activity.

Also included in the invention is a recombinant cell comprising an isolated DNA encoding a plant GS-Xpump polypeptide. In one aspect, the cell is selected from the group consisting of a prokaryotic cell and a eukaryotic cell.

Further included in the invention is a vector comprising an isolated DNA encoding a plant GS-Xpump polypeptide.

The invention also includes an antibody specific for a plant GS-Xpump polypeptide.

In addition, an isolated preparation of a nucleic acid which is in an antisense orientation to all or a portion of a plant GS-Xpump gene is included in the invention and a cell and a vector comprising this isolated preparation of a nucleic acid are further included.

The invention also relates to a transgenic plant, the cells, seeds and progeny of which comprise an isolated DNA encoding a plant GS-X pump.

In addition, the invention relates to a transgenic plant, the cells, seeds and progeny of which comprise an isolated preparation of a nucleic acid which is in an antisense orientation to all or a portion of a plant GS-Xpump gene.

Further, there is included a transgenic plant, the cells, seeds and progeny of which comprise an isolated DNA encoding YCF 1, or any mutants, derivatives, homologs and fragments thereof having YCF 1 activity.

The invention further relates to an isolated DNA comprising a plant GS- X pump promoter sequence. In one aspect, the promoter sequence is selected from the group consisting of an AtMRPl and an AtMRP2 promoter sequence.

Also included in this aspect of the invention is a cell and a vector comprising an isolated DNA comprising a plant GS-Xplant promoter sequence.

The invention additionally relates to a transgenic plant, the cells, seeds and progeny of which comprise a transgene comprising an isolated DNA comprising a GS-Xpump promoter sequence, wherein the GS-Xpump promoter sequence is selected from the group consisting of an AtMRPl, an AtMRP2 and a YCFl promoter sequence.

The promoter sequence may also have operably fused thereto a reporter gene.

There is also included in the invention a method of identifying a compound capable of affecting the expression of a plant GS-X gene. The method comprises providing a cell comprising an isolated DNA comprising a plant GS-X pump promoter sequence having a reporter sequence operably linked thereto, adding to the cell a test compound, and measuring the level of reporter gene activity in the cell, wherein a higher or a lower level of reporter gene activity in the cell compared with the level of reporter gene activity in a cell to which the test compound was not added, is an indication that the test compound is capable of affecting the expression of a plant GS-X pump gene.

In addition, the invention relates to a method of removing xenobiotic toxins from soil. The method comprises growing in the soil a transgenic plant of comprising an isolated DNA encoding a GS-Xpump.

Also included is a method of removing heavy metals from soil comprising growing in the soil a transgenic plant of comprising an isolated DNA encoding a GS-Xpump.

The invention further relates to a method of generating a transgenic pathogen resistant plant comprising introducing to the cells of the plant an isolated DNA encoding a GS-Xpump, wherein the pump is capable of transporting glutathionated isoflavonoid alexins into the cells of the plant.

Additionally, there is included a method of manipulating plant pigmentation comprising modulating the expression of a GS-X pump protein in the plant, wherein the GS-Xpump protein is selected from the group consisting of AtMRP1, AtMRP2 and YCF1.

The invention also relates to a method of alleviating oxidative stress in a plant comprising introducing into the cells of the plant DNA encoding a GS-Xpump, wherein the DNA is selected from the group consisting of DNA encoding AtMRP 1, AtMRP2 and YCF1.

Further included is a method of manipulating the expression of a gene in a plant cell. The method comprises operably fusing a GS-Xpump promoter sequence to the DNA sequence encoding the gene to form a chimeric DNA, and generating a transgenic plant, the cells of which comprise the chimeric DNA, wherein upon activation of the GS-X pump promoter sequence, the expression of the gene is manipulated.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a series of graphs depicting differential sensitivities of DYT165 cells (wild type, Figure 1A) and DTY167 cells (ycflA mutant, Figure 1B) to growth inhibition by 1-chloro-2,4-dinitrobenzene (CDNB). Cells were grown at 30°C for 24 hours to an OD600 nm of approximately 1.4 in YPD medium before inoculation of aliquots into 15 ml volumes of the same medium containing 0-60 pM CDNB.

OD600 nm was measured at the times indicated.

Figure 2 is a graph depicting the time course of [3H]DNP-GS uptake by vacuolar membrane vesicles purified from DTY165 and DTY167 cells. Uptake was measured in the absence (-MgATP) or presence of 3 mM MgATP (+MgATP) in reaction media containing 66.2 IlM [3H]DNP-GS, 10 mM creatine phosphate, 16 units/ml creatine kinase, 50 mM KCl, 0.1% (w/v) bovine serum albumin, 400 mM sorbitol, and 25 mM Tris-MES (pH 8.0) at 25"C. Values shown are means l S.E. (n = 3).

Figure 3 is a series of graphs depicting the kinetics of uncoupler- insensitive [3H]DNP-GS uptake by vacuolar membrane vesicles purified from DTY165 and DTY167 cells. Figure 3A: MgATP concentration-dependence of uncoupler- insensitive uptake. Figure 3B: DNP-GS concentration-dependence of MgATP- dependent uncoupler-insensitive uptake. The MgATP concentration-dependence of uptake was measured with 66.2 µM [3H] DNP-GS. The DNP-GS concentration- dependence of uptake was measured with 3 mM MgATP. Uptake was allowed to proceed for 10 minutes in standard uptake medium containing 5 µM gramicidin D.

The kinetic parameters for vacuolar membrane vesicles purified from DTY165 cells were Km(MgATp) 86.5 + 29.5 I1M, Km(DNpGS) 14 1 i 7 4 µM, Vmax(MgATP) 38.4 # 5.6 nmol/mg/1 0 minutes, Vmax(DNp-Gs) 51.0 i 6.3 nmol/mg/l0 minutes. The lines of best fit and kinetic parameters were computed by nonlinear least squares analysis (Marquardt, 1963, J. Soc. Ind. Appl. Math. 11:431-441). Values shown are means S.E. (n=3).

Figure 4 is a series of graphs depicting sucrose density gradient fractionation of vacuolar membrane-enriched vesicles prepared from DYT165 cells.

One ml (1.1 mg protein) of partially purified vacuolar membrane vesicles derived from vacuoles prepared by the Ficoll flotation technique were applied to a linear sucrose density gradient (10-40%, w/v) and analyzed for protein (Figure 4A), a-mannosidase activity (Figure 4B), V-ATPase activity (Figure 4C), and MgATP-dependent, uncoupler-insensitive [3H]DNP-GS uptake (Figure 4D). [3H]DNP-GS uptake and enzyme activity were assayed as described herein in Table 4 and the accompanying text.

Figure 5 includes a graph (Figure 5A) depicting the effect of transformation with pYCF 1-HA or pRS424 on MgATP-dependent, uncoupler- insensitive [ H]DNP-GS uptake by vacuolar membranes purified from DTY165 and DTY167 cells. Uptake was measured in standard uptake medium containing 66.2 I1M [3H]DNP-GS and 5 M gramicidin D. Also shown (Figure 5B) is an image of a gel depicting immunoreaction of vacuolar membrane proteins prepared from pYCF 1 -HA-

transformed and pRS424-transformed DTY 165 and DTY167 cells with mouse monoclonal antibody raised against the 12CA5 epitope of human influenza hemagglutinin. All lanes were loaded with 25 µg of delipidated membrane protein and subjected to SDS-polyacrylamide gel electrophoresis and Western analysis as described herein. The Mr of YCF 1-HA (boldface type) and the positions of the Mr standards are indicated on the figure.

Figure 6 is a series of graphs depicting transformation with pYCF 1-HA (Figure 6A) or pRS424 (Figure 6B) on the sensitivity of DTY167 cells to growth retardation by CDNB. Cells were grown at 300C for 24 hours to an OD600 nm of approximately 1.4 in AHC medium (Kim et al., 1994, Proc. Natl. Acad. Sci. USA 91:6128-6132) before inoculation of aliquots into 15 ml volumes of the same medium containing 0-60 M CDNB. OD600 nm was measured at the times indicated.

Figure 7 is a series of photomicrographs ofDTY165 (Figures 7A and 7C) and DTY167 cells (Figures 7B and 7D) after incubation with monochlorobimane.

Cells were grown in YPD medium for 24 hours at 30°C and 100 1ll aliquots of cell suspensions were transferred into 15 ml volumes of fresh YPD medium containing 100 µM monochlorobimane. After incubation for 6 hours, the cells were washed and examined in fluorescence (Figures 7C and 7D) or Nomarski mode (Figures 7A and 7B) as described herein.

Figure 8 is a series of graphs depicting uptake of Cd2+ into vacuolar membrane vesicles purified from DTY165 and DTYl67 cells. Uptake of 109Cd2+ by DTY 165 membranes (Figure 8A) or DTY 167 membranes (Figure 8B) was measured in the absence of MgATP plus (O) or minus GSH (1 mM) (#) or in the presence of MgATP (3 mM) plus (#) or minus (#) GSH. 109Cd2SO4 and gramicidin-D were added at concentrations of 80 FaM and 5 I1M, respectively. Figure 8C: Rate of 109Cd2+ uptake by DTY165 membranes plotted as a function ofthe total concentration of Cd2+ ([Cd2+]totai) added to uptake medium containing 1 mM GSH, 3 mM MgATP and 5 I1M gramidicin-D. Values shown are means + SE (n = 3-6).

Figure 9 is a series of graphs depicting purification of cadmium glutathione complexes by gel-filtration (Figure 9A) and anion-exchange chromatography (Figures 9B and 9C). Twenty mM 109Cd2SO4 was incubated with 40 mM GSH at 45 0C for 24 hours and the mixture was chromatographed on Sephadex G- 15 to resolve a high molecular weight 109Cd-labeled component (HMW-l09Cd.Gs) from a low molecular weight component (LMW-109Cd. GS) (Figure 9A). The peaks corresponding to HMW-109Cd. GS and LMW-109Cd. GS were then chromatographed on Mono-Q and eluted with a linear NaCl gradient (---) (Figure 9B and 9C). 109Cd (cpm x 10-3) was determinated on 5 µl aliquots of the column fractions by liquid scintillation counting.

Figure 10 is a series of graphs depicting the kinetics of MgATP- dependent, uncoupler-insensitive '°9Cd.GS2 (HMW-109Cd.GS, Figure 10A) and '09Cd.GS (LMW-'09Cd.GS, Figure 10B) uptake. DNP-GS was added at the concentrations (M) indicated to DTY165 membranes (#,#,#,#,#) or DTY167 membranes (O). A secondary plot of the apparent Michaelis constants for Cd.GS2 uptake (KmaPP/Cd.GS2) as a function of DNP-GS concentration is shown (Figure 10C).

The kinetic parameters for Cd.GS2 transport by DTY165 membranes were Km, 39.1 + 14.1 µM, Vmax, 157.2 # 30.4 nmol/mg/10 minutes and Ki(DNP-GS), 11.3 # 2.1 µM.

Kinetic parameters were computed by nonlinear least squares analysis (Marquardt, 1963, supra). Values shown are means + SE (n = 6).

Figure 11 is a graph depicting matrix-assisted laser desorption mass spectrometry (MALD-MS) of HMW-Cd. GS. MALD-MS was performed on Sephadex G-15-, Mono-Q-purified HMW-Cd. GS as described herein. The molecular structure inferred from a mean m/z ratio of 725.4 # 0.7 (n = 9) and average Cd.GS stoichiometry of 0.5 [bis(glutathionato)cadmium, Cd.GS2, molecular weight 724.6 Da] is shown.

Figure 12 is an image of a gel depicting induction of YCFI expression and YCF1-dependent Cd.GS2 and DNP-GS transport by pretreatment ofDTY165 cells with CdSO4 (Cd², 200 µM) or CDNB (150 µM) for 24 hours. YCFI-specific mRNA and 18S rRNA were detected in the total RNA extracted from control or pretreated

cells (10 pLg/lane) by RNase protection. Uptake of l°9Cd.GS2 (50 pM) or [3H]DNP-GS (66.2 I1M) by vacuolar membrane vesicles was measured in standard uptake medium containing 5 pM gramicidin-D. Values shown are means i SE (n = 3).

Figure 13A and 13B is the sequence of AtMRP2 cDNA (SEQ ID NO:1). Lower case letters correspond to 5'- and 3'-untranslated regions (UTRs).

Figure 14A-D is the genomic sequence of AtMPR2 (SEQ ID NO:2).

Lower case letters at the beginning and end of the sequence correspond to 5'- and 3'- UTRs, respectively; lower case letters nested within the sequence correspond to introns.

Figure 15 is the deduced amino acid sequence of AtMRP2 (SEQ ID NO:3).

Figure 16A and 16B is the sequence ofAtMRPI cDNA (SEQ ID NO:4). Lower case letters correspond to 5'- and 3'-UTRs.

Figure 17A-D is the genomic sequence of AtMRPl (SEQ ID NO:5).

Lower case letters at the beginning and end of the sequence correspond to 5'- and 3'- UTRs, respectively; lower case letters nested within the sequence correspond to introns.

Figure 18 is the deduced amino acid sequence of AtMRP1 (SEQ ID NO:6).

Figure 19 is a series of graphs depicting the time course and concentration-dependence of DNP-GS uptake in AtMRP1-transformed yeast. Figure 19A is a graph depicting the time course of [3H]DNP-GS uptake by membrane vesicles purified from pYES3-AtMRPl-transformed or pYES3-transformed DTY168 cells.

MgATP-dependent uptake was measured in reaction media containing 61.3 pM [3H]DNP-GS, 5 ,uM gramicidin-D, 10 mM creatine phosphate, 16 units/ml creatine kinase, 50 mM KCl, 1 mg/ml bovine serum albumin, 400 mM sorbitol and 25 mM Tris-Mes (pH 8.0) at 25°C. Values shown are means + SE (n = 3). Figure 19B is a graph depicting concentration dependence of MgATP-dependent, uncoupler-insensitive uptake of [3H]DNP-GS by membrane vesicles purified from pYES3-AtMRPI-

transformed DTY168 cells. Uptake was allowed to proceed for 10 minutes in standard uptake medium containing 5 pM gramicidin-D. The kinetic parameters for uptake were Km(DNp.GS) 49.7 + 15.4 µM, V,, 6.0 + 1.7 nmol/mg/10 minutes. The lines of best fit and kinetic parameters were computed by nonlinear least squares analysis (Marquardt, 1963, supra). Values shown are means + SE (n = 3).

Figure 20 is a series of graphs depicting sensitivity of MgATP- dependent, uncoupler-insensitive [3H]DNP-GS uptake by membrane vesicles purified from pYES3-AtMRPl-transformed and pYES3-transformed DTY168 cells. Uptake was measured for 10 minutes in standard uptake medium containing the indicated concentrations of vanadate. In Figure 20A, there is a graph depicting total MgATP- dependent, uncoupler-insensitive [3H]DNP-GS uptake by membrane vesicles purified from pYES3-AtMRP1-transformed and pYES-transformed DTY168 cells. In Figure 20B, there is a graph depicting AtMRP1-dependent uptake. I50 (exclusive of uninhibitable AtMRP1-independent component) = 8.3 + 3.2 I1M. Values shown are means + SE (n= 3).

Figure 21 is a series of graphs depicting the hydropathy alignment of AtMRP2, AtMRP1, S. cerevisiae YCF1 (ScYCF1), human MRP1 (HmMRP1) and rat cMOAT (RtCMOAT).

Figure 22 is a diagram depicting domain comparisons between AtMRP1, ScYCF1, HmMRP1, RtCMOAT, rabbit EBCR (RbEBCR) and HmCFTR.

The domains indicated are the N-terminal extension (NH2), first and second sets of transmembrane spans (TM1 and TM2, respectively), first and second nucleotide binding folds (NBF 1 and NBF2, respectively), putative CFTR-like regulatory domain (R), and the C-terminus (COOH).

Figure 23 is the promoter sequence of the Arabidopsis AtMRPl gene (SEQ ID NO:7). Discrete elements which are present in the promoter sequence are indicated in boldface letters.

Figure 24 is the promoter sequence of the Arabidopsis AtMRP2 gene (SEQ ID NO:8). Discrete elements which are present in the promoter sequence are indicated in boldface letters.

Figure 25 is a graph depicting MgATP-dependence of [3H]medicarpin uptake by vacuolar membrane vesicles before (Medicarpin/GSH) and after maize GST- mediated conjugation with GSH (Medicarpin-GS). [3H]medicarpin or [3H]medicarpin- GS was added at a concentration of 65 ,uM. MgATP was either omitted (-MgATP) or added at a concentration of 3 mM (+MgATP). Values shown are means # SE (n = 3).

Figure 26 is a graph depicting concentration dependence of MgATP- dependent, uncoupler-insensitive [3H]medicarpin-GS uptake into vacuolar membrane vesicles. Uptake was allowed to proceed for 20 minutes in standard uptake medium containing 3 mM MgATP and 5 I1M gramicidin D. The kinetic parameters were Km 21.5 i 15.5 pM and Vmax 77.8 + 23.3 nmol/mg/20 minutes. Values shown are means + SE (n = 3).

Figure 27 is a series of graphs depicting concentration-dependence of MgATP-dependent, uncoupler-insensitive C3G-GS, IAA-GS and ABA-GS uptake by vacuolar membrane vesicles purified from V. radiata (Figure 27A) and Z. mays (Figure 27B). Uptake was allowed to proceed for 10 minutes in reaction medium containing 50 µM GS-conjugate, 400 mM sorbitol, 3 mM MgATP, 50 mM KCl, 0.1% (w/v) BSA, 5 M gramicidin-D and 25 mM Tris-Mes (pH 8.0) at 25°C. Values shown are means + SE (n = 3).

Figure 28 is an image of a photograph depicting the growth of wild type (WT) and YCFl transgenic Arabidopsis (YCF1) seeds on media containing CdSO4 (200 ,uM) or 1-chloro-2,4-dinitrobenzene (CDNB, 25 µM). Transgenic plants were generated as described herein.

DETAILED DESCRIPTION OF THE INVENTION The invention is based upon the molecular identification of a new class of membrane transporter in yeast and plants, the US-Xpump. As a result of the present

invention, new insights into the membrane transport phenomena associated with heavy metal tolerance, herbicide detoxification, plant-pathogen interactions, plant responses to (phyto)hormones, plant pigmentation and bioremediation are evident. These insights provide a means, as is evident from the description of the present invention, for the manipulation of plants and the cells thereof, to affect heavy metal tolerance, herbicide detoxification, plant-pathogen interactions, plant responses to (phyto)hormones, plant pigmentation and bioremediation.

The process of "storage excretion" is a necessity for plants. Whereas mammals have the option of excreting GS-conjugates to the extracellular medium for elimination by the kidneys, plants are nearly totally reliant on the sequestration of noxious compounds in the central vacuole, which frequently accounts for 40-90% of total intracellular volume. Due to the virtual absence of specialized excretory organs and the presence of massive vacuoles in plants, a process (intracellular compartmentation) that is probably only an intermediate step in the elimination of xenobiotics from the cytosol of mammalian cells, is believed to constitute a terminal phase of detoxification in plants.

The data which are described herein establish that the yeast gene YCFl and two plant homologs of YCFI, AtMRP1 and AtMRP2, isolated from Arabidopsis thaliana, each encode a vacuolar GS-Xpump. The data further establish that the GS-X pump participates in herbicide metabolism (exemplified by organic xenobiotic transport), heavy metal sequestration (exemplified by cadmium transport), plant- pathogen interactions (exemplified by vacuolar uptake of medicarpin), plant cell pigmentation (exemplified by transport of glutathionated anthocyanins) and plant hormone metabolism (exemplified by the transport of glutathionated auxins).

The plant AtMRPl and AtMRP2 gene products use MgATP as an energy source for the transport of glutathionated derivatives of both endogenous and exogenous compounds in plants and thus, the discovery of these genes in the present invention is important at three levels. The identification of these genes and their encoded products represents the first identification of ABC transporters in plants for

which a biochemical function is defined. The discovery establishes, contrary to the prevailing chemiosmotic model for solute transport in plants, that many energy- dependent solute transport processes in plants are not driven by a transmembrane H+ electrochemical potential difference. Further, the identification and isolation of these genes and their encoded products permits a plant element, critical for removal of compounds from the cytosol that can form glutathionine S-conjugates, to be manipulated.

It has been discovered in the present invention that two plant genes, AtMRPl and AtMRP2, are the structural and functional homologs of the gene encoding yeast YCF 1. Proteins encoded by plant AtMRPl and AtMRP2 thus represent a new subclass of ATP binding cassette transporters.

It has been further discovered in the present invention that the yeast YCF 1 protein, a GS-X pump, is capable of MgATP-energized transport of organic GS- conjugates and of MgATP-energized transport of cadmium upon complexation with GSH. In addition, when plants have introduced into the cells thereof the YCFI gene (a transgenic plant comprising YCFl), expression of YCF1 therein confers upon the plants resistance to both inorganic and organiC xenobiotics exemplified by cadmium and 1- chloro-2,4-dinitrobenzene, respectively.

Also discovered in the present invention is the fact that AtMRPl and AtMRP2, when expressed in a strain of yeast which is deficient in YCF 1, can substitute for YCFl as a GS-X pump. In addition, transformation of plants by YCF1 confers upon the plant properties which are characteristic of YCFl gene expression. Thus, it appears that YCF1 and the AtMRP genes are essentially functionally interchangeable.

In addition, there is provided as part of the invention the promoter/regulatory sequences which control expression of the plant AtMRPl and AtMRP2 genes of the invention. These promoter sequences are useful for the identification of compounds which affect expression of thEse genes in plants and for conferring on other genes the ability to respond to factors that modulate AtMRPl and/or AtMRP2 expression.

Further discovered in the present invention is the fact that the plant GS- X pump serves to facilitate the vacuolar storage of antimicrobial compounds induced following the hypersensitive response to fungal pathogens in the healthy cells surrounding fungally-induced lesions. Such a process is believed to limit the spread of tissue damage by limiting propagation of the pathogen and spatially delimiting the toxic action of the phytoalexin itself.

Ascription of specific enzymic and regulatory roles to most of the genes of the anthocyanin biosynthetic pathway has been achieved by genetic and biochemical studies of maize with one notable exception, the Bronze-2 gene. It is known that the characteristic coloration of Bronze-2 (bz2) mutants is a consequence of the accumulation of cyanidin-3-glucoside in the cytosol. However, in wild type (Bz2) plants, anthocyanins are transported into the vacuole and become purple or red. In the mutant (bz2) plants, anthocyanin is restricted to the cytoplasm where it is oxidized to a brown ("bronze") pigment. The biochemical basis for the accumulation of anthocyanins in the cytosol is not known. However, Marrs et al., (1995, supra) have discovered that Bz2 encodes a glutathione S-transferase which is responsible for conjugating anthocyanin with GSH. It has now been discovered in the present invention that the plant GS-Xpump is the entity responsible for the delivery of glutathionated anthocyanins into the vacuole.

Identification of the GS-X pump at the molecular level has served to confirm its wide distribution and demonstrate that these transporters constitute a multigene family within the ABC transporter superfamily. The critical finding was that overexpression of the human multidrug resistance-associated protein (MRPl) gene (Cole et al., 1992, supra) confers increased MgATP-dependent GS-conjugate transport (Muller et al., 1994 supra; Leier et al.,, 1994, J. Biol. Chem. 269:27807-27810).

Several other closely related GS-Xpump genes have been characterized.

For example, a liver-specific GS-X pump (cMOAT), mutation of which is believed to cause hereditary hyperbilirubinemia, has been cloned (Paulusma et al., 1996,Science 271:1126-1128). The present invention establishes that YCF1 is a GS-Xpump. In

addition, as will become apparent from a reading of the present description, two plant genes, AtMRP1 and AtMRP2 have been discovered in the present invention to encode homologs of MRP1, YCF 1 and cMOAT.

The identification of YCF1 as a vacuolar GS-Xpump is described in detail in the experimental details section. Similarly, the identification of two plant homologs of YCF1, AtMRP1 and AtMRP2, is also described in detail in the experimental details section. Once armed with the present invention, the skilled artisan will know how to identify and isolate genes encoding other plant GS-Xpumps involved in sequestration of a variety of compounds in plants by following the procedures described herein.

A plant gene encoding a GS-Xpump is isolated using any one of several known molecular procedures. For example, primers comprising conserved regions of the sequences of any of YCFl, AtMRP1 or AtMRP2, or in fact primers comprising conserved regions of any MRP subclass (i.e., probes directed to human MRP1 cMOAT, and other MRP genes) may be used as probes to isolate, by polymerase chain reaction (PCR) or by direct hybridization, as yet unknown YCFl, AtMRP1 or AtMRP2 homologs in a DNA library comprising specific plant DNAs. Alternatively, antibodies directed against YCF 1, AtMRP1 or AtMRP2 may be used to isolate clones encoding a GS-Xpump from an expression library comprising specific plant DNAs. The isolation of primers, probes, molecular cloning and the generation of antibodies are procedures that are well known in the art and are described, for example, in Sambrook et al. (1989, Molecular Cloning. A Laboratory Manual, Cold Spring Harbor, New York) and in Harlow et al. (1988, Antibodies, A Laboratory Manual, Cold Spring Harbor, New York).

The invention includes an isolated DNA encoding a plant GS-Xpump capable of transporting a glutathionated compound across a biological membrane.

Preferably, the membrane is derived from a cell. Preferably, the DNA encoding a plant GS-Xpump is at least about 40% homologous to at least one of YCF1, AtMRP1 or AtMRP2. More preferably, the isolated DNA encoding a plant GS-X pump is at least

about 50%, even more preferably, at least about 60%, yet more preferably, at least about 70%, even more preferably, at least about 80%, yet more preferably, at least about 90% homologous, and more preferably, at least about 99% homologous to at least one of YCFl, AtMRP1 or AtMRP2. More preferably, the isolated DNA encoding a plant GS-Xpump is Arabidopsis AtMRP1 or AtMRP2. Most preferably, the isolated DNA encoding a plant GS-Xpump is SEQ ID NOS:1, 2, 4 or 5.

Thus, the invention should be construed to include genes which encode Arabidopsis AtMRP1 and AtMRP2 and Arabidopsis AtMRPl and AtMRP2-related genes.

By "GS-Xpump" as used herein, is meant a protein which transports a glutathione-conjugated compound across a biological membrane.

By the term "DNA encoding a GS-Xpump" as used herein is meant a gene encoding a polypeptide capable of transporting a glutathionated compound across a biological membrane.

By "AtMRP-related gene" as used herein, is meant a gene encoding a GS-X pump which is a member of the MRP/YCFl/cMOA T family of genes. An AtMRP1 or AtMRP2-related gene may be present in a cell which also encodes an AtMRP gene or it may be present in a different cell and in a different plant species.

As described in the Experimental Detail section, AtMRP genes encode proteins which have specific domains located therein, namely, the N-terminal extension, transmembrane spans, TOM 1 and TM2, nucleotide binding folds, NBF 1 and NBF2, putative CFTR-like regulatory domain (R) and the C terminus. An AtMRP- related gene is therefore also one in which selected domains in the related protein share significant homology (at least about 40% homology) with the same domains in either of YCF l, AtMRP1 or AtMRP2. For example, when the R-domain in the AtMRP- related protein shares at least about 40% homology with the R domain in YCF 1, AtMRP 1 or AtMRP2, and when the product of that is a GS-X pump, then that gene is an AtMRP-related gene. Similarily, when the N-terminal extension in the AtMRP- related protein shares at least about 40% homology with the N-terminal extension in

YCF 1, AtMRP 1 or AtMRP2, and when the product of that is a GS-X pump, then that gene is an AtMRP-related gene. It will be appreciated that the definition of an AtMRP- related gene encompasses those genes having at least about 40% homology in any of the described domains contained therein with the same or a similar domain in either of YCF1, AtMRP1 or AtMRP2. In addition, when the term homology is used herein to refer to the domains of these proteins, it should be construed to be applied to homology at both the nucleic acid and the amino acid levels.

While a significant homology between similar domains in AtMRP- related genes or their protein products is considered to be at least about 40%, preferably, the homology between domains is at least about 50%, more preferably, at least about 60%, even more preferably, at least about 70%, even more preferably, at least about 80%, yet more preferably, at least about 90% and most preferably, the homology between similar domains is about 99% between a domain in an AtMRP- related gene or protein product thereof, and at least one of YCF1, AtMRPl or AtMRP2 or the protein products thereof.

Plants from which AtMRPl, AtMRP2 or YCF1 related genes may be isolated include any plant in which the GS-Xpump is found, including, but not limited to, soybean, castor bean, maize, petunia, potato, tomato, sugar beet, tobacco, oats, wheat, barley, pea, faba bean and alfalfa.

By the term "glutathionated-conjugated compound" as used herein is meant a compound, e.g., a metal, a xenobiotic, a isoflavonoid phytoalexin, anthocyanin or auxin, which is chemically conjugated to glutathionine. Conjugation of compounds to glutathione occurs naturally within cells and organisms and may also be accomplished enzymatically or non-enzymatically in vitro as described herein in the experimental details section.

Also included in the invention is an isolated DNA encoding a biologically active polypeptide fragment of a plant GS-Xpump. Preferably, the isolated DNA encoding a biologically active polypeptide fragment of a plant GS-X pump is at least about 40% homologous to a biologically active polypeptide fragment

of at least one of YCF1, AtMRP1 or AtMRP2. More preferably, the isolated DNA encoding a biologically active polypeptide fragment of a plant GS-X pump is at least about 50%, even more preferably, at least about 60%, yet more preferably, at least about 70%, even more preferably, at least about 80%, yet more preferably, at least about 90%, and even more preferably, at least about 99% homologous to a biologically active polypeptide fragment of at least one of YCF 1, AtMRP 1 or AtMRP2. Most preferably, the isolated DNA encoding a biologically active polypeptide fragment of a plant GS-Xpump is a biologically active polypeptide fragment of Arabidopsis AtMRP1 or AtMRP2.

Preferably, the isolated DNA encoding a biologically active polypeptide fragment of a plant GS-Xpump is about 200 nucleotides in length. More preferably, the isolated DNA encoding a biologically active polypeptide fragment of a plant GS-X pump is about 400 nucleotides, even more preferably, at least about 600, yet more preferably, at least about 800, even more preferably, at least about 1000, and more preferably, at least about 1200 nucleotides in length.

The invention further includes a vector comprising a gene encoding a plant GS-Xpump and a vector comprising nucleic acid sequence encoding a biologically active fragment thereof. The procedures for the generation of a vector encoding a plant GS-Xpump, or fragment thereof, are well known in the art once the sequence of the gene is known, and are described, for example, in Sambrook et al.

(supra). Suitable vectors include, but are not limited to, disarmed Agrobacterium tumor-inducing (Ti) plasmids (e.g., pBIN19) containing the target gene under the control of the cauliflower mosaic virus (CaMV) 35S promoter (Lagrimini et al., 1990, Plant Cell 2:7-18) or its endogenous promoter (Bevan, 1984, Nucl. Acids Res.

12:8711-8721).

Also included in the invention is a cell comprising an isolated DNA encoding a plant GS-Xpump and a cell comprising an isolated DNA encoding a biologically active fragment thereof. Such a cell is referred to herein as a "recombinant cell."

The procedures for the generation of a cell encoding a plant GS-X pump or fragment thereof, are well know in the art once the sequence of the gene is known, and are described, for example, in Sarnbrook et al. (supra). Suitable cells include, but are not limited to, yeast cells, bacterial cells, mammalian cells, and baculovirus- infected insect cells transformed with the gene for the express purpose of generating GS-X polypeptide. In addition, plant cells transformed with the gene for the purpose of producing cells and regenerated plants having increased resistance to and increased capacity for heavy metal accumulation, increased resistance to organic xenobiotics and increased capacity for organic xenobiotic accumulation or altered coloration.

The invention also includes an isolated preparation of a polypeptide comprising a plant GS-X pump capable of transporting a glutathionated compound across a biological membrane. Preferably, the isolated preparation of a polypeptide comprising a plant GS-Xpump is at least about 30% homologous to at least one of YCF1, AtMRP 1 or AtMRP2. More preferably, the isolated preparation of a polypeptide comprising a plant GS-Xpump is at least about 40%, even more preferably, at least about 50%, yet more preferably, at least about 60%, even more preferably, at least about 70%, more preferably, at least about 80%, even more preferably, at least about 90% and more preferably, at least about 99% homologous to at least one of YCF 1, AtMRP1 or AtMRP2. More preferably, the isolated preparation of a polypeptide comprising a plant GS-Xpump is Arabidopsis AtMRP1 or AtMRP2.

Most preferably, the isolated preparation of a polypeptide comprising a plant GS-X pump is SEQ ID NOS: 3 or 6.

Also included in the invention is an isolated preparation of a biologically active polypeptide fragment of a plant GS-Xpump. Preferably, the isolated preparation of a biologically active polypeptide fragment of a plant GS-X pump is at least about 30% homologous to a biologically active polypeptide fragment of at least one of YCF 1, AtMRP 1 or AtMRP2. More preferably, the isolated preparation of a biologically active polypeptide fragment of a plant GS-X pump is at least about 40%, even more preferably, at least about 50%, yet more preferably, at least

about 60%, even more preferably, at least about 70% and yet more preferably, at least about 80%, even more preferably, at least about 90% and more preferably, at least about 99% homologous to a biologically active polypeptide fragment of at least one of YCF 1, AtMRP 1 or AtMRP2. Most preferably, the isolated preparation of a biologically active polypeptide fragment of a plant GS-Xpump is a biologically active polypeptide fragment of Srabidopsis AtMRP1 or AtMRP2.

Preferably, the polypeptide in the isolated preparation of a biologically active polypeptide fragment of a plant GS-Xpump is about 60 amino acids in length.

More preferably, the polypeptide in the isolated preparation of a biologically active polypeptide fragment of a plant US-X pump is about 130 amino acids, even more preferably, at least about 200, yet more preferably, at least about 300, even more preferably, at least about 350, and more preferably, at least about 400 amino acids in length.

As used herein, the term "homologous" refers to the subunit sequence similarity between two polymeric molecules e.g., between two nucleic acid molecules, e.g., between two DNA molecules, or two polypeptide molecules. When a subunit position in both of the two molecules is occupied by the same monomeric subunit, e.g., if a position in each of two polypeptide molecules is occupied by phenylalanine, then they are homologous at that position. The homology between two sequences is a direct function of the number of matching or homologous positions, e.g., if half (e.g., 5 positions in a polymer 10 subunits in length) of the positions in two polypeptide sequences are homologous then the two sequences are 50% homologous; if 70% of the positions, e.g., 7 out of 10, are matched or homologous, the two sequences share 70% homology. By way of example, the polypeptide sequences ACDEFG and ACDHIK (SEQ ID NOS:9 and 10, respectively) share 50% homology and the nucleotide sequences CAATCG and CAAGAC share 50% homology.

An "isolated DNA," as used herein, refers to a DNA sequence which has been separated from the sequences which flank it in a naturally occurring state, e.g., a DNA fragment which has been removed from the sequences which are normally

adjacent to the fragment, e.g., the sequences adjacent to the fragment in a genome in which it naturally occurs. The term also applies to nucleic acids which have been substantially purified from other components which naturally accompany the nucleic acid (e.g., RNA, DNA or protein) in its natural state. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or which exists as a separate molecule (e.g., as a cDNA or a genomic or cDNA fragment produced by PCR or restriction enzyme digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence.

As used herein, the term "isolated preparation of a polypeptide" describes a polypeptide which has been separated from components which naturally accompany it. Typically, a polypeptide is isolated when at least 10%, more preferably at least 20%, more preferably at least 50%, more preferably at least 60%, even more preferably at least 75%, more preferably at least 90%, and most preferably at least 99% of the total material (by volume, by wet or dry weight, or by mole per cent or mole fraction) of a sample is the polypeptide of interest. The degree of isolation of the polypeptide can be measured by any appropriate method, e.g., by column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis. For example, a polypeptide is isolated when it is essentially free of naturally associated components or when it is separated from the native compounds which accompany it in its natural state.

As used herein, by the term "biologically active" as it refers to GS-X pump activity as used herein, is meant a polypeptide, or a fragment thereof, which is capable of transporting a glutathionated compound across a biological membrane.

In summary, the invention should be construed to include DNA comprising AtMRPl and AtMRP2, and any mutants, derivatives, homologs and fragments thereof, which encode GS-Xpump biological activity.

The invention further features an isolated preparation of a nucleic acid which is antisense in orientation to a portion or all of a plant GS-X pump gene, wherein the nucleic acid is capable of inhibiting expression of the GS-Xpump gene when introduced into cells comprising the GS-Xpump gene. The nucleic acid is antisense to either a portion or all of a plant GS-X pump gene, which gene is preferably Arabidopsis AtMRP1, Arabidopsis AtMRP2 or a homolog thereof. The "isolated preparation of a nucleic acid" and the "portion" of the gene to which the nucleic acid is antisense, should be of a sufficient length so as to inhibit expression of the desired target gene.

The actual length of the isolated preparation of the nucleic acid may vary, and will depend on the particular target gene and the region of that gene which is targetted.

Typically, the isolated preparation of the nucleic acid will be at least about 15 contiguous nucleotides; more typically, it will be between about 15 and about 50 contiguous nucleotides, or it may even be more than 50 contiguous nucleotides in length.

As used herein, a sequence of a nucleic acid is "antisense" to a portion or all of a GS-Xpump gene when the sequence of nucleic acid does not encode a GS-X polypeptide. Rather, the sequence which is being expressed in the cells is identical to the non-coding strand of the GS-X pump gene and thus, does not encode a GS-X pump polypeptide.

"Complementary," as used herein, refers to the subunit sequence complementarity between two nucleic acids, e.g., two DNA molecules. When a nucleotide position in both of the molecules is occupied by nucleotides normally capable of base pairing with each other, then the nucleic acids are considered to be complementary to each other at this position. Thus, two nucleic acids are complementary to each other when a substantial number (at least 50%) of corresponding positions in each of the molecules are occupied by nucleotides which normally base pair with each other (e.g., A:T and G:C nucleotide pairs).

In yet another aspect of the invention, there is provided an antibody directed against a plant GS-Xpump, preferably AtMRP1 or AtMRP2, which antibody

is specific for the whole molecule or either the N-terminal or the C-terminal or internal portions of AtMRP 1 or AtMRP2. Methods of generating such antibodies are well known in the art and are described, for example, in Harlow et al. (supra).

The present invention also provides for analogs of proteins or peptides encoded by AtMRP1 or AtMRP2. Analogs can differ from naturally occurring proteins or peptides by conservative amino acid sequence differences or by modifications which do not affect sequence, or by both.

For example, conservative amino acid changes may be made, which although they alter the primary sequence of the protein or peptide, do not normally alter its function. Conservative amino acid substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; phenylalanine, tyrosine.

Modifications (which do not normally alter primary sequence) include in vivo, or in vitro chemical derivatization of polypeptides, e.g., acetylation, or carboxylation. Also included are modifications of glycosylation, e.g., those made by modifying the glycosylation patterns of a polypeptide during its synthesis and processing or in further processing steps; e.g., by exposing the polypeptide to enzymes which affect glycosylation, e.g, mammalian glycosylating or deglycosylating enzymes. Also embraced are sequences which have phosphorylated amino acid residues, e.g., phosphotyrosine, phosphoserine, or phosphothreonine.

Also included are polypeptides which have been modified using ordinary molecular biological techniques so as to improve their resistance to proteolytic degradation or to optimize solubility properties or to render them more suitable as a

therapeutic agent. Analogs of such polypeptides include those containing residues other than naturally occurring L-amino acids, e.g, D-amino acids or non-naturally occurring synthetic amino acids. The peptides of the invention are not limited to products of any of the specific exemplary processes listed herein.

The invention further includes a transgenic plant comprising an isolated DNA encoding a plant GS-Xpump polypeptide or a fragment thereof, capable of transporting a glutathionated compound across a biological membrane. The transgenic plant of the invention may comprise a transgene encoding a plant GS-X pump polypeptide or a fragment thereof, or it may comprise a transgene encoding a yeast GS- X pump polypeptide or a fragment thereof, which yeast transgene is expressed in the plant to yield a biologically active US-X pump protein product. By way of example, there is provided herein in the experimental examples section a transgenic Arabidopsis plant comprising a yeast YCF1 transgene, which when the transgene is expressed in the transgenic plant, confers upon the plant the ability to grow on media containing concentrations of heavy metal (cadmium) or organic xenobiotic (CDNB) that otherwise prevent of nontransgenic plants.

The invention also includes a transgenic plant comprising an isolated DNA comprising the sequence of a plant GS-X pump polypeptide or a fragment thereof, which plant GS-Xpump is capable of transporting a glutathionated compound across a membrane derived from a cell, wherein the sequence of the isolated DNA is positioned in an antisense orientation with respect to the direction of transcription of the DNA.

Thus, included in the invention is a transgenic plant comprising an isolated DNA encoding a yeast YCF 1 or a fragment thereof, capable of transporting a glutathionated compound across a membrane derived from a cell.

In addition, the invention includes a transgenic plant comprising an isolated DNA comprising the sequence of a yeast YCF1 gene or a fragment thereof, wherein the sequence of the isolated DNA is positioned in an antisense orientation with respect to the direction of transcription of the DNA.

By "transgenic plant" as used herein, is meant a plant, the cells, the seeds and the progeny of which comprise a gene inserted therein, which gene has been manipulated to be inserted into the cells of the plant by recombinant DNA technology.

The manipulated gene is designated as a "transgene." By the term "nontransgenic but otherwise substantially homozygous wild type plant" as used herein, is meant a nontransgenic plant from which the transgenic plant was generated.

"Positioned in an antisense orientation with respect to the direction of transcription of the DNA" as used herein, means that the transcription product of the DNA, the resulting mRNA, does not encode a GS-Xpump. Rather, the mRNA comprises a sequence which is complementary to an mRNA which encodes a GS-X pump.

If vacuolar transport rate limits xenobiotic detoxification and if the amount of GS-Xpump is rate limiting on the overall rate of vacuolar uptake, transgenic plants with increased YCF1, AtMRP1 orAtMRP2 expression are expected to be more resistant to the toxic effects of glutathione-conjugable xenobiotics and capable of accumulating higher vacuolar conjugate levels than non-transgenic plants. The former property permits the sustained growth of transgenic plants in the presence of xenobiotic concentrations that would retard the growth of plants exhibiting normal levels of transporter expression. The latter property confers on the plants the ability for hyperaccumulation of glutathionated xenobiotics.

Increased resistance to xenobiotics has application in herbicide technology and plant growth in habitats polluted with organics. Hyperaccumulation has application in the extraction of organic pollutants from contaminated ground soils.

The closest known similar technologies to those described herein (a) involve the isolation of mutants or the engineering of plants in which the target for xenobiotic action is no longer sensitive, (b) involve the generation of mutants with elevated cellular levels of glutathionine (GSH) or with increased glutathione-S- transferase activities, or (c) involve the application of chemical agents ("safeners") that

elevate GSH and/or glutathione-S-transferase levles or activities. These known technologies differ from the strategy proposed herein in three respects: (i) The utility of mutated target gene products is limited in its application to those xenobiotics that directly interact with the target in question. In contrast, the vacuolar GS-Xpump is of broad substrate specificity. (ii) Technologies based on elevated cellular GSH levels or increased glutathione-S-transferase catalytic efficiencies are limited by the capacity of cells to subsequently metabolize and/or sequester the conjugates generated. The success of these latter technologies eventually depends on delivery of GSH-conjugates into the vacuole and in turn, depends on the activity of the vacuolar GS-Xpump. (iii) Since the plant vacuole frequently constitutes 40-90% of total intracellular volume and the GS-X pump mediates the uptake of xenobiotics into this compartment, the potential for hyperaccumulation on a tissue weight basis is great. Hyperaccumulators may therefore be used for the fixation/sequestration of toxins and their removal from soils.

None of the other known technologies have this characteristic.

The generation of transgenic plants comprising sense or antisense DNA having the sequence of a GS-X pump or a fragment thereof, may be accomplished by transformation of the plant with a plasmid encoding the desired DNA sequence.

Suitable vectors include, but are not limited to, disarmed Agrobacterium tumor- inducing (Ti) plasmids containing a sense or antisense strand placed under the control of the strong constitutive CaMV 35S promoter or under the control of an inducible promoter (Lagrimini et al., 1990, supra; van der Krol et al., 1988, Gene 72:45-50).

Methods for the generation of such constructs, plant transformation and plant regeneration are well known in the art once the sequence of the desired gene is known and are described, for example, in Ausubel et al. (1993, Current Protocols in Molecular Biology, Greene and Wiley, New York).

Suitable vector and plant combinations will be readily apparent to those of skill in the art and can be found, for example, in Maliga et al. (1994, Methods in Plant Molecular Biology: A Laboratory Manual, Cold Spring Horbor, New York).

Transformation of plants may be accomplished using the Agrobacterium-mediated leaf disc transformation method described by Horsch et al.

(1988, Leaf Disc Transformation, Plant Molecular Biology Manual A5:1).

A number of procedures may be used to assess whether the transgenic plant comprises the desired DNA. For example, genomic DNA obtained from the cells of the transgenic plant may be analyzed by Southern blot hybridization or by PCR to determine the length and orientation of any inserted, transgenic DNA present therein.

Northern blot hybridization analysis or PCR may be used to characterize mRNA transcribed in cells of the transgenic plant. In situations where it is expected that the cells of the transgenic plant express GS-Xpolypeptide or a fragment thereof, Western blot analysis may be used to identify and characterize polypeptides so expressed using antibody raised against the GS-Xpump or fragments thereof. The procedures for performing such analyses are well known in the art and are described, for example, in Sambrook et al. (supra).

The transgenic plants of the invention are useful for the manipulation of xenobiotic detoxification, heavy metal detoxification, control of plant pathogens, control of plant coloration, herbicide metabolism and phytohormone metabolism. For example, a transgenic plant encoding an AtMRPl or an AtMRP2 gene or an AtMRPI- or AtMRP2-related gene, or a yeast YCF1 or YCFl-related gene, is useful for xenobiotic detoxification and heavy metal detoxification when grown on soil containing xenobiotics or heavy metals. Such plants are capable of removing xenobiotic toxins or heavy metals from the soil thereby generating soil which has reduced levels of compounds that are detrimental to the overall health of the environment.

Accordingly, the invention includes a method of removing xenobiotic toxins from soil comprising generating a transgenic plant having a transgene encoding a GS-X pump and planting the plant or the seeds of the plant in the soil wherein xenobiotic toxins in the soil are sequestered within the plant during growth of the plant in the soil.

Similarly, the invention includes a method of removing heavy metals from soil comprising generating a transgenic plant having a transgene encoding a GS-X pump and planting the plant or the seeds of the plant in the soil wherein heavy metals in the soil are sequestered within the plant during growth of the plant in the soil.

When the levels of xenobiotic toxins or heavy metals in the soil have been sufficiently reduced, the transgenic plant may be removed from the soil and destroyed or discarded in an environmentally safe manner. For example, the harvested plants can be reduced in volume and/or weight by thermal, microbial, physical or chemical means to decrease handling, processing and potential subsequent land filling costs (Cunningham et al., 1996, Plant Physiol. 110:715-719). In the case of valuable metals, subsequent smelting and recovery of the metal may be cost-effective (Raskin, 1996, Proc. Natl. Acad. Sci. USA 93:3164-3166).

This technique of remediating soil is more efficient, less expensive and easier than most chemical or physical methods. The estimated costs of remediation are as follows: U.S. $10-100 per cubic meter of soil for removal of volatile or water soluble pollutants by in situ remediation using plants; U.S. $60-300 per cubic meter of soil for landfill or low temperature thermal treatment remediation of soil contaminated with the same compounds; and, U.S. $200-700 per cubic meter of soil for remediation of soil contaminated with materials requiring special landfilling arrangements or high temperature thermal treatment (Cunningham et al., 1995, Trends Biotechnol. 13:393- 397).

Preferably, the transgene in the transgenic plant of the invention is AtMRP1, AtMRP2, YCFI or genes encoding fragments or analogs of AtMRPl, AtMRP2 or YCF1, or the transgene is a gene which is related to AtMRP1, AtMRP2, YCF1.

The types of plants which are suitable for use in this method of the invention include, but are not limited to, high yield crop species for which cultivation practices have already been perfected, or engineered endemic species that thrive in the area to be remediated.

In certain situations, it may be necessary to prevent the removal of substances such as xenobiotic toxins and heavy metals from the soil. In such situations, transgenic plants are generated comprising a transgene comprising a GS-X pump sequence which is in the antisense orientation with respect to transcription. Such transgenes therefore serve to inhibit the function of a GS-X pump expressed in the plants thereby preventing removal of xenobiotics or heavy metals from the soil.

The production of plants having GS-X pump antisense sequences has application in the manipulation of plant/food coloration and in the diminution of organic xenobiotic (e.g., herbicide) or heavy metal accumulation by crop species. For example, ingestion by animals or humans of low organic toxin/low heavy metal crops will likely contribute to an improvement in the overall health of animals and humans.

Accordingly, the invention includes a method of preventing the removal of xenobiotic toxins or heavy metals from soil comprising generating a transgenic plant having a transgene comprising a GS-Xpump sequence which is in the antisense orientation with respect to transcription and planting the plant or the seeds of the plant in the soil, wherein removal of xenobiotics and heavy metals from the soil is prevented during growth of the plant in the soil.

The antisense sequences which are useful for the generation of transgenic plants having antisense GS-Xpump sequences are those which will inhibit expression of a resident GS-X gene in the plant.

The types of plants which are suitable for use in this method of the invention using antisense sequences include, but are not limited to, plants for which anthocyanins contribute to flower, fruit or leaf coloration and food crops for which decreased organic xenobiotic and/or heavy metal accumulation is desirable.

In a similar manner to that described herein, a transgenic plant may be generated which exhibits increased accumulation and/or resistance to isoflavonoid alexins by introducing into the cells of the plant a transgene encoding a GS-X pump capable of transporting glutathionated isoflavonoid alexins into vacuoles in the plant, thereby isolating the isoflavonoid alexins from the cytoplasm of the cells of the plant.

Preferably, the transgene is AtMRPI, AtMRP2, YCFl or genes encoding fragments or analogs of AtMRP1, AtMRP2 or YCF1, or the transgene is a gene which is related to AtMRPl, AtMRP2 or YCFl.

The invention thus includes a method of generating a pathogen-resistant transgenic plant comprising introducing into the plant a transgene encoding a GS-X pump capable of transporting glutathionated isoflavonoid alexins into vacuoles in the plant.

The types of plants suitable for the introduction of the desired transgene include, but are not limited to, plants which are leguminous plants, for example, alfalfa, cashew nut, castor bean, faba bean, french bean, mung bean, pea, peanut, soybean and walnut.

As discussed herein, it has also been discovered in the present invention that the Bz2 gene which encodes a glutathione-S-transferase, glutathionates anthocyanins and possibly other compounds for transport by the GS-Xpump. The anthocyanin-derivatives so generated are subsequently transported across biological membranes by the vacuolar GS-Xpump. Vacuolar anthocyanins are responsible for the red and purple hues of many plant organs (petals, leaves, stems, seeds, fruits, etc.).

Vacuolar anthocyanins are found in most flowering plants. However, they are not solely responsible for plant coloration. Rather, plant coloration is determined by the relative amounts and combinations in which these various pigments are accumulated.

Thus, it is possible to manipulate plant coloration by generating transgenic plants with increased (sense DNA) or decreased (antisense DNA) expression of the GS-Xpump.

Transgenic plants having GS-Xpump sense sequences are expected to contain more red/purple pigmentation that their nontransgenic but otherwise homozygous counterparts and transgenic plants having GS-X pump antisense sequences are expected to contain less red/purple pigmentation and possibly more brown pigmentation that their nontransgenic but otherwise homozygous counterparts. The generation of such types of transgenic plants may be accomplished following the procedures described herein.

With respect to the aforementioned information regarding anthocyanins, it is important to note that accumulating evidence from studies of the MRP-subclass members from non-plant sources reveals that the group of transporters formerly referred to an GS-X pumps because of their affinity toward GS-conjugates, GSSG and cysteinyl leukotrienes, do not transport GS-conjugates exclusively (Ishikawa et al., 1997, Bioscience Reports 17:189-208). Investigation of the human MRP1 protein, cMOAT and ScYCF 1 establish that these proteins are capable of transporting a broad range of compounds in addition to GS-conjugates and GSSG Jedlitschky et al., 1996, Cancer Res. 56:988-994; Paulusma et al., 1996, Science 271:1126-1128; Jansen et al., 1987, Hepatol. 7:71-76; Sathirakul et al., 1993, J. Pharmacol. Exp. Therap. 268:65-73).

Thus, these proteins transport non-glutathionated compounds.

It has been discovered in the present invention that the plant proteins, AtMRP1 and AtMRP2, differ in their substrate preferences. For example, no only does AtMRP2 exhibit a much higher transport capacity than does AtMRP 1, but AtMRP2 has the capacity to transport chlorophyll breakdown products in leaf senescence, which breakdown products are not glutathionated. Thus, according to the present invention, it is possible to manipulate plant coloration by changing the relative levels of expression of various members of this class of transporters in a plant cell. It is possible, using the information provided herein, to affect the rate of breakdown of chlorophyll, for example, by manipulating the expression of AtMRP2 in a plant cell.

In addition to the above, there is provided as part of the invention, AtMRP1 and AtMRP2 promoter sequences. By operably coupling the AtMRP1 or AtMRP2 promoters to other genes, it may be possible to confer on these other genes expression characteristics similar to those of AtMRP1 or AtMRP2, namely, modulation by xenobiotics, plant pathogens, etc. The data which are presented herein include the promoter sequences of these genes, which promoter sequences are useful in a variety of applications in plants. For example, GS-Xpump activity which is associated with herbicide metabolism (exemplified by organic xenobiotic transport), heavy metal sequestration (exemplified by cadmium transport), plant-pathogen interactions

(exemplified by vacuolar uptake of medicarpin), plant cell pigmentation (exemplified by transport of glutathionated anthocyanins) and plant hormone metabolism (exemplified by the transport of glutathionated auxins) may be examined as a result of the present invention. The present invention facilitates the identification of plants and cells therein which are capable of GS-X pump activity, and further facilitates the exploitation of plant cell GS-X pump activity for the purpose of affecting plant function with respect to herbicide metabolism, heavy metal sequestration, plant-pathogen interactions, plant cell pigmentation and plant hormone metabolism.

The invention includes an isolated DNA comprising a plant GS-Xpump promoter sequence capable of driving expression of a plant GS-X pump gene, which gene is capable of transporting a glutathionated compound across a biological membrane. Preferably, the membrane is derived from a cell.

Preferably, the isolated DNA comprising a plant GS-Xpump promoter sequence is at least about 40% homologous to at least one of the AtMRP1 or AtMRP2 promoter sequences presented herein in Figures 23 and 24, respectively. More preferably, the isolated DNA comprising a plant GS-Xpump promoter sequence is at least about 50%, even more preferably, at least about 60%, yet more preferably, at least about 70%, even more preferably, at least about 80%, yet more preferably, at least about 90% homologous, and more preferably, at least about 99% homologous to at least one of AtMRP1 or AtMRP2 promoter sequences presented herein in Figures 23 and 24, respectively. Most preferably, the isolated DNA comprising a plant GS-X pump promoter sequence is Arabidopsis AtMRP1 or AtMRP2 as shown in Figures 1 and 2, respectively.

Thus, the invention should be construed to include isolated DNA sequences comprising promoter sequences which in their natural form drive expression of genes which encode Arabidopsis AtMRP1 and AtMRP2 and Arabidopsis AtMRP1 and AtMRP2-related genes. Once armed with the present invention, it is a simple matter to isolate sequences which are related to those shown in Figures 1 and 2. For example, conventional hybridization technology and/or PCR technology may be

employed, primers may be designed using the sequences provided herein, data bases may be searched and the like. Procedures for the isolation of promoter sequences which are related to those described herein are described in Sambrook et al. (1989, Molecular Cloning. A Laboratory Manual, Cold Spring Harbor, NY) and in Ausubel et al. (1993, Current Protocols in Molecular Biology, Greene and Wiley, New York).

By the term "promoter sequence" as used herein, is meant a DNA sequence which is required for expression of a gene which is operably linked thereto.

In some instances, this sequence may be a core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements which are required for expression of the gene in a tissue-specific manner.

Thus, a promoter sequence must include an RNA polymerase binding site and may include appropriate transcription factor binding sites as are necessary for activation of transcription and expression of the gene to which the promoter sequence is attached at the 5' end of the gene.

Typically, the promoter sequence of the invention comprises at least about 150 bp in length. More typically, the promoter sequence comprises at least about 300 bp in length. More typically, the promoter sequence comprises at least about 400 bp, even more typically, at least about 500 bp, yet more typically, at least about 600 bp, even more typically, at least about 800 bp, yet more typically, at least about 1000 bp and even more typically, at least about 1200 or more bp in length.

The promoter sequence of the invention may also comprise discrete sequences (elements) which function to regulate the activity of the promoter.

Frequently, such elements respond to the presence or absence of environmental factors, thereby controlling gene expression in direct response to factors which are associated with the environmental mileau of the plant. The response of the plant to these factors affects the overall well-being of the plant. Elements which may be present in the promoter sequence of the invention include, but are not limited to, a Myb recognition sequence, a xenobiotic regulatory element, an antioxidant response element, a bZIP recognition sequence, and the like.

Plants from which AtMRP1- or AtMRP2-related genes and therefore promoter sequences, may be isolated include any plant in which the GS-Xpump is found, including, but not limited to, soybean, castor bean, maize, petunia, potato, tomato, sugar beet, tobacco, oats, wheat, barley, pea, faba bean and alfalfa.

The invention further includes a vector comprising a plant GS-Xpump promoter sequence operably fused to a reporter gene and capable of driving expression of the reporter gene. The procedures for the generation of a vector comprising a plant GS-Xpump promoter sequence are well know in the art once the sequence of the gene is known, and are described, for example, in Sambrook et al. (supra) . Suitable vectors include, but are not limited to, disarmed Agrobacterium tumor-inducing (Ti) plasmids (e.g., pBIN19) (Lagrimini et al., 1990, Plant Cell 2:7-18; Bevan, 1984, Nucl. Acids Res. 12:8711-8721).

Also included in the invention is a cell comprising a plant GS-Xpump promoter sequence operably fused to a reporter gene. The procedures for the generation of a cell encoding a plant GS-Xpump or fragment thereof, are well know in the art once the sequence of the gene is known, and are described, for example, in Sambrook et al. (supra). Suitable cells include, but are not limited to, plant cells, yeast cells, bacterial cells, mammalian cells, and baculovirus-infected insect cells. In addition, plant cells transformed with the promoter/reporter gene construct, for the purpose of assessing the effect of various compounds on promoter activity are also contemplated in the invention. Normal plant cells and those plant cells having increased resistance to and increased capacity for heavy metal accumulation, increased resistance to organic xenobiotics and increased capacity for organic xenobiotic accumulation or altered coloration, which cells comprise the promoter sequence of the invention operably fused to a reporter gene, are all contemplated as part of the invention. When the promoter is fused to a reporter gene, the promoter is said to be operably linked to the reporter gene.

A "reporter gene" as used herein, is one which when expressed in a cell, results in the production of a detectable product in the cell. The level of expression the

product in the cell is proportional to the activity of the promoter sequence which drives expression of the reporter gene.

By describing two nucleic acid sequences as "operably linked" as used herein, is meant that a single-stranded or double-stranded nucleic acid moiety comprises each of the two nucleic acid sequences and that the two sequences are arranged within the nucleic acid moiety in such a manner that at least one of the two nucleic acid sequences is able to exert a physiological effect by which it is characterized upon the other.

Suitable reporter genes include, but are not limited to, -glucuronidase (GUS) and green fluorescent protein (GFP), although any reporter gene capable of expression and detection in plant cells which are either known or heretofore unknown, may be fused to the plant GS-Xpromoter sequences of the invention.

The invention further includes a transgenic plant comprising an isolated DNA comprising a plant GS-Xpump promoter sequence as defined herein.

The generation of transgenic plants comprising a plant GS-Xpump promoter sequence operably fused to a reporter gene, may be accomplished by transformation of the plant with a plasmid comprising the desired DNA sequence.

Suitable vectors include, but are not limited to, disarmed Agrobacterium tumor- inducing (Ti) plasmids (Lagrimini et al., 1990, supra; van der Krol et al., 1988, Gene 72:45-50). Methods for the generation of such constructs, plant transformation and plant regeneration are well known in the art once the sequence of the desired nucleic acid is known and are described, for example, in Ausubel et al. (1993, Current Protocols in Molecular Biology, Greene and Wiley, New York).

Suitable vector and plant combinations will be readily apparent to those of skill in the art and can be found, for example, in Maliga et al. (1994, Methods in Plant Molecular Biology: A Laboratory Manual, Cold Spring Harbor, New York).

Transformation of plants may be accomplished using the Agrobacterium-mediated leaf disc transformation method described by Horsch et al.

(1988, Leaf Disc Transformation, Plant Molecular Biology Manual A5:1).

A number of procedures may be used to assess whether the transgenic plant comprises the desired DNA. For example, genomic DNA obtained from the cells of the transgenic plant may be analyzed by Southern blot hybridization or by PCR to determine the length and orientation of any inserted, transgenic DNA present therein.

Northern blot hybridization analysis or RT-PCR may be used to characterize mRNA transcribed in cells of the transgenic plant. In situations where it is expected that the cells of the transgenic plant express GS-Xpolypeptide or a fragment thereof, Western blot analysis may be used to identify and characterize polypeptides so expressed using antibody raised against the GS-Xpump or fragments thereof. The procedures for performing such analyses are well know in the art and are described, for example, in Sambrook et al. (supra).

The transgenic plants of the invention are useful for the examination of xenobiotic detoxification, heavy metal detoxification, control of plant pathogens, control of plant coloration, herbicide metabolism and phytohormone metabolism. For example, a transgenic plant comprising an AtMRPl or an AtMRP2 promoter sequence fused to a reporter gene is useful for the examination of xenobiotic detoxification and heavy metal detoxification when grown on soil having xenobiotic toxins or heavy metals. Such plants are useful to an understanding of the mechanisms by which GS-X pump gene expression is activated and are therefore useful for the eventual generation of plants which are capable of removing xenobiotic toxins or heavy metals from the soil thereby generating soil which has reduced levels of compounds that are detrimental to the overall health of the environment.

The types of plants which are suitable for use include, but are not limited to, high yield crop species for which cultivation practices have already been perfected, or engineered endemic species that thrive in the area to be remediated. In addition plants for which anthocyanins contribute to flower or leaf coloration and food crops for which decreased organic xenobiotic and/or heavy metal accumulation is desirable are also suitable for use in the invention. Further useful plants are those in which it is desirable that they are capable of increased accumulation and/or resistance

to isoflavonoid alexins. Plants for which pathogen resistance is desired are also useful in the invention. Such plants include, but are not limited to, plants which are leguminous plants, for example, alfalfa, cashew nut, castor bean, faba bean, french bean, mung bean, pea, peanut, soybean and walnut. In addition, plants for which it is desirable to manipulate plant coloration are also useful in the invention.

The promoter sequences of Arabidopsis GS-Xpump genes AtMRP1 and AtMRP2 are shown in Figures 23 and 24, respectively. The following should be noted.

bZIP transcription factor recognition elements have the sequences CACGTG or TGACG(T/C). One of these is present in the AtMRP2 promoter sequence, but none are present in the AtMRPl promoter sequence. Myb transcription factor recognition elements having the sequences A(a/D)(a/D)C(G/C) and AGTTAGTTA, wherein a/D = A, G or T with A being preferred, are present in the AtMRPl promoter sequence, but are not present in the AtMRP2 promoter sequence. Xenobiotic regulatory elements (ARE) having the core sequence GCGTG are found in multiple copies in the promoters of cytochrome P450 monooxygenase genes and glutathione S-transferase genes (Rushmore et al., 1993, J. Biol. Chem. 268:11475-11478). One XRE is found in the promoter sequence of AtMRPI. Antioxidant response elements (AREs) consist typically of two core sequences GTGACA(A/T)(A/T)GC (SEQ ID NO:1 1) that are binding sites for Activator Protein (AP-1) transcription factor complex (Daniel, 1993, CRC Crit. Rev. Biochem 25:173-207; Friling et al., 1992, Proc. Natl. Acad. Sci. USA 89:668-672). There is only one ARE in the AtMRPI promoter sequence shown in Figure 23. It has been proposed that GST genes containing an ARE are induced by electrophiles and conditions that generate oxidative stress (Daniel, supra). RNA instability determinants having the sequence ATTTA have been found in several plant GSTs. These sequences, considered to target RNAs for degradation by RNases are usually found in the 3'-UTRs of genes (Takahashi et al., 1992, Proc. Natl. Acad. Sci.

USA 89:56-59). Several of these sequences are found in both the AtMRP1 and AtMRP2 promoter sequences presented herein. However, it is not clear whether these sequences merely reflect the AT-richness of the sequences.

To assess GS-X pump gene expression in a plant cell whether the cell is contained within a plant or whether the cell is separated from the plant, a plasmid may be generated which comprises the -glucuronidase (GUS) reporter gene fused to a plant GS-Xpromoter sequence. Preferably, the promoter sequence is either AtMRPl or AtMRP2. The appropriate restriction fragment is subcloned into the GUS expression vector pBIl0l.3 (Jefferson et al., 1987, EMBO J., 6:3901-3907). After confirming the correct reading frame by sequencing, Agrobacterium or any other suitable vector, is transformed with the expression construct and is then used to used to transform the plant, or the cells thereof (Valvekens et al., 1988, Proc. Natl. Acad. Sci.

USA 85:5536-5540).

Expression of GUS may be localized histochemically by staining with 5-bromo-4-chloro-3-indoyl -D-glucuronide (X-Gluc) (Jefferson et al., supra).

Sections are obtained from the plant, they are incubated in X-Gluc, cleared by boiling in ethanol and are examined under the microscope. To eliminate or enumerate complications arising from the transfer of GUS reaction product between cells, the distribution of GUS expression is then further examined both immunologically and biochemically. -glucuronidase protein is assessed using standard dot-blotting and immunolocalization techniques (Harlow et al., 1988, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, NY) using rabbit anti- -glucuronidase serum (Clontech). Direct estimates of GUS activity are be made fluorimetrically using 4-methyl-umbelliferyl glucuronide as substrate (Jefferson et al., supra) after dissection and extraction of explants.

GUS reporter gene analyses enable examination of plant responses to oxidative stress and pathogens as well as herbicides. In addition, GUS reporter gene analyses enable tests of whether certain pigment-rich cell types also exhibit high levels of AtMRP expression.

The AtMRPI and AtMRP2 promoter sequences are also useful for manipulating the expression of other genes in plants in that, transgenic plants may be generated which contain a desired plant gene operably fused to a GS-X pump promoter

sequence. The GS-Xpump promoter sequence may be an AtMRPl or an AtMRP2 promoter sequence or a YCF1 promoter sequence positioned in an orientation such that the promoter sequence drives expression of the desired gene. The desired gene may be a plant or a non-plant gene. The generation of such transgenic plants confers upon the plants the ability to respond to the presence of xenobiotics and other compounds which influence the promoter activity In considering transport substrates for GS-X pumps, the status of GSSG as an endogenous GS-conjugate (of GSH with itself) and its involvement in cellular responses to active oxygen species (AOS) should not be overlooked. The sulfhydryl group of GSH confers strong nucleophilicity and the facility for reacting with AOS, such as superoxide radicals (°2 ) hydroxyl radicals (OH ) and hydrogen peroxide.

GSH is found in the majority of eukaryotes but in prokaryotes (eubacteria) it appears to be restricted to the cyanobacteria and purple bacteria (Fahey and Sundquist 1991, Adv. Enzymol. Relat. Mol. Biol. 64:1-53). Since the cyanobacteria are considered to be the first group of organisms capable of oxygenic photosynthesis and these and the purple bacteria probably gave rise to plant chloroplasts and mitochondria, respectively, it has been proposed that the emergence of the capacity for GSH biosynthesis was associated with the appearance of oxygenic and oxytrophic metabolism (approximately 4 x 109 years ago) to combat the attendant problem of AOS production. Most, if not all, of the factors known to elicit GST induction - pathogen attack, heavy metals, certain organic xenobiotics, wounding and ethylene - promote AOS production (Inze and Montagu 1995, Current Opinion in Biotech.

6:153-158). Intriguing, therefore, is the possibility that GS-Xpumps arose from the need to detoxify AOS and the products of their action.

The feasibility of such a scheme has yet to be investigated systematically but a number of disparate observations are at least consistent with a close connection between oxidative stress and GS-Xpump function: (i) All identified MRP-subclass transporters, including AtMRP1 and AtMRP2 recognize GSSG as a substrate. Studies of GS-X pumps originated from the discovery of ATP-dependent

GSSG efflux from erythrocytes (Srivastava and Beutler 1969, J. Biol. Chem.

244:9-16). (ii) In S. cerevisiae, overexpression of yAP 1, a bZIP transcription factor, not only activates the YCFl and GSH1 genes (Wemmie et al 1994, supra; Wu and Moye-Rowley 1994, supra), the latter of which encodes y-glutamylcysteine synthetase, but also a panoply of oxidoreductases (DeRisi et al 1997, Science 278:680-686). Of the 17 genes whose mRNA levels are found to be increased by more than threefold on DNA microarrays by yAP1, more than two-thirds contain canonical upstream yAP binding sites (TTACTAA or TGACTAA), five bear homology to aryl-alcohol oxidoreductases and four to the general class of <BR> <BR> <BR> dehydrogenases/oxidoreductases (DeRisi et al 1997, supra). In view of the capacity of yAP 1 overexpression to confer increased resistance to hydrogen peroxide, o-phenanthroline and heavy metals (Hirata et al 1994, Mol. Gen. Genet. 242:250-257), the fact that an appreciable fraction of the yAP 1 -regulated target genes identified against the yeast genome project database are oxidoreductases and coregulated with both YCFl and GSHl, suggests that all of these genes play a protective role during oxidative stress. (iii) Two particularly harmful and early effects of AOS production are membrane lipid peroxidation. and oxidative DNA damage which yield highly toxic 4-hydroxyalkenals (Esterbauer et al 1991, Biochem. J. 208:129-140) and base propanols (Berhane et al 1994, Proc. Natl. Acad. Sci. USA 91:1480-1484), respectively. Although such a,b-unsaturated aldehydes (and their GS-conjugates) have not yet been screened against the GS-Xpumps from plant sources, they are established <BR> <BR> <BR> substrates for mammalian GSTs (Berhane et al 1994, supra) and their glutathionated derivatives are transported at high efficiency by mammalian GS-X pumps (Ishikawa 1989, J.Biol. Chem. 264:17343-17348).

There is therefore also included in the invention a method of alleviating oxidative stress in a plant comprising intorducing into the cells of the plant DNA encoding a GS-Xpump.

The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of

illustration only, and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

Experimental Examples The experimental examples described herein provide procedures and results for the isolation and characterization of yeast YCF1 and Arabidopsis AtMRPl and AtMRP2 genes, gene products and various functions ascribed thereto. Further there is described data which establish that the Bz2 gene product exerts its effects on plant coloration via the GS-Xpump.

The data which are now described establish that YCFJ is a vacuolar glutathione S-conjugate pump. The data establish that YCF 1 is a membrane protein which is responsible for catalyzing MgATP-dependent, uncoupler-insensitive uptake of glutathione S-conjugates into the vacuole of wild type S. cerevisiae.

YCF1 encodes a protein responsible for resistance of yeast to the effects of cadmium. However, the mechanism by which resistance to Cd2+ is effected was not understood until the present invention. The data presented herein demonstrate that YCFl confers Cd2+ resistance to yeast by effecting transport of Cd2+ out of the cytosol via a YCF1 encoded vacuolar glutathione S-conjugate pump. Further, since YCF1 confers resistance to Cd2+ through the transport of Cd.GS complexes or derivatives thereof, it is likely also capable of transporting other metal.GS-complexes. Examples of these other complexes include, but are not limited to, mercury (Hg), zinc (Zn), platinum (Pt) and arsenic (Ar). Both Hg2+ and Zn2+ form complexes with GSH which are analogous to those formed by Cd2+ (Li et al., 1954, J. Am. Chem. Soc. 76:225-229; Kapoor et al., 1965, Biochem. Biophys. Acta 100:376-383; Perrin et al., 1971, Biochem. Biophys. Acta 230:96-104). In addition, MRP1 eliminates the Pt2+ glutathionine complex bis(glutathionato)platinum from cancer cells (Ishikawa et al., 1994, J. Biol. Chem. 269:29085-29093). Further, the MRP1 gene is overexpressed in cisplatin-resistant human leukemia HL-60 cells, which overexpression is associated

with increased resistance to arsenite (Ishikawa et al., 1996, J. Biol. Chem. 271:14981- 14988). Both Hg2+ and Ar'+ are common environmental contaminants and Zn2+ is an essential micronutrient.

According to the results of the present study, vacuolar membrane vesicles from wild type S. cerevisiae catalyze high rates of MgATP-dependent, uncoupler-insensitive S-conjugate transport, and the kinetics of the transporter involved are similar to those of the mammalian and plant vacuolar US-Xpumps. In addition, vacuole-deficient mutants of S. cerevisiae exhibit markedly increased sensitivity to cadmium, leading to the belief that one requirement for efficient elimination or detoxification of this metal is maintenance of a sizable vacuolar compartment.

It is known that S. cerevisiae yAP-1 transcription factor transcriptionally activates both the YCF1 gene and the GSH1 gene (Wemmie et al., 1994, J. Biol. Chem. 269:32592-32597; Wu et al., 1994, Mol. Cell. Biol. 14:5832- 5839). Since GSH1 encodes y-glutamylcysteine synthetase, an enzyme critical for GSH synthesis, expression of the YCF1 gene and fabrication of one of the precursors for transport by the GS-X pump are coordinately regulated.

In the first set of experiments described below, transport of the model compounds DNP-GS and bimane-GS by isolated membrane vesicles and intact cells was examined.

Yeast Strains and Plasmids Two strains of S cerevisiae were used in these studies: DTY165 (MA Ta ura3-52 his6 leu2-3,-112 his3-A200 trpl-901 Iys2-801 suc2-A) and the isogenicycflA mutant strain, DTYl67 (MATa ura 3-52 his6 leu2-3,-112 his3-200 trp 1-901 Iys2-801 suc2-A, ycgl ::hisG). The strains were routinely grown in rich (YPD) medium, or, when transformed with plasmid containing functional YCF1 gene, in synthetic complete medium (Sherman et al., 1983, Methods in Yeast Genetics, Cold Spring Harbor Laboratory, New York) or ABC medium (Kim et al., 1994, supra) lacking the appropriate amino acids. Escherichia coli strains XL1-blue (Stratagene)

and DH 1S were employed for the construction and maintenance of plasmid stocks (Ausubel et al., 1987, Current Protocols in Molecular Biology, Wiley, New York).

Plasmid pYCF 1-HA, encoding epitope-tagged YCF1, was constructed in several steps. A 1.4-kb SalI-HindIII fragment, encompassing the carboxyl-terminal segment of the open reading frame of YCF1, from pIBIYCF 1 (Szczypka et al., 1994, supra), was subcloned into pBluescript KS-. Single-stranded DNA was prepared and used as template to insert DNA sequence encoding the human influenza hemagglutinin 12CA5 epitope immediately before the termination codon of the YCF1 gene by oligonucleotide-directed mutagenesis. The sequence of the primer for this reaction, with the coding sequence for the 12CA5 epitope underlined, was 5'- <BR> <BR> <BR> GTTTCACAGTTTAAAGCGTAGTCTGGGACGTCGTATGGGTAATTTTCATTG ACC-3' (SEQ ID NO: 12). After confirming the boundaries and fidelity of the HA-tag coding region by DNA sequencing, the 1.4-kb SalI-HindlII DNA fragment was exchanged with the corresponding wild type segment ofpJAW50 (Wemmie et al., 1994, supra) to generate pYCF1-HA.

Isolation of Vacuolar Membrane Vesicles For the routine preparation of vacuolar membrane vesicles, 15 ml of stationary phase cultures ofDTYl65 or DTY167 were diluted into 1-liter volumes of fresh YPD medium, grown for 24 hours at 300C to an OD600 nm Of approximately 0.8 and collected by centrifugation. After washing with distilled water, the cells were converted to spheroplasts with Zymolyase 20T (ICN) (Kim et al., 1994, supra) and intact vacuoles were isolated by flotation centrifugation of spheroplast lysates on Ficoll 400 step gradients as described by Roberts et al. (1991, Methods. Enzymol.

194:644-661). Both the spheroplast lysis buffer and Ficoll gradients contained 2 mg/ml bovine serum albumin, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 llg/ml pepstatin, and 1 mM PMSF to minimize proteolysis. The resulting vacuole fraction was vesiculated in 5 mM MgCl2, 25 mM KC1, 10 mM Tris-Mes (pH 6.9) containing 2 mg/ml bovine serum albumin, 1 pg/ml aprotinin, 1 )lg/ml leupeptin, 1 llg/ml pepstatin, and 1 mM PMSF, pelleted by centrifugation at 37,000 x g for 25 min, and resuspended

in suspension medium (1.1 M glycerol, 2 mM dithiothreitol, 1 mM Tris-EGTA, 2 mg/ml bovine serum albumin, 1 llg/ml aprotinin, 1 pg/ml leupeptin, 1 pg/ml pepstatin, 1 mM PMSF, 5 mM Tris-Mes, pH 7.6) (Kim etna?., 1995, J. Biol. Chem. 270:2630- 2635).

In experiments involving cadmium transport, dithiothreitol and EGTA were removed from the suspension medium to prevent the attenuation of YCF 1- dependent Cd2+ transport otherwise exerted by these compounds. Vesiculated vacuolar membranes were subjected to three cycles of 50-fold dilution into simplified suspension medium (1.1 M glycerol, 5 mM Tris-Mes, pH 8.0), centrifugation at 100,000 x g for 35 minutes and resuspension in the same medium before use.

For the experiment shown in Figure 4, 1 ml of partially purified vacuolar membrane vesicles (1.1-1.2 mg of protein), prepared by Ficoll flotation, were subjected to further fractionation by centrifugation through a 30-ml linear 10-40% (w/v) sucrose density gradient at 100,000 x g for 2 hours. Successive fractions were collected from the top of the centrifuge tube and, after determining sucrose concentration refractometrically, the fractions were diluted with suspension medium.

The diluted fractions were sedimented at 100,000 x g and resuspended in 100All aliquots of suspension medium for assay. For the immunoblots shown in Figure 5 and the marker enzyme analyses shown in Table 4, crude microsomes were prepared by homogenization of spheroplasts in suspension medium and the sedimentation of total membranes at 100,000 x g for 35 minutes.

Microsomes and purified vacuolar membranes that were to be employed for SDS-polyacrylamide gel electrophoresis and immunoblotting were washed free of bovine serum albumin by three rounds of suspension in suspension medium minus bovine serum albumin and centrifugation at 100,000 x g for 35 minutes. The final membrane preparations were either used immediately or frozen in liquid nitrogen and stored at -85 0C.

Measurement of Marker Enzvme Activities

a-Mannosidase was determined according to Opheim (1978, Biochem.

Biophys. Acta 524:121 - 125) using p-nitrophenyl-a-D-mannopyranoside as substrate.

NADPH-cytochrome c reductase was estimated as FMN-promoted reduction of NADPH (Kubota et al., 1977, J. Biol. Chem. 81:197-201). GDPase was measured as the rate of liberation of Pi from GDP (Yanagisawa et al., 1990, J Biol. Chem.

265:19351-19355) in reaction buffer containing 0.05% (w/v) Triton X-100.

V-ATPase, F-ATPase, and P-ATPase were assayed as bafilomycin A1 (1 µM), azide (1 mM), and vanadate (100 CLM) inhibited ATPase activity, respectively, at pH 8.0 (V-ATPase, F-ATPase) or pH 6.5 (P-ATPase) (Rea and Turner, 1990, Methods Plant Biochem. 3:385-405).

Measurement of DNP-GS Uptake Unless otherwise indicated, [3H]DNP-GS uptake was measured at 25"C in 200 pl reaction volumes containing 3 mM ATP, 3 mM MgSO4, 5 µM gramicidin- D, 10 mM creatine phosphate, 16 units/ml creatine kinase, 50 mM KC 1, 1 mg/ml bovine serum albumin, 400 mM sorbitol, 25 mM Tris-Mes (pH 8.0), and 66.2 pM [3H]DNP-GS (8.7 mCi/mmol) (Li et al., 1995, supra). Gramicidin D was included in the uptake medium to abolish the H+ electrochemical potential difference ( pH+) that would otherwise be established by the V-ATPase in medium containing MgATP.

Uptake was initiated by the addition of vacuolar membrane vesicles (10-15 pg of membrane protein), brief mixing of the samples on a vortex mixer and uptake was then allowed to proceed for 1-60 minutes. Uptake was terminated by the addition of 1 ml of ice-cold wash medium (400 mM sorbitol, 3 mM Tris-Mes, pH 8.0) and vacuum filtration of the suspension through prewetted Millipore HA cellulose nitrate membrane filters (pore diameter, 0.45 µm). The filters were rinsed twice with 1 ml of ice-cold wash medium and air-dried, and radioactivity was determined by liquid scintillation counting in BCS mixture (Amersham Corp.). Nonenergized [3H]DNP-GS uptake and extravesicular solution trapped on the filters were enumerated by the same procedure except that ATP and Mg2+ were omitted from the uptake medium.

Fluorescence Microscopy

Cells were grown in YPD medium for 24 hours at 30°C to an OD60oX of approximately 1.4, and 100 Cll aliquots of the suspensions were transferred to 15 ml volumes of fresh YPD medium containing 100 I1M syn-(ClCH2,CH3)-1,5- diazabicyclo-[3.3.0]-octa-3,6-dione-2,8-dione (monochlorobimane) (Kosower et al., 1980, J. Am. Chem. Soc. 102:4983-4993). After incubation for 6 hours, the cells were pelleted by centrifugation, washed twice with YPD medium lacking monochlorobimane, and viewed without fixation under an Olympus BH-2 fluorescence microscope equipped with a BP-490 UV excitation filter, AFC-0515 barrier filter, and Nomarski optics attachment.

Electrophoresis and Immunoblotting Membrane samples were subjected to one-dimensional SDS- polyacrylamide gel electrophoresis on 7-12% (w/v) concave exponential gradient gels after delipidation with acetone:ethanol (Parry et al., 1989, J. Biol. Chem. 264:20025- 20032). The separated polypeptides were electrotransferred to 0.45 ,um nitrocellulose filters at 60 V for 4 hours at 40C in a Mini Trans-Blot transfer cell (Bio-Rad) and reversibly stained with Ponceau-S (Rea et al., 1992, Plant PhysioL 100:723-732). The filters were blocked and incubated overnight with mouse anti-HA monoclonal antibody (20 Fug/ml) (Boehringer-Mannheim). Immunoreactive bands were visualized by reaction with horseradish peroxidase-conjugated goat anti-mouse IgG (1/1000 dilution) (Boehringer-Mannheim) and incubation in buffer containing H2O2 (0.03% w/v), diaminobenzidine (0.6 mg/ml) andNiCl2 (0.03% w/v) (Rea et al., 1992, supra).

Purification of Cadmium-Glutathione Complexes Singly radiolabeled l09Cd.GSn and doubly radio-labeled 109Cd[3H].GSn complexes were prepared by sequential gel-filtration and anion- exchange chromatography of the reaction products generated by incubating 20 mM 109CdSO4 (78.4 mCi/mmol) with 40 mM GSH or 40 mM [3H]GSH (240 mCi/mmol) in 15 ml 10 mM phosphate buffer (pH 8.0) containing 150 mM KNO3 at 450C for 24 hours. For gel-filtration, 2 ml aliquots of the reaction mixture were applied to a column (40 x 1.5 cm ID) packed with water-equilibrated Sephadex G-15, eluted with

deionized water and 109Cd and/or 3H in the fractions was measured by liquid scintillation counting. The fractions encompassed by each of the two l09Cd.GSn peaks identified were pooled, lyophilized and redissolved in 4 ml of loading buffer (5 mM Tris-Mes, pH 8.0). For anion-exchange chromatography, 0.5 ml aliquots of the resuspended lyophilizates from gel-filtration chromatography were applied to a Mono- Q HR5/5 column (Pharmacia) equilibrated with the same buffer. Elution was with a linear gradient of nazi (0.5 ml/minute; 0-500 mM) dissolved in loading buffer. The individual fractions corresponding to the major peaks of 109Cd obtained from the Mono-Q column (one each for the peaks resolved by gel-filtration chromatography) were pooled, lyophilized and resuspended in 4 ml deionized water after liquid scintillation counting. Buffer salts were removed before transport measurements or mass spectrometry by passing the samples down a column (120 x 1.0 cm ID) packed with water-equilibrated Sephadex G-15.

Measurement of 109Cd2 Uptake MgATP-energized, uncoupler-insensitive 109Cd2+ uptake by vacuolar membrane vesicles was measured at 25"C in 200 1ll reaction volumes containing 3 mM ATP, 3 mM MgSO4, 5 pM gramicidin-D, 10 mM creatine phosphate, 16 units/ml creatine kinase, 50 mM KCl, 400 mM sorbitol, 25 mM Tris-Mes (pH 8.0) and the indicated concentrations of 109CdSO4, GSH or 109Cd- and/or 3H-labeled purified Cd.GSn complexes as described herein except that the wash media contained 100 pM CdSO4 in addition to sorbitol (400 mM) and Tris-Mes (3 mM, pH 8.0).

Pretreatment of DTY165 Cells with Cd2+ or 1-Chloro-2.4- dinitrobenzene For studies on the inducibility of YCF1 expression and YCF1- dependent transport, DTY165 cells were grown in YPD medium (Sherman etna?., 1983, supra) for 24 hours at 30DC to an OD600 nm Of 1.0-1.2, pelleted by centrifugation and resuspended in fresh YPD medium containing CdSO4 (200 ,uM) or 1-chloro-2,4-dinitrobenzene (CDNB). After washing in distilled water, total RNA was extracted and vacuolar membrane vesicles were prepared from the pretreated cells.

Control RNA and membrane samples were prepared from DTY165 cells treated in an identical manner except that CdSO4 and CDNB were omitted from the second incubation cycle.

RNase Protection Assavs Cd- and CDNB-elicited increases in YCF1 mRNA levels were assayed by RNase protection using 18S rRNA as an internal control. YCFI-specific probe was generated by PCR amplification of the full-length YCF1. . HA gene, encoding human influenza hemagglutinin 12CA5 (HA) epitope-tagged YCF1, using plasmid pYCF1- HA as template. The forward YCFl-specific primer and backward primer containing the HA-tag coding sequence had the sequences 5'- AAACTGCAGATGGCTGGTAATCTTGTTTC-3' (SEQ ID NO:13) and 5'- <BR> <BR> <BR> GCCTCTAGATCAAGCGTAGTCTGGGACGTCGTATGGGTAATTTTCATTGA-3 ' (SEQ ID NO: 14), respectively. An 18S rRNA-specific probe was synthesized by PCR of S. cerevisiae genomic DNA using sense and antisense primers having the sequences 5'-AGATTAAGCCATGCATGTCT-3' (SEQ ID NO:15) and 5'- TGCTGGTACCAGACTTGCCCTCC-3' (SEQ ID NO:16), respectively. Both PCR products were individually subcloned into pCRTMII vector (Invitrogen) to generate plasmids pCR-YCFl and pCR-Y18S. After linearization of pCR-YCFl and pCR- Y18S with AflI and NcoI, a 320-nucleotide YCFl-specific RNA probe and 220- nucleotide 18S rRNA-specific probe were synthesized using T7 RNA polymerase and SP6 RNA polymerase, respectively. Aliquots of total RNA, prepared as described (Kohrer et al., 1991, Methods in Enzymol. 194:390-398)), from control, CdSO4- or CDNB-pretreated DTY165 cells were hybridized with a mixture of32P-labeled YCF1 antisense probe (1 x 106 cpm) and 18S rRNA antisense probe (5 x 102 cpm) and RNase protection (Teeter et al., ,1990, Mol. Cell. Biol. 10:5728-5735) was assayed using an RPAII kit (Ambion).

Matrix-Assisted Laser Desorption Mass Spectrometry (MALD-MS) The 109Cd.GSn complexes purified by gel-filtration and anion- exchange chromatography were adjusted to a final concentration of 2-5 mM (as Cd)

with deionized water, mixed with an equal volume of sinapinic acid (10 mg/ml) dissolved in acetonitrile/H20/trifluoroacetic acid (70:30:0.1 % (v/v)) and applied to the ion source of a PerSeptive Biosystems Voyager RP Biospectrometry Workstation.

The instrument, which was equipped with a 1.3 m flight tube and variable two-stage ion source set at 30 kV, was operated in linear mode. Mass/charge (m/z) ratio was measured by time-of-flight after calibration with external standards.

Protein Estimations Protein was estimated by a modification of the method of Peterson (1977, Anal. Biochem. 83:346-356).

Chemicals S-(2,4-dinitrophenyl)glutathione (DNP-GS) was synthesized from 1- chloro-2,4-dinitrobenzene (CDNB) and GSH by the procedure of Kunst et al. (1983, Biochem. Biophys. Acta 983:123-125) and (Li et al., 1995, supra). [3H]DNP-GS (specific activity, 8.7 mCi/mmol) and bimane-GS were synthesized enzymatically and purified by a modification of the procedure of Kunst et al. (1983, supra) according to Li et al. (1995, supra). Metolachlor-GS was synthesized by general base catalysis and purified by reverse-phase high performance liquid chromatography (Li et al., 1995, supra).

GSH and CDNB were purchased from Fluka; AMP-PNP, aprotinin, ATP, creatine kinase (type I from rabbit muscle, 150-250 units/mg of protein), creatine phosphate, FCCP, oxidized glutathione (GSSG), S-methylglutathione, cysteinylglycine, cysteine, glutamate and gramicidin D, leupeptin, PMSF, verapamil, and vinblastine were from Sigma; monochlorobimane was from Molecular Probes; cellulose nitrate membranes (0.45-pm pore size, HA filters) were from Millipore; [3H]glutathione[(glycine-2-3H]-L-Glu-Cys-Gly; 44 Ci/mmol) was from DuPont NEN; and 109CdSO4 (78.44 Ci/mmol) was from Amersham Corp. Metolachlor was a gift from CIBA-Geigy, Greensboro, NC. All other reagents were of analytical grade and purchased from Fisher, Fluka, or Sigma.

Sensitivitv to CDNB

If the YCFI gene product were to participate in the detoxification of S- conjugable xenobiotics, mutants deleted for this gene would be expected to be more sensitive to the toxic effects of these compounds than wild type cells. This is what was found (Figure 1).

The isogenic wild type strain DTY 165 and the ycfl A mutant strain, DTY167, were indistinguishable during growth in YPD medium lacking CDNB; both strains grew at the same rate after a brief lag. However, the addition of CDNB to the culture medium caused a greater retardation of the growth of DTY167 cells (Figure 1B) than DTY 165 cells (Figure 1A). Inhibitory concentrations of CDNB resulted in a slower, more linear, growth rate for at least 24 hours for both strains, but DTY167 underwent growth retardation at lower concentrations than did DTY165. The optical densities ofthe DTY167 cultures were diminished by 65, 82, 85, and 91% by 40, 50, 60, and 70 M CDNB, respectively, after 24 hours of incubation (Figure 1B), whereas the corresponding diminutions for the DTY165 cultures were 14, 31, 59, and 92% (Figure 1A). The increase in sensitivity to CDNB conferred by deletion of the YCF1 gene was similar to that seen with cadmium.

Impaired Vacuolar DNP-GS Transport Vacuolar membrane vesicles purified from DTY165 cells exhibited high rates of MgATP-dependent [3H]DNP-GS uptake (Figure 2). Providing that creatine phosphate and creatine kinase were included in the uptake media to ensure ATP regeneration, addition of 3 mM MgATP increased the initial rate of DNP-GS uptake by 122-fold to a value of 12.2 nmol/mg/minute. The same membrane fraction from DTY 167 cells, although capable of similar rates of MgATP-independent DNP- GS uptake, was only 17-fold stimulated by MgATP and capable of an initial rate of uptake of only 1.7 nmol/mg/minute (Figure 2).

Selective Impairment of Uncoupler-Insensitive Transport Direct comparisons between vacuolar membrane vesicles from DTY165 and DTY167 cells demonstrated that deletion of the YCF1 gene selectively abolished MgATP-energized, hIlH+-independent DNP-GS transport.

Agents that dissipate both the pH (ApH) and electrical (A) components of the #µH+ established by the V-ATPase (FCCP, gramicidin D) or directly inhibit the V-ATPase, itself (bafilomycin Al), decreased MgATP-dependent DNP-GS uptake by vacuolar membrane vesicles from DTY165 cells from 77.7 + 1.0 nmol/mg/10 minutes to between 43.2 + 1.0 and 47.4 i 1.7 nmol/mg/l0 minutes (Table 1). Ammonium chloride, which abolishes ApH while leaving At unaffected, on the other hand, did not inhibit DNP-GS uptake (Table 1). On the basis of these characteristics, the inability of uncouplers to markedly increase the inhibitions caused by V-ATPase inhibitors, alone, and the resistance of 50-60% of total uptake to inhibition by any one of these compounds (Table 1), DNP-GS uptake by vacuolar membranes from wild type cells is concluded to proceed via two parallel mechanisms: a V-ATPase inhibitor- and uncoupler-insensitive pathway that is directly energized by MgATP, and a /lH+-dependent, V-ATPase inhibitor-sensitive and uncoupler- sensitive pathway that is primarily driven by the inside-positive At established by the V-ATPase.

Of these two pathways, the lt-dependent pathway predominated in membranes from DTY167 cells (Table 1). FCCP, gramicidin D, and bafilomycin A1 diminished net DNP-GS uptake by DTY167 vacuolar membranes from 15.4 + 0.4 nmol/mg/10 minutes to between 4.3 i 0.3 and 6.4 + 0.3 nmol/mg/10 minutes.

Moreover, although the effects of FCCP or gramicidin D and V-ATPase inhibitors in combination were slightly greater than those seen when these agents were added individually, the transport remaining was only about 10% of that seen with wild type membranes and only 2-4-fold stimulated by MgATP. In conjunction with the negligible inhibitions seen with NH4Cl, alone, indicating that Allr, not ApH, is the principal driving force for the transport activity remaining in their vacuolar membranes, DTY167 cells are inferred to be preferentially impaired in MgATP- energized, lpLH+-independent DNP-GS transport.

The nonhydrolyzable ATP analog, AMP-PNP, did not promote DNP- GS uptake by vacuolar membrane vesicles from either DTY165 or DTY167 cells (Table 1), indicating a requirement for hydrolysis of the y-phosphate of ATP regardless of whether uptake was via the YCF1- or At-dependent pathway.

Table 1. Effects of MgATP MgAMP-PNP nrotononhores. ionophores and V-ATPase inhibitors on [ HDNP-GS uptake bv vacuolar membrane vesicles purified from DTY165 and DTY167 cells.

Uptake was measured for 10 minutes in standard uptake medium described herein containing 66.2 µM t3H]DNP-GS plus the compounds indicated. MgATP (3 mM) was present throughout unless otherwise indicated. MgAMP-PNP, bafilomycin AI, FCCP, gramicidin D, and NH4CI were added at concentrations of3 mM, 0.5 µM, 5 pM, 5 pM and 1 mM, respectively. Values outside parentheses are means + SE (n = 3-6); values inside parentheses are rates of uptake expressed as percentage of control. DNP-GS UPTAKE ADDITIONS DTY165 DTY167 (nmol/mg/10 minutes) Control 77.7 # 1.0 (100) 15.4 # 0.4 (100) -MgATP 2.2 i 0.4 (2.8) | 1.5 + 0.6 (9.7) MgAMP-PNP(-MgATP) 2.5 + 0.5 (3.2) 1.4 + 0.3 (9.1) FCCP 47.4 + 1.7 (61.0) 6.4 + 0.3 (41.8) Gramicidin D 45.8 # 1.4 (58.9) 5.8 # 0.1 (37.7) NH4Cl 69.1 # 2.9 (88.9) 14.9 # 0.7 (96.8) NH4Cl+ gramicidin D 42.6 # 1.8 (54.8) 4.1 # 0.2 (26.6) Bafilomycin A1 43.2 # 1.0 (55.6) 4.3 # 0.3 (27.9) Bafilomycin A1 + gramicidin D 39.2 i 2.6 (50.5) 3.8 + 0.1 (24.7) Abolition of High Affinitv. Uncoupler-insensitive Uptake Examination of the concentration dependence of [3H]DNP-US uptake revealed a near total abolition of high affinity, MgATP-dependent, uncoupler- insensitive transport by vacuolar membrane vesicles from the ycflA mutant strain (Figure 3). When measured in the presence of uncoupler (gramicidin D), the rate of DNP-GS uptake by vacuolar membrane vesicles purified from DTY165 cells increased as a simple hyperbolic function of MgATP (Figure 3A) and DNP-GS concentration (Figure 3B) to yield Km values OF 86.5+29.5 IlM (MgATP) and 14.1 i 7.4 I1M

(DNP-GS) and a Vmax of 51.0 + 6.3 nmol/mg/10 minutes (DNP-GS). By contrast, uncoupler-insensitive uptake by the corresponding membrane fraction from DTY 167 cells was more than 15-fold slower over the entire concentration range, showed no evidence of saturation and increased as a linear function of both DNP-GS and MgATP concentration (Figure 3).

Selective Inhibitors ofYCFl-mediated Transport MgATP-dependent, uncoupler-insensitive DNP-GS uptake by vacuolar membrane vesicles purified from DTY165 cells was sensitive to inhibition by vanadate, vinblastine, verapamil, GSSG and glutathione S-conjugates other than DNP- GS (Tables 2 and 3). One hundred pM concentrations of metolachlor-GS, azidophenacyl-GS and bimane-GS and 1 mM GSSG inhibited uptake by about 50% (Table 2), while vanadate, vinblastine, and verapamil exerted 50% inhibitions at concentrations of 179, 89 and 203 I1M, respectively (Table 3). None of these agents significantly inhibited residual MgATP-dependent, uncoupler-insensitive DNP-GS uptake by vacuolar membrane vesicles from DTYl67 cells (Tables 2 and 3).

TABLE 2. Effects of GSH. GSSG. and glutathione S-coniuzates other that DNP-GS on MgATP- dependent, uncoupler-insensitive [ H]DNP-GS uptake by vacuolar membrane vesicles purified from DTY165 and DTY167 cells. Uptake was measured as described for Table 1 except that 5 gM gramicidin D was included in all of the uptake media. Values outside parentheses are means + SE (n = 3-6); values inside parentheses are rates of uptake expressed as percentage of control. DNP-GS UPTAKE COMPOUND DTY165 DTY167 (nmol/mg/10 minutes) Control 47.9 # 2.5 (100) 6.5 # 0.8 (100) GSH (1 mM) 50.6 + 2.3 (105.6) 4.6 + 1.1(70.8) GSSG (1 mM) 26.0 # 0.9 (54.3) 4.4 # 0.4 (67.7) Metolachlor-GS (100 µM) 27.6 # 0.9 (57.7) 4.8 # 0.7 (73.8) Azidophenacyl-GS (100 µM) 16.0 # 1.4 (33.5) 5.2 # 0.3 (80.0) | Bimane-GS (100 pM) 25.2 i 1.1 (52.6) 4.5 + 0.4 (69.2) TABLE 3. Sensitivity of MgATP-dependent, uncoupler-insensitive [ H]DNP-GS uptake by vacuolar membrane vesicles purified from DTY165 cells to inhibition by vanadate.

vinblastine. and verapamil. Uptake was measured as described in Table 1 except that 5 pM gramicidin D was included in all of the uptake media. The concentrations of the compounds causing 50% inhibition of uptake (I50 values) were estimated by nonlinear least squares analysis after fitting the the data to a single negative exponential (Marquardt, 1963, supra). I50 Addition DTY165 DTY167 pM Vanadate 179,1 Insensitive Vinblastine 88.8 > 500 Verapamil 202.6 Insensitive Vacuolar Membrane Localization The capacity for MgATP-dependent, uncoupler-insensitive [3H]DNP- GS uptake strictly copurified with the vacuolar membrane fraction (Table 4). By

comparison with crude microsomes (total membranes) prepared from whole spheroplast homogenates of DTY165 cells, vacuolar membrane vesicles derived from vacuoles purified by the Ficoll flotation technique were coordinately enriched for DNP- GS uptake and for both of the vacuolar membrane markers assayed, a-mannosidase and bafilomycin A1-sensitive ATPase (V-ATPase) activity. The respective enrichments of MgATP-dependent, uncoupler-insensitive DNP-GS uptake, a-mannosidase and bafilomycin Al-sensitive ATPase activity were 28-, 53- and 22-fold. By contrast, the vacuolar membrane fraction was 4.5-, 6.3-, 11.1- and 4.3-fold depleted of NADPH cytochrome c reductase (endoplasmic reticulum), latent GDPase (Golgi), vanadate- sensitive ATPase (plasma membrane), and azide-sensitive ATPase activity (mitochondrial inner membrane), respectively. Accordingly, when vacuolar membrane vesicles derived from Ficoll-flotated vacuoles were subjected to further fractionation on linear 10-40% (w/v) sucrose density gradients, MgATP-dependent, uncoupler- insensitive [3H]DNP-GS uptake, a-mannosidase and bafilomycin A1-sensitive ATPase activity were found to comigrate and exhibit identical density profiles (Figure 4).

TABLE 4. Comparison of rates of MgATP-dependent, uncoupler-insensitive [ H]DNP-GS transport.

and specific activities of marker enzvmes in crude microsomes and vacuolar membrane vesicles prepared from DTYl65 cells. Microsomes and vacuolar membrane vesicles were prepared from spheroplasts and the marker enzymes were assayed as described herein. Values shown are means # SE (n=3). ACTIVITY PREPARATION DNP-GS UPTAKE a-mannosidase NADPH-cyt c reductase nmol/mg/10 min nmol/mg/min nmol/mg/min Microsomes 2.5 # 0.3 6.3 i 0.3 88.0 i 1.3 Vacuolar 69.9 j 1.0 329.3 j 3.2 19.3 # 0.6 membrane Enrichment (- 27.96 52.27 0.22 fold) ACTIVITY PREPARATION V-ATPase GDPase P-ATPase F-ATPase µmol/mg/h µmol/mg/h Microsomes 11.76.3 z 6.3 35.0 i 1.1 37.1 + 4.6 155.63.0 Vacuolar | 253.1 # 15.8 | 5.5 # 0.1 | 3.2 # 1.6 | 35.1 # 8.4 membrane Enrichment (- 21.63 0.16 0.09 0.23 fold) Plasmid-encoded YCF1 Mediates Vacuolar DNP-GS Transport and CDNB Resistance Immunoblots of vacuolar membranes from pYCF 1 -HA-transformed DTY165 or DTY167 cells, probed with mouse anti-HA monoclonal antibody, demonstrated incorporation of YCF 1-HA polypeptide into the vacuolar membrane fraction (Figure SB). Immunoreaction with the 12CA5 epitope was not detectable in lanes loaded with membranes from pRS424-transformed cells but the same quantities of membranes prepared from pYCF 1 -HA-transformed cells yielded a single intensely immunoreactive band with an electrophoretic mobility (M# = 156,200) commensurate

with a computed mass of 172 kDa for the fusion protein encoded by YCF1-HA (Figure 5B).

Direct participation of the plasmid-borne YCF1-HA gene product in DNP-GS transport and CDNB detoxification was verified by the finding that vacuolar membrane vesicles purified from pYCF1-HA-transformed DTY167 cells exhibited a 6- fold enhancement of MgATP-dependent, uncoupler-insensitive [3H]DNP-GS uptake (Figure 5A) which was accompanied by a decrease in the susceptibility of such transformants to growth retardation by exogenous CDNB (Figure 6). Whereas pYCF1- HA-transformed DTY167 cells exhibited a similar resistance to growth retardation by CDNB as untransformed DTY165 cells (compare Figure 6B with Figure 1A), the same mutant strain showed neither increased vacuolar DNP-GS transport in vitro nor decreased susceptibility to CDNB in vivo after transformation with parental plasmid pRS424, lacking the YCF 1-HA insert (Figure 6B).

Vacuolar Accumulation of Bimane-GS In Vivo Monochlorobimane, a membrane-permeant, nonfluorescent compound, is specifically conjugated with GSH by cytosolic glutathione S-transferases (GSTs) to generate the intensely fluorescent, membrane-impermeant S-conjugate, bimane-GS (Shrieve et al., 1988, J. Biol. Chem. 263:14107-12114; Oude Elferink et al., 1993, Hepatology 17:343-444; Ishikawa etna?., 1994, J. Biol. Chem. 269:29085-29093). The GS-X pumps of both animal and plant cells exhibit activity toward a broad range of S- conjugates, including bimane-GS (Ishikawa et al., 1994, supra; Martinoia et al., 1993, supra), and DNP-GS uptake by the yeast enzyme is shown herein to be reversibly inhibited by this compound (Table 2). These data suggest competition between bimane-GS and DNP-GS for a common uptake mechanism. Exogenous monochlorobimane therefore satisfies the minimum requirements of a sensitive probe for monitoring the intracellular transport and localization of its S-conjugate.

Fluorescence microscopy ofDTY165 and DTY167 cells after incubation in growth medium containing monochlorobimane provides direct evidence that YCF 1 contributes to the vacuolar accumulation of its glutathione S-conjugate by

intact cells (Figure 7). DTY165 cells exhibited an intense punctate fluorescence, corresponding to the vacuole as determined by Nomarski microscopy, after 6 hours of incubation with monochlorobimane (Figures 7A and 7C). The fluorescence associated with vacuolar bimane-GS was by comparison severely attenuated in most, and completely absent from many, DTYl67 cells (Figures 7B and 7D).

vcfl# Mutants are Defective in GSH-Dependent Cd- Transport Physiological (1 mM) concentrations of GSH (Kang, 1992, Drug Metabolism and Disposition 20:714-718) promoted Cd2+ uptake by vacuolar membrane vesicles purified from the wild type strain DTY165 but not the yctl A mutant strain DTY167 (Figure 8). Addition of Cd2+ (80 µM) to GSH-containing media elicited MgATP-dependent, uncoupler-insensitive 109Cd2+ uptake rates of 4.5 and 0.8 nmol/mg/minute by DTY165 and DTY167 membranes, respectively (Figures 8A and 8B). Uptake by DTY165 membranes was diminished more than 9-fold by the omission of GSH (Figure 8A) whereas uptake by DTY167 membranes was slightly stimulated (Figure 8B).

GSH maximally stimulated uptake within minutes (t1/2 < 5 minutes) of the addition of Cd2+ to the uptake medium and uptake was sigmoidally dependent on Cd2+ concentration, achieving half-maximal velocity at 120 µM (Figure 8C).

Specific Requirement for GSH The stimulatory action of GSH was abolished by the omission of MgATP from the assay medium (Figure 8 and Table 5) and 1 mM concentrations of GSSG, S-methylglutathione, cysteinylglycine, cysteine or glutamate did not promote MgATP-dependent, uncoupler-insensitive Cd2+ uptake by vacuolar membrane vesicles from either strain (Table 5).

TABLE 5, Effects of different GSH-related compounds on uncoupler-insensitive 109Cd uptake by vacuolar membrane vesicles purified from DTY 165 or DTY 167 cells. GSH, oxidized glutathione (GSSG), S-methylglutathione (GS-CH3), cysteinylglycine, cysteine and glutamate were added at concentrations of 1 mM. MgATP, 109CdSO4 and gramicidin-D were added at concentrations of 3 mM, 80 µM and 5 µM, respectively. Values shown are means # SE (n = 3-6). 109CdUPTAKE (nmol/mg/10 minutes) COMPOUND DTY165 DTY167 -MgATP +MgATP -MgATP +MgATP Cd2+ 5.8 # 2.4 5.6 # 1.5 4.3 # 1.3 4.6 # 2.1 Cd2+ + GSH 4.2 # 1.2 37.4 # 4.5 3.3 # 1.1 8.3 # 2.7 Cd2+ + GSSG - 5.1 # 3.2 - 3.8 # 2.3 Cd2+ + GS-CH3 - 4.5 # 1.9 - 3.7 # 3.1 Cd2+ + Cys-Gly - 5.6 # 3.2 - 6.9 # 1.4 Cd2+ + Cys - 7.0 # 1.2 - 3.9 # 1.0 Cd2+ + Glu - 5.7 # 1.1 - 5.2 # 1.3 Purification of Transport-Active Complex To determine the mode of action of GSH and the form in which Cd2+ is transported, reaction mixtures initially containing Cd2+ and GSH were fractionated and YCFI-dependent uptake was assayed.

Incubation of 109Cd2+ with GSH and gel-filtration of the mixture on Sephadex G-15 yielded two major 109Cd-labeled peaks: a low molecular weight peak (LMW-Cd. GS) and a high molecular weight peak (HMW-Cd. GS) (Figure 9A). When rechromatographed on Mono-Q, LMW-Cd. GS and HMW-Cd. GS eluted at 0 (Figure 9C) and 275 mM NaCl, respectively (Figure 9B). Of these two 109Cd-labeled components, HMW-Cd.GS alone, underwent YCF1-dependent transport. MgATP-dependent, uncoupler-insensitive HMW-109Cd.GS uptake by DTY165 membranes increased as a single Michaelian function of concentration (Km, 39.1 # 14.1 µM; Vmax, 157.2 + 60.7 nmol/mg/10 minutes) (Figure 10A). By contrast, uptake of LMW-109Cd.GS by DTYl 65 membranes was negligible at all of the concentrations examined (Figure 10B). Vacuolar membranes from DTY167 cells transported neither HMW-109Cd.GS nor LMW-109Cd.GS (Figures 10A and lOB).

Bis(glutathionato!cadmium Is the Transport-Active Complex The transport-active complex, HMW-Cd.GS, was identified as bis(glutathionato)cadmium (Cd.GS2) by three criteria: (i) The average Cd:GS molar ratio of the transported species, estimated from the 109Cd:3H ratios of the HMW-Cd.GS peaks obtained after chromatography of reaction mixtures initially containing 109Cd2+ and [ H]GSH on Sephadex G-15 and Mono-Q were 0.44 # 0.09 and 0.49 # 0.17, respectively (Table 6). (ii) DTY165 membranes accumulated 109Cd and [ H]GS in a molar ratio of 0.49 i0.01 when incubated in media containing HMW-109Cd.[ H]GS, MgATP and gramicidin-D (Table 6). (iii) The principal ion peak detected after MALD-MS of HMW-Cd. GS had an m/z ratio of 725.4 i 0.7, consistent with the molecular weight of bis(glutathionato)cadmium (724.6 Da, Figure 11). The transport- inactive complex, LMW-Cd.GS, on the other hand, was tentatively identified as mono(glutathionato)cadmium on the basis of its smaller apparent molecular size (Figure 9A), failure to bind Mono-Q (Figure 9C) and Cd:GS ratio of 0.67 + 0.04 and 0.86 i 0.07 after chromatography on Sephadex G-15 and Mono-Q (Table 6), respectively.

While an m/z ratio of 725 for HMW-Cd. GS would be equally compatible with the transport of Cd.GSSG, this is refuted by two findings: (i) GSSG alone does not promote YCF1-dependent uptake (Table 5). (ii) The transport-active complex is probably a mercaptide. Pretreatment of HMW-Cd. GS with 2- mercaptoethanol inhibits MgATP-dependent, uncoupler-insensitive Cd2+ uptake by DTY165 membranes by more then 80% (Table 6) and S-methylation abolishes the stimulatory action of GSH (Table 5).

TABLE 6. Molar Cd:GS ratios of LMW-Cd.GS and HMW-Cd.GS complexes fractionated bv Sephadex G-15 and Mono-O chromatoeraphv (Figure 9) before and after MgATP-dependent, uncouDler-insensitive uptake bv vacuolar membrane vesicles purified from DTY165 and DTY167 cells. Cd:GS ratios were estimated from the 109Cd:[3H] radioisotope ratios of samples prepared from 109CdSO4 and [ H]GSH. HMW-109Cd.[ H]GS was pretreated with 2-mercaptoethanol (2- ME) by heating a 1:4 mixture of HMW-109Cd.[ H]GS with 2-ME at 60°C for 10 minutes before measuring 109Cd2+ uptake. Uptake was measured using 50 µM concentrations (as Cd) of the complexes indicated in standard uptake medium containing 5 µM gramicidin-D. Values shown are means # SE (n = 3-6). 109Cd UPTAKE (nmol/mg/10 min) MOLAR RATIO Cd:GS FRACTION DTY165 DTY167 BEFORE AFTER UPTAKE UPTAKE Sephadex G-15 HMW-Cd.GS - - 0.44 # 0.09 - LMW-Cd.GS - - 0.67 # 0.04 - Mono-Q HMW-Cd.GS 66.3 + 2.7 5.6 + 2.6 0.49 + 0.17i 0.49 + 0.01 LMW-CdGS 4.4 + 0.8 3.9 # 1.4 0.86 + 0.0i - After 2-ME HMW-Cd.GS 11.9 # 2.4 4.4 # 3.0 - - Cd.GS2 Transport is Directlv Energized bv MgATP Purification of Cd.GS2 enabled the energy requirements of YCF 1 - dependent transport to be examined directly and confirmed that more than 83% of the MgATP-dependent, uncoupler-insensitive Cd2+ transport measured using DTY165 membranes was mediated by YCF 1. Agents that dissipate both the ApH and AT components of the H+-electrochemical gradient established by the V-ATPase (FCCP, gramicidin-D) or directly inhibit the V-ATPase, itself (bafilomycin Al), decreased MgATP-dependent Cd-GS2 uptake by vacuolar membrane vesicles from DTY165 cells by 22% (Table 7). Ammonium chloride which abolishes ApH while leaving AT unaffected, on the other hand, inhibited uptake by only 15% (Table 7). From these results and the inability of uncouplers to markedly increase the inhibitions caused by V-ATPase inhibitors alone (Table 7), Cd.GS2 uptake by wild type membranes is

inferred to proceed via a YCF1-dependent, MgATP-energized pathway that accounts for most of the transport measured and a YCF 1-independent pathway, primarily driven by the H -gradient established by the V-ATPase, that makes a minor contribution to total uptake.

Table 7. Effects of uncouplers and V-ATPase inhibitors on uptake of kis(lutathionato)cadmium (Cd.GS2) bv vacuolar membrane vesicles purified from DTY 165 and DTY 167 cells. Uptake was measured in standard uptake medium containing 50 µM purified 109Cd.GS2. Bafilomycin A1, FCCP, gramicidin-D and NH4CI were added at concentrations of 0.5 µM, 5 'LM, 5 M, and 1 mM, respectively. Values outside parentheses are means + SE (n = 3-6); values inside parentheses are rates of uptake expressed as percentage of control. 109Cd.GS2 UPTAKE (nmol/mg/10 minutes) ADDITION DTY165 | DTY167 Control 105.8 i 12.4 (100) 17.3 + 2.7 (100) Gramicidin-D 77.8 + 6.4 (73.5) 9.3 i 2.0 (56.6) FCCP | 62.2 # 11.4 (58.8) | 10.2 # 1.6 (59.0) NH4CI 89.8 + 8.2 (84.8) 10.0 + 1.7 (57.8) NH4CI+ gramicidin-D 69.8 i 12.0 (66.0) 8.8 t 2.2 (50.9) Bafilomycin Al 81.8 + 6.0 (76.6) 12.8 + 3.6 (74.0) Bafilomycin A1+ gramicidin-D 70.2 + 12.2 (66.4) 7.2 + 2.4 (41.6) Cd. US2 Competes with DNP-GS for Transport As would be predicted if Cd.GS2 and the model organic GS-conjugate DNP-GS follow the same transport pathway, the Ki for inhibition of MgATP- dependent, uncoupler-insensitive Cd.GS2 uptake by DNP-GS (11.3 + 2.1 pLM; Figures 10A and 10C) coincided with the Km for DNP-GS transport (14.1 # 7.4 I1M).

Pretreatment with Cd2+or CDNB Increases YCFI Expression RNase protection assays of YCF1 expression in DTY165 cells and measurements of MgATP-dependent, uncoupler-insensitive 109Cd.US2 and [3H]DNP- GS uptake by vacuolar membranes prepared from the same cells after 24 hour of growth in media containing CdSO4 (200 µM) or the cytotoxic DNP-GS precursor, CDNB (150 M), demonstrated a parallel increase in all three quantities. YCFl-

specific mRNA levels were increased by 1.9- and 2.5-fold by pretreatment of DTY165 cells with CdSO4 and CDNB, respectively (Figure 12). The same pretreatments increased MgATP-dependent, uncoupler-insensitive 109Cd.GS2 uptake into vacuolar membrane vesicles by 1.4- and 1.7-fold and [3H]DNP-GS uptake by 1.6- and 2.8-fold (Figure 12).

These investigations provide the first indication of the mechanism by which YCF1 confers Cd2+ resistance in S. cerevisiae and its relationship to the transport of organic GS-conjugates by demonstrating that the integral membrane protein encoded by this gene specifically catalyzes the MgATP-energized uptake of bis(glutathionato)cadmium by vacuolar membrane vesicles.

The codependence of Cd-GS2 and organic GS-conjugate transport on YCF1 is evident at multiple levels: (i) The ycflA mutant strain, DTY167, is hypersensitive to Cd2+ and CDNB in the growth medium and both hypersensitivities are alleviated by transformation with plasmid-borne YCF1. (ii) Vacuolar membrane vesicles purified from DTY 167 cells are grossly impaired for MgATP-energized, uncoupler-insensitive organic GS-conjugate and GSH-promoted Cd2+ uptake. (iii) Cd.GS2, and organic GS-conjugates compete for the same uptake sites on YCFi. (iv) Factors that increase YCF1 expression elicit a parallel increase in Cd.GS2 and organic GS-conjugate transport. Thus, a number of ostensibly disparate observations, the strong association between cellular GSH levels and Cd2+ resistance (e.g., Singhal et al., 1987, FASEB J. 1:220-223), the markedly increased sensitivity of vacuole deficient S. cerevisiae strains to Cd2+ toxicity, and the coordinate regulation of the yeast YCF1 and GSH1 genes, the latter of which encodes y-glutamylcysteine synthetase (Wemmie et al., 1994, supra; Wu et al., 1994, supra), are now explicable in terms of a model in which YCF 1 catalyzes the GSH-dependent vacuolar sequestration of Cd2+.

Further, at the biochemical level, YCF 1 specifically catalyzes the transport of Cd.GS2 as the data provided herein establish. In addition, at the cellular level, YCF1 confers resistance to and is induced by a spectrum of xenobiotics.

Expression of YCF1 is increased by exposure of cells to glutathione-conjugable

xenobiotics and Cud2+. The close resemblance ofYCF1 to MRP1, the capacity of YCF 1 for both organic toxin and heavy metal transport, and its discovery in one of the most tractable and thoroughly molecularly characterized eukaryotes, S. cerevisiae, establishes that YCF 1 is useful for manipulation of the transport of organic toxins and heavy metals in plants, mammals and yeast. Thus, according to the present invention, methods for overcoming, or at least diminishing, heavy metal contamination through bioremediation using native species or genetically engineered organisms are now possible.

Cloning of plant MRP1/YCF1 homologs As described herein, the data presented herein establish that YCF1 encodes a protein functionally equivalent to human MRP1. There is next described the discovery that two plant genes, AtMRP1 and AtMRP2, from Arabidopsis encode MRP1/YCF1 homologs.

To isolate genes likely involved in glutathione S-conjugate transport from Arabidopsis thaliana, degenerate PCR primers corresponding to appropriate portions of human MRPI (Cole et al., 1992, supra) and YCFI (Szczypka et al., 1994, supra) were designed. Four degenerate primers were synthesized but only two of these yielded amplification products of the appropriate size that hybridized with MRP1 and YCF1. The sequences of the two primers were: 5'-GARAARGTIGGIATHGTIGGIMGIACIGGIGC-3'(MRP2) (SEQ ID NO: 17) and 5'-TCCATDATIGTRTTIARICKTGIGC-3'(MRP4) (SEQ ID NO: 18), where I = inosine, K = T or G, M = C or A and R = A or G. MRP2 corresponds to positions 1321-1331 and 1300-1310 in MRP1 and YCF1, respectively; MRP4 corresponds to positions 1486-1494 and 1466-1474. Database searches confirmed that the sequences of the peptides specified by MRP2 and MRP4 were specific to MRP1 and YCF1 but not any other ABC transporter in GenBank database release 90 (Altschul et al., 1990, J.

Mol. Biol., 215: 403-410).

Degenerate PCR was performed using Arabidopsis genomic DNA as template. Amplification was for 45 cycles using the following thermal profile: 94"C

for 30 seconds, 50°C for 30 seconds and 72°C for 1 minute. A 0.6 kb PCR product was isolated, shown to hybridize strongly with a mixed probe encompassing the second NBF domain of MRP1 and YCF1, and was cloned into pCRII vector (Invitrogen).

Sequence analysis verified that the deduced translation product of the Arabidopsis PCR product exhibited greatest similarity to YCF 1 and MRP 1 plus an unidentified 1.7 kbArabidopsis EST (ATTS 1246; Hofte metal., 1993, Plant 4:1051- 1061). In order to increase the likelihood of obtaining positive clones, a mixed probe consisting of the 0.6 kb PCR product and 1.6 kb EST was employed for further screens.

Eleven independent positive clones were obtained after screening approximately 3 x 105 plaques of a size-fractionated (3-6 kb) Arabidopsis cDNA library constructed in #ZAPII (Kieber et al., 1993, Cell, 72: 427-441) with the mixed probe. Restriction mapping confirmed that all 11 isolates corresponded to the same gene. The longest of these inserts, a 3.5 kb insert, designatedAtMRP2, was subcloned and sequenced (Figure 13).

Since this isolate of AtMRP2 was estimated to be missing approximately 1.5 kb of the 5' sequence of the ORF (assuming that the complete ORF of AtMRP2 is similar in size to the ORFs of human MRP1 and yeast YCF1), 500 bp of the most 5' sequence of AtMRP2 was used to probe two Arabidopsis bacterial artificial chromosome (BAC) libraries, UCD and TAMU (Choi et al., 1995, Weeds World, 2: 17-20) to isolate clones containing the missing sequence. This procedure yielded 8 BAC clones: U1L22, U8C12, U12A2, U23J22, U419, T9C22, T1B17 and T4K22.

After digestion with Hindlil, those fragments that hybridized with the 3.5 kb cDNA insert were introduced into pBluescript SK and sequenced. Two of these BAC clones (TlB17, T4K22) comprise a second MPR1 plant homolog, designated AtMRP1, while the remainder (U1L22, U8C12, U12A2, U23J22, U419, T9C22) comprise AtMRP2 (see below).

After establishing that an approximately 10 kb HindIII fragment from BAC clone U1L22 encompassed sequences identical to AtMRP2, a BglII restriction fragment comprising the first 3 kb of the BAC clone was used to rescreen

approximately 2 x 106 plaques from the Arabidopsis AZAPII cDNA library. Twenty six independent positive clones were obtained and the one containing the longest cDNA insert, 5.2 kb, was sequenced.

Sequence analysis demonstrated that the 5.2 kb cDNA was not identical to AtMRP2 but instead a very closely related gene. Designated AtMEP1 (Figure 16), the 5.2 kb cDNA was 84.3% and 88.2% identical to AtMRP2 at the nucleotide and amino acid levels, respectively. Importantly, AtMRP1 is a full-length cDNA.

Having determined the complete sequence of the AtMRP1 cDNA, it was possible to identify the initiation codon of the AtMRP2 genomic clone, design a specific 5'-UTR primer and amplify the remaining 5' end of AtMRP2 to generate a full- length cDNA. Thus, full-length cDNAs encoding AtMRP2 and AtMRP1 (Figures 13 and 16, respectively) and genomic clones corresponding to AtMRP2 and AtMRP1 have been generated (Figure 14 and 17, respectively). The deduced amino acid sequences of AtMRP2 and AtMRP1 are presented in Figures 15 and 18, respectively.

Expression ofAtMRPI in Saccharomvces cerevisiae The experiments described below establish that AtMRP 1 mediates the MgATP-dependent transport of GS-conjugates. The results of similar experiments on AtMRP2 demonstrate that this gene product has the same transport capability.

The data presented herein establishes that YCF1 from Saccharomyces cerevisiae encodes a 1,515 amino acid ATP-binding cassette (ABC) transporter protein which localizes to the vacuolar membrane and catalyzes MgATP-dependent GS- conjugate transport. Membrane vesicles from wild type (DTY165 ) cells contain two pathways for transport of the model GS-conjugate, DNP-US: an MgATP-dependent, uncoupler-insensitive pathway and an electrically driven pathway. Membranes from the mutant strains DTY 167 and DTY168, harboring a deletion of the YCF1 gene, are by contrast more than 90% impaired in MgATP-dependent, uncoupler-insensitive DNP-GS transport. Yeast strains lacking a functional YCF1 gene therefore represent a model system for probing the GS-conjugate transport function of plant YCF1/MRP1 homologs.

To test the transporter capacity of AtMRP1 (the first clone for which a full-length cDNA was obtained) for conferring GS-conjugate transport, yeast strain DTY168 (disrupted for the YCF1 gene) was transformed with an expression vector engineered to contain the coding sequence of AtMRP1. After selection of the transformants, membranes were prepared and assayed for MgATP-dependent, uncoupler-insensitive DNP-GS transport as described herein. The results establish that AtMRP 1 catalyzes GS-conjugate transport in a manner indistinguishable from the vacuolar GS-X pump.

Construction of the expression vector In order to constitutively express the AtMRP1 gene in S. cerevisiae, a derivative of the yeast-E. coli shuttle vector, pYES2 (Invitrogen), was constructed.

Essentially, the 831 bp Xbal/Notl fragment encompassing the 3-phosphoglycerate kinase (PGK) promoter of plasmid pFL61 (Minet et al., 1992, Plant J. 2:417-422) was inserted between the Spel/Notl restriction sites of pYES2. In so doing, the galactose- inducible yeast GAL1 promoter of pYES2 was replaced by the constitutive yeast PGK promoter, pPGK. This plasmid, designated pYES3, is otherwise identical to pYES2.

The gene to be expressed is inserted into the multiple cloning site located between the PGK promoter and CYC1 termination sequences.

Preliminary experiments had established that the 5' untranslated region (UTR) of the original AtMRP1 cDNA isolate diminished expression of the open reading frame in yeast. Thus, to maximize expression, the 127 bp 5'-UTR of AtMRP1 was removed. For this purpose, pBluescript SK--AtMRP1 was digested with HpallSnaBI to delete 3045 bp of the internal sequence. The remaining 5 kb fragment from this digest was gel-purified and self-ligated to generate truncated AtMRP1 cDNA as a template for PCR. One hundred pmol of AtMRP1-Nco primer (5'- AAACCGGTGCGGCCGCCATGGGGTTTGAGCCGT-3') (SEQ ID NO:19) and 100 pmol of T3 primer (5'-AATTAACCCTCACTAAAGGG-3') (SEQ ID NO:20) were used to amplify a 2002 bp fragment of AtMRPl using Pfu DNA polymerase

(Stratagene). Amplification was for 30 cycles using the following thermal profile: 94°C for 15 seconds; 50°C for 15 seconds; and 72°C for 3.5 minutes.

The PCR product was gel-purified, digested with SpeI and cloned into the EcoRV/SpeI sites of pBluescript 5K to generate pSK- AtMRP1-Nco2. The 1227 bp SphI/SpeI fragment of this construct was then exchanged with the 4363 bp SphI/SpeI fragment of pBluescript pSK AtlWRPl to generate pSK- AtMRPI-Nco5. pYES- AtMRP 1, lacking the 5' UTR, was constructed by digesting pSK- AtMRP1-Nco5 with XhoI/SpeI to obtain a 5049 bp truncated AtMRP1 gene fragment which was cloned into the XhoI/XbaI sites of pYES3. One kb of the 5' sequence of the AtMRP1 insert of pYES3-AtMRPl was analyzed and was found to match exactly the sequence of the original cDNA clone.

Transformation of Yeast S. cerevisiae strain DTY168 (MATa his6, leu2-3, -112, ura3-52 ycfl::hisG) was transformed with pYES3-AtMRPl or empty vector lacking the AtMRP1 insert (pYES3) by the LiOAc/PEG method (Giest et al., 1991, Yeast 7:253- 263) and selected for uracil prototrophy by plating on AHC medium containing tryptophan (Kim et al., 1994, supra).

Isolation of membrane vesicles For the preparation of membrane vesicles, 15 ml volumes of stationary phase cultures of the transformants were diluted into 1 L of fresh AHC medium and grown to an OD600 nm Of about 1.2. Membrane vesicles were purified as described herein and in Kim et al. (1995, supra).

Measurement of DNP-GS uptake DNP-GS uptake was measured as described herein in 200 iji reaction volumes containing 3 mM ATP, 3 mM MgSO4, 5 µM gramicidin-D, 10 mM creatine phosphate, 16 units/ml creatine kinase, 50 mM KCl, 1 mg/ml BSA, 400 mM sorbitol, 25 mM Tris-Mes (pH 8.0) and the indicated concentrations of [3H]DNP-GS (17.4 mCi/mmol). Gramicidin-D (uncoupler) was included to abolish the H+

electrochemical potential difference that would otherwise be established by the V- ATPase in media containing MgATP.

The results of this studv Membrane vesicles purified from pYES3-A tMRPl -transformed DTY168 cells exhibit an approximately 4-fold increase in MgATP-dependent, uncoupler-insensitive [3H]DNP-GS uptake by comparison with membrane vesicles purified from DTY168 cells transformed with empty vector (Figure 19). When measured at a DNP-GS concentration of 61.3 pLM, the initial rates of uptake by membrane vesicles purified from pYES3-AtMRPl-transformed and pYES 3- transformed cells were 0.4 rrrnol/mg/minute and 0.1 nmol/mg/minute, respectively (Figure 19).

The concentration dependence and vanadate inhibitility of uptake verify direct participation of AtMRP 1. MgATP-dependent, uncoupler-insensitive uptake by membrane vesicles purified from the pYES3-AtMRP1 transformants increases as a single hyperbolic function of DNP-GS concentration to yield Km and Vmax values of 48.7 + 15.4 pM and 6.0 + 1.7 nmol/mg/10 minutes, respectively (Figure 19). pYES3- AtMRP1-dependent DNP-GS uptake decreases as a single exponential function of the concentration of the phosphoryl transition state analog vanadate, to yield an I50 of 8.3 i 3.3 pM (Figure 20). By contrast, the apparent Km for DNP-GS uptake by membrane vesicles purified from pYES3-transformed DTY168 cells is in excess of 500 I1M and uptake is insensitive to vanadate.

On the basis of its sequence characteristics and the results of these experiments, AtMRP1 encodes the vacuolar GS-Xpump. The increases in uptake following the introduction of plasmid borne AtMRP1 into yeast (ca. 4 nmol/mg/20 minutes) are cornnensurate with the rates of MgATP-dependent, uncoupler-insensitive DNP-GS uptake measured in vacuolar membrane vesicles purified from plant sources (2.3, 3.8, 18.2, 5.8, and 2.1 nmol/mg/20 minutes for Arabidopsis leaf, Arabidopsis root, Beta vulgar its storage root, Vigna radiata hypocotyl and Zea mays root, respectively) (Table III in Li et al., 1995, supra). The Km for DNP-GS transport by heterologously

expressed AtMRP1 is similar to that reported for the endogenous GS-Xpump of plant vacuolar membranes (81.3 + 41.8 I1M, Li et al., 1995, supra). The I50 for inhibition of AtMRP l-dependent DNP-GS transport by vanadate coincides with the I50 for inhibition of the endogenous vacuolar GS-X pump of plant cells (7.5 i 3.9 I1M, Li et al., 1995, supra).

Having confirmed that the endogenous vacuolar GS-Xpump of S.

cerevisiae is lacking in theyctlA mutant strains, DTY168 and DTY167 (Li et al., 1995, supra), and in any case has a markedly lower Km for DNP-GS and is 6 to 8-fold less sensitive to vanadate than the plant cognate, these findings establish that AtMRPl per se is responsible for the MgATP-dependent, uncoupler-insensitive transport measured in these experiments. Given that heterologous expression of AtMRP1 alone is sufficient for DNP-GS transport in DTY168 cells, it is concluded that one of the GS- Xpumps of Arabidopsis has been cloned in its entirety.

Sequence comparisons of MRP1, cMOAT, YCF1, AtMRP1 and AtMRP2 with other members of the ABC transporter superfamily reveal two major subgroups. One group contains MRP1, cMOAT, YCF1, AtMRPl, AtMRP2 and the Leishmania P-glycoprotein-related molecule (Lei/PgpA). The other group contains the MDRs, the major histocompatibility complex transporters and STE6. However, of all the ABC transporters defined to date, cMOAT, YCF1, AtMRP1 and AtMRP2 exhibit the closest resemblance to each other. Unlike the similarities between the GS-Xpump subgroup, Lei/PgpA and CFTR, which center on the nucleotide binding folds (NBFs), the similarities between the GS-Xpump members cMOAT, YCF1, AtMRP1 and AtMRP2 are found throughout the sequence. GS-Xfamily members are 40-45% identical (60-65% similar) at the amino acid level, possess NBFs with an equivalent spacing of conserved residues and are colinear with respect to the location, extent and alteration of putative transmembrane spans and extramembrane domains. Two features of members of the GS-Xpump family that distinguish them from other ABC transporters are their possession of a central truncated CFTR-like regulatory domain

rich in charged amino acid residues and an approximately 200 amino acid residue N- terminal extension.

A hydropathy aligment of AtMRP 1, AtMRP2, YCF 1, HmMRP 1, and RtCMOAT is shown in Figure 21. Note the following: (i) The almost exact equivalence of AtMRP 1 and AtMRP2 with respect to the alternation of hydrophobic and hydrophilic stretches. (ii) The close correspondence of AtMRP 1 and AtMRP2 with all of the other members of the MRPl/YCFl/cMOAT subclass of ABC transporters in terms of the overall hydropathy profiles. (iii) The "signature" profile for the N-terminal 200 amino acid residues of all of the sequences shown, which is unique to the MRP1/YCF1/cMOAT subclass. Hydropathy was computed according to Kyte and Doolittle (1982, J. Mol. Biol. 46:105-132) over a running window of 15 amino acid residues. Hydrophobic stretches of sequence fall below the line and hydrophilic stretches fall above the line.

In Figure 22 there is depicted domain comparisons between AtMRP1, ScYCF 1, HmMRP 1, RtCMOAT, RbEBCR and HmCFTR. The domains indicated are the N-terminal extension (NH2), first and second transmembrane spans (TM1 and TM2, respectively), first and second nucleotide binding folds (NBF 1 and NBF2, respectively), putative CFTR-like regulatory domain (R), and the C-terminus (COOH).

This comparison is also tabulated in Tables 8 and 9.

TABLE 8: Identity and similarity analysis of putative domains of AtMRP1 against AtMRP2 ScYCF1, HmMRP1, RtCMOAT; HmCFTR and RbEBCR ScYCF1, Saccharomyces cerzvisiae YCF1; HmMRP1, human MRP1; RtCMOAT, rat cMOAT; HMCFTR, human CFTR; RbEBCR, rabbit EBCR.

The domains identified are N-terminal extension (NH2), transmembrane segments 1 and 2 (TM1 and TM2, respectively), CFTR-like regulatory domain (R), nucleotide binding folds 1 and 2 (NBF1 and NBF2, respectively) and C-terminus (COOH). Similarity was calcuted as described herein over the sequence segments indicated in Table 9. SEQUENCE DOMAIN OVERALL NH2 TM1 NBFI AtMRP2 Identity 87.0 74.4 90.4 92.1 Similarity 93.7 85.2 96.1 96.7 ScYCF1 Identity 36.1 13.3 32.2 50.0 SEQUENCE DOMAIN OVERALL NH2 TM1 NBFI Similarity 55.4 32.9 52.6 75.0 HmMRP1 Identity 41.5 16.2 37.4 58.0 Similarity 63.3 34.8 57.5 78.7 RtCMOAT Identity 38.6 19.6 33.8 58.7 Similarity 60.2 36.7 61.0 80.0 HmCFTR Identify 29.2 0 22.8 40.7 Similarity 55.1 0 47.8 62.0 RbEBCR Identity 38.9 17.2 34.5 62.4 Similarity 60.4 34.9 61.6 82.6 SEQUENCE DOMAIN R TM2 NBF2 COOH AtMRP2 Identity 80.5 86.9 91.3 89.4 Similarity 89.8 94.2 96.5 94.4 ScYCF1 Identity 33.9 34.7 34.7 58.1 Similarity 59.5 57.9 57.9 71.8 HmMRP1 Identity 31.6 31.9 61.9 48.3 Similarity 50.4 56.3 72.8 69.0 RtCMOAT Identity 33.9 34.4 60.7 50.0 Similarity 50.0 58.8 75.7 67.2 HmCFTR Identity 45.7 22.3 39.5 28.1 Similarity 75.0 51.3 61.6 58.4 RbEBCR Identity 35.6 34.1 61.9 43.0 Similarity 51.7 59.1 74.0 62.7 |TABLE 9. Positions and sizes of segments of sequence analyzed in Table 8. SEQUENCE DOMAIN OVERALL NH2 TM1 NBF1 AtMRP1 Position 1-223 224-631 634-782 Size 1622 223 407 148 AtMRP2 Position 1-223 224-631 634-782 SEQUENCE DOMAIN OVERALL NH2 TM1 NBF1 Size 1622 223 407 148 ScYCF 1 Position 1-210 211-645 646-787 Size 1515 210 435 142 HmMRP 1 Position 1-240 241-660 661-810 Size 1531 240 420 150 RtCMOAT Position 1-192 193-648 649-799 Size 1540 192 456 151 HmCFTR Position 0 1-440 441-590 Size 1481 0 440 150 RbEBCR Position 1-193 194-651 652-800 Size 1562 193 458 149 SEQUENCE DOMAIN R TM2 NBF2 COOH AtMRP1 Position 783-900 901-1244 1245-1417 1418-1622 Size 117 343 172 205 AtMRP2 Position 783-905 906-1249 1250-1422 1423-1622 Size 122 343 172 200 ScYCF 1 Position 788-936 937-1279 1280-1453 1454-1515 Size 149 343 174 163 HmMRP1 Position 811-960 961-1300 1301-1473 1474-1531 Size 150 340 173 59 RtcMOAT Position 800-960 961-1302 1303-1476 1477-1541 Size 161 342 174 65 HmCFTR Position 591-847 848-1217 1218-1389 1390-1481 Size 256 371 172 92 RbEBCR Position 801-961 962-1304 1305-1477 1478-1562 Size 161 343 173 86

As is apparent from the data presented above, there is significant homology between similar domains among AtMRP-related proteins. In particular, the N-terminal and R domains share significant homology among the AtMRP-related proteins tested. These data establish that in addition to primary sequence, the secondary structure of the molecule plays a significant role in GS-X pump function.

It should be appreciated that AtMRP1 and AtMRP2 constitute a family of genes in Arabidopsis, wherein various members of the family have different substrate specificities as demonstrated by the next set of experiments.

Substrate Preferences of AtMRP 1 and AtMRP2 To examine the substrate preferences of AtMRP 1 and AtMRP2, the following experiments were performed.

Isolation of Bn-NCC- 1 [l4C]Bn-NCC-1 (33.3 mCi/mmol) was extracted from senescent cotyledons of rape (Brassica nap us) and was purified by preparative HPLC (Krautler et al., 1992, Plant Physiol. Biochem. 30:333-346). Determination of the purity of the final preparation by analytical HPLC and enumeration of concentration and specific radioactivity (33.3 mCi/mmol) were performed according to Hinder et al. (1966, J.

BioL Chem. 271:27233-27236). Unlabeled Bn-NCC-1 was isolated from fully senescent cotyledons of excised shoots that had been maintained in complete darkness for 1 week.

Measurement of transport Cells were grown and vacuolar membrane-enriched vesicles were prepared as described (Kim et al., 1995, J. Biol. Chem. 270:2630-2635-). Uptake of ['4C]Bn-NCC-1, [3H]C3G-GS, [3H]DNP-GS, [3H]GSSG, [t4C]metolachlor or [3H]taurocholate was measured routinely in 200,ul reaction volumes containing membrane vesicles (10-20 ,ig protein), 3 mM ATP, 3 mM MgSO4, 5 MM gramicidin- D, 10 mM creatine phosphate, 16 units/ml creatine phosphate kinase, 50 mM KC1, lmg/ml BSA, 400 mM sorbitol, 25 mM Tris-Mes (pH 8.0) and the indicated

concentrations of transport substrate. Uptake was terminated by the addition of 1 ml ice-cold wash medium (400 mM sorbitol/3 mM Tris-Mes, pH 8.0) and vacuum filtration of the suspension through prewetted Millipore HA cellulose nitrate filters (pore size 0.45 m). The filters were rinsed twice with wash medium, and the retained radioactivity was determined by liquid scintillation counting. Nonenergized uptake was estimated by the same procedure except that ATP was omitted from the uptake medium.

The effect of taurocholate on the release of [3H]DNP-GS from membrane vesicles that had been allowed to mediate AtMRP2-dependent accumulation of this compound during a preceding uptake period was determined. This was accomplished by rapid depletion of ATP from the uptake medium using a hexokinase trap (glucose + ATP -> glucose-6-phosphate + ADP) and measurements of the decrease in vesicular radiolabel in the presence or absence of taurocholate. Membranes from DTY168/pYES3-AtMRP2 cells were incubated for 10 minutes in standard uptake medium containing 61.3 M [3H]DNP-GS after which time 200 mM glucose and 50 units/ml hexokinase (Type F-300 from baker's yeast) were added. After incubation for a further 2 minutes, taurocholate (50 M) or Triton X-100 (9.01% v/v) was added and release of vesicular [3H]DNP-GS was measured as described. Control samples were treated identically except that no additions were made after the initial 10 minute incubation period.

Substrate Preferences The absence of AtMRP2-dependent transport from DTY168 and DTY168/pYES3 membranes and the selective inhibition of this system by micromolar concentrations of vanadate, established that AtMRP2-dependent transport may be measured in two ways. This may be accomplished by assessing the difference between the rates of MgATP-dependent, uncoupler-insenstitive uptake by DTY168/pYES3- AtMRP2 membranes by comparison with DTY168 or DTY168/pYES3 membranes, or by assessing the vanadate-sensitive component of MgATP-dependent, uncoupler- insensitive uptake by DTY168/pYES3-AtMRP2 membranes. Because the results were

qualitatively and quantitatively similar whichever method was used, "AtMRP2- dependent" transport as used in this section refers to uptake which is measured as the increment consequent on transformation of DTY 168 cells with pYES3-AtMRP2 versus pYES3.

Application of this methodology to vacuolar membrane-enriched vesicles purified from pYES3-AtMRP2- versus pYES3-transformed DTY168 cells and expansion of the transport assays to measurements of the concentration dependence of [ H]DNP-GS, [ H]GSSG, [14C]metolachlor-GS, [14C]Bn-NCC-1 and [ H]taurocholate uptake, demonstrated that the substrate preferences and maximal transport capacities of AtMRP2 and AtMRP 1 differed markedly. While uptake of all of the GSH derivatives examined conformed to Michaelis-Menten kinetics, the Vma, values for AtMRP2- dependent uptake were consistently serveralfold greater than those for AtMRP 1 - dependent uptake. The Vmax values for AtMRP2-dependent uptake of [3H]DNP-GS, [3H]GSSG and ['4C]metolachlor-GS were 16.3 + 3.1, 38.1 + 3.2 and 136.0+28.1 nmol/mg/10 min, respectively; the corresponding values for AtMRP1 were 8.2 + 1.6, 6.8 # 1.1 and 17.5i5.2 nmol/mg/10 min. With the exception of [3H]GSSG whose Km for AtMRP1-dependent uptake (21.9.2 + 58.3 µM) was three times greater than that for AtMRP2-dependent uptake (73.0 + 15.1µM), the Km values estimated for AtMRP2 and AtMRP1 were very similar (65.7+29.8 versus 63.6 + 36.5 µM for metolachlor-GS).

Single concentration (50 µM) measurements of uptake of the glutathionated anthocyanin, cyanin-3-glucoside-GS (C3G-GS), demonstrated an approximately 6-fold greater capacity of AtMRP2 for transport of this compound (rate = 48.4 i 2.2 nmol/mg/10 min) by comparison with AtMRP1 (rate = 7.9 + 0.7 nmol/mg/l Omin).

In no case was MgATP-dependent, uncoupler-insensitive uptake of the unconjugated precursors of the GS-compounds, DNP, GSH, metolachlor and C3U detectable.

Neither AtMRP2 nor AtMRP1 catalyzed the uptake of [3H]taurocholate.

Transformation of DTY168 cells with either pYES3-AtMRP2 or pYES3-AtMRP1

conferred little or no increase in the capacity of vacuolar membrane-enriched vesicles for [3H]taurocholate uptake over that measured with vesicles prepared from pYES3- transformed cells. The results of these experiments are presented in Table 10.

Table 10. Kinetic parameters for uncoupler-insensitive AtMRP 1 - and AtMRP2- dependent transport of GS-derivatives, Bn-NCC- 1 and taurocholate.

AtMRP 1 AtMRP2 Compound Km Vmax Km Vmax DNP-GS 73.8 + 18.8 8.2 + 1.6 65.7 + 29.8 16.3 + 3.1 GSSG 219.2 + 58.3 6.8 + 1.1 73.0 + 15.1 38.1 + 3.2 Metolachlor-GS 63.6 + 36.5 17.5 + 5.2 75.1 + 31.6 136.0 + 28.1 Bn-NCC-1 ------Linear------ 15.2 + 2.3 63.1 + 2.5 Taurocholate ------Linear------ ------Linear------ MgATP-dependent, uncoupler-insensitive uptake by DTY 1 68/pYES3-AtMRP 1, DTY168/pYES3-AtMRP2 and DTY168/pYES3 membranes was measured as described herein. The Km and Vmax values were estimated by fitting the data to a single Michaelis-Menten function by nonlinear least squares analysis. Values shown are means + SE.

The 2- to 8-fold greater capacity of AtMRP2 versus AtMRP 1 for transport of the compounds examined was not attributable to differences in the levels of expression of their cDNAs from the PGK gene promoter of pYES3. Quantitative RT- PCR of equivalent amounts of total RNA extracted from DTY168/pYES3-AtMRP2 and DTY168/pYES3-AtMRP1 cells yielded similar levels of the 800 bp PCR

amplification product predicted from the sequences of the oligonucleotide primers used. Since neither amplification product was generated when PCR was performed without reverse transcription or when total RNA from DTY168/pYES3 cells was employed as template, contamination by genomic DNA and/or RT-PCR of transcripts other than those from AtMRP2 or AtMRP1, respectively, was not responsible for the observed results.

Anomalous interactions between candidate transport substrates Two critical properties of AtMRP2 were its capacity for the simultaneous transport of GS-conjugates and Bn-NCC-1 and its pronounced sensitivity to inhibition by taurocholate. Simultaneous measurements of [l4C]Bn-NCC-1 and [3H]DNP-GS uptake by membrane vesicles purified from DTY168/pYES3-AtMRP2 cells revealed parallel accumulation of both compounds with little or no interference of the transport of one by the other. AtMRP2-dependent uptake of [14C]Bn-NCC-1 at an extravesicular concentration equivalent to its Km value (1 SMM,) was nearly three times less sensitive to DNP-GS than would be predicted if this GS-conjugate were a competitior. If DNP-GS were a simple competitive inhibitor such that its Km value (66M) approximated its K value for the inhibition of BN-NCC-1 uptake, 120 MM DNP-GS would be expected to inhibit [l4C]Bn-NCC-1 uptake by 48% but this was not observed. DNP-GS concentrations in excess of 120 µM decreased [14C]Bn-NCC-1 uptake by less than 18%. Reciprocally, the concentration-dependence of AtMRP2- mediated [3H]DNP-GS uptake was not affected appreciably by Bn-NCC- 1. The Km and V values for AtMRP2-dependent [ H]DNP-GS. uptake in the presence of 15 MM Bn- NCC-1 (80.5 # 28.6 MM and 18.3 i 1.6 nmol/mg/10/min) were similar to those measured in its absence.

Although neither AtMRP2 nor AtMRP 1 transported taurocholate, AtMRP2-mediated transport was selectively inhibited by this compound. AtMRP1- dependent [3H]DNP-GS uptake was relatively insensitive to taurocholate (I50> 250 MM) but the uptake of both [3H]DNP-GS and [l4C]Bn-NCC-1 by AtMRP2 was strongly inhibited (15o(DNP.Gs uptake) = 27 # 1.3 MM; I50(Bn-NCC-1 uptake) = 49.5 0.3 MM).

Taurocholate at the concentrations employed appeared to exert is effect on AtMRP2-mediated transport by inhibiting pump activity directly rather than by increasing background membrane permeability and decreasing net influx by increasing passive DNP-GS efflux. Addition of taurocholate at a concentration (50 MM) sufficient to inhibit AtMRP2-dependent [3H]DNP-GS uptake by 70% to DTY168/pYES3- AtMRP2 vesicles that had accumulated [3H]DNP-GS for 10 minutes before arresting pump action by ATP depletion using a hexokinase trap, did not accelerate the efflux of intravesicular 3H-label over that measured on vesicles subject to a hexokinase trap in the absence of taurocholate. Imposition of a hexokinase trap and addition of a concentration of detergent (Triton X-100; 0.01% v/v) known to permeate these membranes (Zhen et al., 1997, J. Biol. Chem. 272:22340-22348), on the other hand, increased the rate and extent of release of the [3H]DNP-GS accumulated during the preceding 10 minute uptake period by more than 3-fold versus DTY168/pYES3- AtMRP2 vesicles treated with hexokinase alone or hexokinase plus taurocholate.

The high capacity of AtMRP2 for the transport of large amphipathic anions other than GS-conjugates (i.e., Bn-NCC demonstrates that one pump can assume more than one of the several ABC transporter-like functions identified in plants to date. In the case of AtMRP2, this includes transport activity directed to a broad- range GS-conjugate pump and a chlorophyll metabolite pump. Thus, one the one hand, the high capacity of heterologously expressed AtMRP2, and to a lesser extent AtMRP1, for the transport of metolachlor-GS, and by extension GS-conjugates of other herbicides to glutathionation, is consistent with the molecular identification of transporters capable of removing these and related compounds from the cytosol. On the other hand, the high capacity of AtMRP2 for the transport of Bn-NCC is consistent with the identification of an element capable of contributing to the further metabolism and eventual removal of tetrapyrrole derivatives generated during leaf senescence from the cytosol.

Vacuolar uptake of glutathionated medicarpin bv the glutathione conjugate pump

A key event in the disease resistance response of legumes is the rapid and localized accumulation of isoflavonoid phytoalexins. Accordingly, most studies of plant-pathogen interactions in the Leguminosae have centered on the enzymology and molecular biology of the isoflavonoid biosynthetic pathway (Dixon et al., 1995, Physiol. Plant 93:385). However, the mechanism and sites of intracellular accumulation of these compounds is not understood. Since many isoflavonoid phytoalexins are as toxic to the host plant as they are to its pathogens, it is essential that they are accumulated in the plant in a site which is sequestered (i. e., isolated) from the cytoplasm.

The following experiments describe uptake of free [3H]medicarpin by vacuolar membrane vesicles purified from etiolated hypocotyls of mung bean (Vigna radiata). This uptake is slow and relatively insensitive to MgATP. However, after incubation with glutathione and a total glutathione-S-transferase preparation from maize (Zea mays), [3H]medicarpin uptake occurs at a rate which is 8-fold faster in the presence, as opposed to the absence of MgATP. MgATP-dependent uptake of glutathione/glutathione-S-transferase pretreated [3Hjmedicarpin is only slightly inhibited by uncoupler (gramicidin D), but is strongly inhibited by vanadate and the model glutathione-S-conjugate, S-(2,4-dinitrophenyl)glutathione. These results demonstrate that the MgATP-energized glutathione-conjugate pump identified herein in the membrane preparation is capable of high affinity, high capacity transport of glutathionated isoflavonoid phytoalexins. The experimental procedures and results of these experiments are now described.

Preparation of [3H]medicarpin [3H]medicarpin was produced by base-catalyzed tritium exchange from 3H2O using unlabeled medicarpin isolated from fenugreek (Trigonella foenumgraecum) seedlings exposed to 3 mM Curl2.

GST purification and conjugation of medicarpin Two-week old maize (Zea mays) B73N seedlings were grown under continuous light at 21 °C. Twenty four hours prior to harvesting, the seedlings were exposed to a mild treatment with 2,4-dichlorophenoxyacetic acid and atrazine to stimulate GST expression (Timmerman, 1989, Physiol. Plant 77:465). Two-gram samples of root and shoot tissue were ground to homogeneity in 50 ml of 500 mM sodium phosphate buffer, pH 7.8 (Buffer A). The extract was centrifuged at 7,000 x g for 10 minutes at 4 0C and the resulting supernatant was filtered through Miracloth and mixed with 2 ml of S-hexylglutathione-conjugated agarose beads (Sigma). After incubation for 5 minutes at 21 °C, the beads were sedimented by centrifugation at 500 x g at 4°C. The supernatant was discarded and the beads were resuspended in 2.5 ml of prechilled Buffer A and centrifuged again. Bound GST was eluted by resuspension of the beads in 2 ml Buffer B (20 mM GSH, 500 mM sodium phosphate, pH 7.8) and incubation for 5 minutes at 21 °C. The beads were sedimented by centrifugation at 500 x g and the supernatant was assayed for GST activity (Mannervick et al., 1981, Methods Enzymol., 77:231).

[ H]medicarpin (0.5 IlCi, 4.5 Ci/mol) was conjugated with GSH by incubation with 25 µl of total purified maize GSTs for 3 hours at 21 °C in the dark.

Control, unconjugated samples were prepared by mixing [3H]medicarpin (0.5 pCi) with cold Buffer B and immediately freezing the mixture in liquid nitrogen.

Svnthesis of S-(2 .4-dinitrophenvl)lutathione (DNP-GS) DNP-GS was synthesized from 1 -chloro-2,4-dinitrobenzene and GSH by a modification of the enzymatic procedure of Kunst et al., (1989, Biochim. Biophys.

Acta 983:123; Li et al., 1995, supra).

Preparation of vacuolar membrane vesicles Vacuolar membrane vesicles were purified from etiolated hypocotyls of V. radiata cv. Berken as described (Li et al., Plant PhysioL 109: 1257, Li et al., 1995, supra).

Measurement of uptake

Unless otherwise indicated, [3H]medicarpin or [3H]medicarpin-GS uptake was measured at 25 CC in 200 ul reaction volumes containing 3 mM ATP, 3 mMMgSO4, 10 mM creatine phosphate, 16 U/ml creatine kinase, 50 mM KCl, 0.1% (w/v) BSA, 400 mM sorbitol and 25 mM Tris-Mes buffer, pH 8.0. Uptake was initiated by the addition of 12 ptI membrane vesicles (30-40 Fug protein) and brief mixing of the samples on a vortex mixer. Uptake was terminated by the addition of 1 ml ice-cold wash medium (400 mM sorbitol, 3 mM Tris-Mes, pH 8.0) and vacuum filtration of the suspension through prewetted HA cellulose nitrate filters (pore diameter 0.45 µm). The filters were rinsed twice with a 1 ml ice-cold wash medium, air-dried and radioactivity was determined by liquid scintillation counting.

Protein estimations and source of commercial chemicals was as described herein.

Results Appreciable MgATP-dependent uptake of [3H]medicarpin by vacuolar membrane vesicles purified from etiolated hypocotyls of mung was dependent on preincubation of this compound with GSH and GSTs. Free [3H]medicarpin incubated in the presence of GSH in the absence of GSTs was taken up at 16.7 + 3.6 and 7.4 # 1.3 nmol/mg/20 minutes in the presence and absence of MgATP, respectively (Figure 25).

In contrast, [3H]medicarpin-GS synthesized by incubating [3H]medicarpin with GSH in the presence of affinity-purified maize GSTs, was taken up at 81.0 + 13.3 and 11.3+0.4 nmol/mg/20 minutes in the presence and absence of MgATP, respectively (Figure 25).

MgATP-dependent [3H]medicarpin-GS uptake was strongly inhibited by vanadate and DNP-GS but was relatively insensitive to uncouplers. Whereas inclusion of vanadate (10 pM) or DNP-GS (100 pM) in the assay medium inhibited [3H]medicarpin uptake by more than 85%, addition of the ionophore, gramicidin D, diminished uptake by only 17% (Table 11).

Table 11. Effects of different inhibitors on [ H]medicarpin-GS uptake by vacuolar membrane vesicles. [3H]medicarpin-GS was added at a concentration of 65 I1M. MgATP was either omitted (- MgATP) or added at a concentration of 3 mM (+MgATP). Gramicidin-D, vanadate and DNP-GS were added at concentrations of 5 1M, 10 uM and 100 uM, respectively. Values outside parentheses are means + SE (n = 3); values inside parentheses are rates of uptake expressed as percentage of control. control. [ H]Medicarpin-GS Uptake (nmol/mg/10 minutes) TREATMENT +MgATP I -MgATP Control 85.6 + 13.3 (100) 16.7 i 6.0 (100) + Gramicidin-D 71.2 i 3.0 (83.2) 13.2 # 2.1 (79.0) + Gramicidin-D + vanadate 12.9 + 0.9 (15.1) 17.4 i 0.5 (104.2) + Gramicidin-D + DNP-GS 11.7 + 2.8 (13.7) 5.6 + 3.1 (33.5) MgATP-dependent, uncoupler-insensitive uptake increases as a single Michaelian function of [3H]medicarpin-GS concentration to yield Km and Vmax values of21.5 # 15.5 pM and 77.8 +23.3 nmol/mg/20 minutes, respectively (Figure 26).

Direct involvement of the GS-X pump in the accumulation of [3H]medicarpin-GS by vacuolar membrane vesicles is therefore evident at three levels: (i) Glutathionation of medicarpin selectively increases MgATP-dependent uptake.

MgATP-independent uptake is marginally affected by glutathionation but MgATP- dependent uptake is stimulated by approximately six-fold confirming that medicarpin- GS is the transported species and MgATP is the energy source. (ii) Uptake is directly energized by MgATP. The inability of uncoupler to markedly inhibit [3H]medicarpin- GS uptake implies that the H+-electrochemical gradient that would otherwise be established by the vacuolar H+-ATPase in the presence of MgATP does not drive uptake. Rather, the pronounced inhibition of MgATP-dependent uptake exerted by vanadate agrees with the notion that GS-X-mediated uptake is strictly dependent on ATP hydrolysis and formation of a phosphoenzyme intermediate (Martinoia et al.

1993, supra; Li et al., 1995, supra), (iii) [3H]medicarpin-GS and the model GS-Xpump substrate DNP-US, whose transport has been exhaustively analyzed in this system as

described herein, compete for uptake indicating that both are transported by the same moiety.

The efficacy of medicarpin-GS as a substrate for the vacuolar GS-X pump is striking. Even though the Km for medicarpin-GS uptake is undoubtedly an overestimate, since the yield from the conjugation reaction was not enumerated, it is nevertheless 2 to 25-fold lower than those estimated previously for DNP-US, C3G-GS (80 and 46 I1M in this system), glutathione-S-N-ethylmaleimide (500 ,uM) and metolachlor-GS (40-60 ,uM, barley vacuoles; Martinoia etc?., 1993, supra). Moreover, the capacity of the GS-Xpump for medicarpin-GS uptake is high (Vmax = 78 nmol/mg/20 minutes) versus DNP-GS (Vmax = 12 nmol/mg/20 minutes) and comparable to that estimated for C3G-GS (Vmax = 45 nmol/mg/minute). Thus, while maize anthocyanin was the first natural substrate shown to be vacuolarly sequestered through the concerted actions of cystolic GSTs and the vacuolar GS-Xpump in plants (Marrs et al., 1995, Nature, 375:397 and data contained herein), medicarpin, and presumably other isoflavonoid phytoalexins, is equally strong a candidate.

These data suggest that the GSTs which are induced following the hypersensitive response to avirulent fungal pathogens likely serve to facilitate the vacuolar storage of antimicrobial compounds in the healthy cells surrounding the lesion.

Transport of glutathionated anthocvanins and auxins bv the vacuolar GS-X pump of plant cells The data which are now described demonstrate that the vacuolar GS-X pumps of corn (Zea mays) roots and etiolated hypocotyls of mung bean (Vigna radiata) transport the anthocyanin cyanidin-3-glucoside (C3G), and the phytohormone, indole- 3-acetic acid (IAA), after conjugation with glutathione. Whereas the unconjugated forms of these compounds undergo negligible uptake into vacuolar membrane vesicles, both C3G-GS and IAA-GS are subject to high rates of MgATP-dependent, uncoupler- insensitive uptake (Figure 27 and Table 12). IAA-GS and C3G-GS uptake approximates Michaelis-Menten kinetics to yield Km values in the micromolar range and Vmax values 7- to 40-fold greater than those measured with the artificial transport substrate, DNP-GS (Table 12 and Li et al., 1995, supra). Uptake of both conjugates is inhibited by DNP-GS and vanadate in a manner consistent with mediation by the GS-X pump (Figure 27 and Table 13). In contrast, glutathionated abscissic acid (ABA-GS) is a poor substrate for the GS-Xpump: uptake is relatively slow and only saturates at high substrate concentrations (Figure 27 and Table 13).

Table 12. Summarv of kinetic Darameters for MgATP-dependent uncoupler- insensitive uptake of C3G-GS. IAA-GS and ABA-GS bv vacuolar membrane vesicles purified from etiolated hvpocotvls of V. radiata and roots of 7. mays.

Kinetic parameters (Km, MM; Vmax, nmoUmg/10 min) were computed from the data shown in Figure 27 by nonlinear least squares analysis. Values shown are means + SE. C3G-GS IAA-GS PARAMETER V. radiata Z. mays V. radiata Km 45.7 # 14.0 39.5 # 16.6 36.0 # 16.7 Vmax 45.3 # 6.5 79.1 # 14.7 17.7 # 5.8 IAA-GS ABA-GS PARAMETER Z. mays V. Radiata Z. mays Km 47.7 # 19.6 >1000 128.8 # 79.1 Vmax 30.0 # 4.9 22.9 # 9.2 4.0 # 1.4 It has been known for some time that the characteristic bronze coloration of Bronze-2 (bz2) mutants is a consequence of the accumulation of cyanidin- 3-glucoside in the cytosol. In wild type (Bz2) plants, anthocyanins are transported into the vacuole and become purple or red whereas in bz2 plants, anthocyanin is restricted to the cytoplasm where it is oxidized to a brown ("bronze") pigment. However, until the present invention, the exact molecular basis of this lesion was unknown. Since Bz2 encodes a GST responsible for conjugating anthocyanin with GSH (Marrs et al., 1995) and glutathionated anthocyanins are transported by the vacuolar GS-Xpump, the

experiments described herein explain the bronze phenotype. Being defective in the glutathionation of anthocyanins, bz2 mutants are unable to pump these pigments from the cytosol into the vacuole lumen; a conclusion borne out by the ability of the GS-X pump inhibitor, vanadate, to phenocopy the bz2 lesion in wild type protoplasts and the efficacy of cyanidin-3-glucoside-GS as a substrate for the plant vacuolar GS-Xpump in vitro, as the data presented herein establish.

The concept underlying the above-described experiments on phytohormones is that they may be subject to metabolic interconversions and compartmentation analogous to those deduced for anthocyanins. On the one hand, it is now established that C3U must be glutathionated before it can be transported into the vacuole. On the other hand, it is evident that most of the vacuolar anthocyanins of intact plants are not stored in this form. Instead, they are subject to long term storage as their malonyl derivatives. It is therefore apparent that while C3G-GS is a short-lived but necessary intermediate for vacuolar anthocyanin compartmentation, it is not the terminal product of this process. The experiments with auxins further illustrate this principle by demonstrating that IAA is susceptible to glutathionation and that the resultant IAA-GS conjugate is transported by the vacuolar GS-Xpump in a MgATP- dependent, uncoupler-insensitive, vanadate-inhibitible manner. Thus, even though IAA-GS derivatives have not been detected in planta, this does not exclude the possibility that they are short-lived transport intermediates necessary for subsequent vacuolar processing of this class of compounds.

Table 13. Concentrations of DNP-GS and vanadate required to inhibit MgATP- dependent uncoupler-insensitive uptake of C3G-GS. IAA-GS and ABA-GS bv 50% ( Iso values bv vacuolar membrane vesicles Durified from etiolated hynocotyls of V.

radiata and roots of 7. mays. ISo values (pM) were estimated by nonlinear least squares analysis after fitting the data to a single negative exponential. ND, not determined. C3G-GS IAA-GS ABA-GS COMPOUND V. radiata Z. mays V. radiata Vanadate 7.9 8.2 6.5 DNP-GS 103.5 112.4 124.2 C3G-GS IAA-GS ABA-GS COMPOUND Z. mays V. radiata Z. mays Vanadate 5.5 ND >150 DNP-GS 109.8 ND 231.3 Generation of a Transgenic Plant Comprising a Transgene Encoding a GS-X Pump To generate a transgenic plant comprising a gene encoding YCF 1, the following experiments were performed. The binary vector pROK-YCF1, encoding wild type YCF 1 was constructed. The sense orientation of the inserts with respect to the CaMV 35S promoter of pROK (Baulscombe et al., 1986, Nature 321:446-449) was confirmed and these constructs, as well as empty vector (pROK) controls, were transformed into Agrobacterium strain C58 by electroporation (Ausubel et al., 1992, Current Protocols in Molecular Biology, pp 27-28).

Kanamycin-resistant Agrobacterium transformants were isolated, the integrity of the constructs in the bacterial recipient was established by PCR and Arabidopsis roots were inoculated with the transformants (Huang et al., 1992, Plant Mol. Biol. 10:372-384). The resulting rosette shoots generated on selective medium

were transferred to root-inducing medium for regeneration. Stable insertion of the sense strands and constitutive expression of YCF1 and YCF1. :HX4 was demonstrated in the kanamycin-resistant Arabidopsis transformants, by probing Southern blots with YCF1 and pROK sequences and by Northem analyses, respectively.

An association between YCF1 expression and altered xenobiotic resistance was tested by screening multiple T2 generation pROK-YCFl, pROK-YCFl- HA and pROK empty vector transformant lines for tolerance to cadmium salts and the GS-conjugable xenobiotic 1-chloro-2,4-dinitrobenzene (CDNB). CDNB has three advantages for studies of this type: (i) It is an established plant toxin (Li et al., 1995, Plant Physiol. 109:117-185); (ii) The kinetics of transport of its glutathionated derivative, DNP-US, as well characterized for YCF1 (Li et al., 1996, J. Biol. Chem.

271:6509-6517) and the endogenous GS-X pump (Li et al., 1995, Plant. Physiol.

107:1257-1268; Li et al., 1995, Plant Physiol. 109:117-185). (iii) DNP-GS is the only known immediate metabolite of CDNB in vivo (Li et al., 1995, Plant Physiol. 109:117- 185).

Methods similar to those described by Howden et al. (Howden et al., 1992, Plant Physiol. 99:100-107; Howden et al., 1995, Plant Physiol. 107:1059- 1066; Howden metal., 1995, PlantPhysiol. 107:1067-1073) were applied to the initial characterization of the transfonnants. T2 seeds were first sown in rows on Cd2+-free and CDNB-free medium in Petri dishes standing on edge so that the roots grew vertically down the surface of the agar. Three to 4 days after germination, the seeds were transferred, again in rows, to media containing a range of CdSO4 or CDNB concentrations. After rotating the Petri dishes though 1800 and allowing growth for another 24-48 hours, the seedlings were scored for hook length. The results of this study are shown in Figure 28. It is evident from the data presented therein that transgenic Arabiposis plants comprising YCF1 acquire increased resistance to cadmium salts and the organic cytotoxin, CDNB.

The disclosures of each and every patent, patent application and publication cited herein are hereby incorporated herein by reference in their entirety.

While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: Rea, Philip A Lu, Yu-Ping Li, Ze-Sheng (ii) TITLE OF INVENTION: GLUTATHIONE-S-CONJUGATE TRANSPORT IN PLANTS (iii) NUMBER OF SEQUENCES: 20 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: PANITCH SCHWARZE JACOBS & NADEL, P.C.

(B) STREET: One Commerce Square, 2005 Market Street, 22nd Fl.

(C) CITY: Philadelphia (D) STATE: Pennsylvania (E) COUNTRY: US (F) ZIP: 19103-7086 (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 #1.30 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: Not yet assigned (B) FILING DATE: 18-NOV-1997 (C) CLASSIFICATION: (vii) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: US 60/031,040 (B) FILING DATE: 18-NOV-1996 (viii) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: US 60/061,328 (B) FILING DATE: 08-OCT-1997 (ix) ATTORNEY/AGENT INFORMATION: (A) NAME: Doyle Leary Ph.D., Kathryn (B) REGISTRATION NUMBER: 36,317 (C) REFERENCE/DOCKET NUMBER: 9596-12PC (x) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 215-965-1284 (B) TELEFAX: 215-567-2991 (C) TELEX: 831-494 (2) INFORMATION FOR SEQ ID NO:1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 5232 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: TTATGAAAAT TTATTATTTT TGTTGCTATG GTTTTTTGGA ATTAGAAGCT CATTTCAAAG 60 TTGTTGATTT TCTTTGCAGG GTAGGGAATT GGTGTGGTAG CTTGTGATGC ACTGTGTTTG 120 AGGGAAAGGA AAGGATAACG ATGGGGTTTG AGTTTATTGA ATGGTATTGT AAGCCGGTGC 180 CTAATGGTGT GTGGACTAAA ACAGTGGCTA ATGCATTTGG TGCATACACG CCTTGTGCTA 240 CTGACTCTTT TGTGCTTGGT ATCTCTCAAC TGGTTCTGTT GGTTCTGTGC CTGTATCGTA 300 TATGGCTCGC CTTAAAGGAT CACAAGGTGG AGAGGTTCTG TTTGAGGTCG AGATTGTATA 360 ACTATTTCCT GGCTTTGTTG GCTGGTATGC TACTGCTGAG CCTTTGTTTA GATTGATCAT 420 GGGGATTTCA GTTTTAGATT TTGATGGACC TGGACTTCCT CCTTTTGAGG CATTCGGATT 480 GGGTGTCAAA GCTTTTGCTT GGGGCGCTGT AATGGTCATG ATTTTAATGG AAACTAAAAT 540 TTACATCCGT GAACTCCGTT GGTATGTCAG GTTTGCTGTC ATATATGCTC TTGTGGGGGA 600 TATGGTCTTG TTAAATCTTG TTCTCTCAGT CAAGGAGTAC TATAGCAGTT ATGTTCTGTA 660 TCTCTACACA AGCGAAGTGG GAGCTCAGGT TCTGTTTGGA ATTCTCTTGT TTATGCATCT 720 TCCCAATTTG GATACTTACC CTGGCTACAT GCCAGTGCGG AGTGAAACTG TGGATGATTA 780 TGAGTATGAA GAGATTTCTG ATGGACAACA AATATGCCCT GAGAAGCATC CAAATATATT 840 TGACAAAATC TTCTTCTCAT GGATGAATCC CTTGATGACT TTGGGATCTA AAAGGCCTCT 900 AACAGAGAAG GATGTGTGGT ATCTAGACAC TTGGGATCAG ACTGAAACTC TGTTCACGAG 960 TTTCCAGCAT TCCTGGGATA AGGAACTACA AAAGCCGCAA CCGTGGCTGT TGAGAGCATT 1020 GAACAATAGC CTGGGAGGAA GGTTTTGGTG GGGAGGATTT TGGAAGATCG GGAATGATTG 1080 CTCACAGTTT GTGGGACCTC TTTTACTGAA TCAACTCTTA AAGTCAATGC AAGAGGATGC 1140 GCCAGCTTGG ATGGGTTACA TCTATGCGTT CTCAATCTTT GGTGGAGTGG TGTTCGGGGT 1200 GCTATGTGAA GCTCAATATT TCCAGAATGT CATGCGTGTT GGTTACCGAC TGAGATCTGC 1260 TCTGATTGCT GCTGTGTTCC GCAZATCGTT GAGGTTAACT AATGAAGGTC GTAGAAAGTT 1320 TCAAACAGGA AAGATAACCA ACTTAATGAC GACTGATGCC GAATCTCTTC AGCAAATATG 1380 CCAATCACTT CATACCATGT GGTCGGCTCC ATTTCGTATA ATTATAGCAC TGATTCTCCT 1440 CTATCAGCAA TTGGGTGTTG CCTCGCTCAT TGGTGCATTG TTGTTGGTCC TTATGTTCCC 1500 TTTACAGACT GTTATTATAA GCAAAATGCA GAAGCTGACA AAGGAAGGTC TGCAGCGTAC 1560 TGACAAGAGA ATTGGCCTTA TGAATGAAGT TTTAGCTGCA ATGGATACAG TAAAGTGTTA 1620 TGCTTGGGAA AACAGTTTCC AGTCCAAGGT CCAAACTGTA CGTGATGATG AATTATCTTG 1680 GTTCCGGAAA TCACAGCTCC TGGGAGCGTT GAATATGTTC ATACTGAATA GCATTCCTGT 1740 TCTTGTGACT ATTGTTTCAT TTGGTGTGTT CACATTACTT GGAGGAGACC TGACCCCTGC 1800 AAGAGCATTT ACGTCACTCT CTCTCTTTGC TGTGCTTCGT TTCCCTCTCT TCATGCTTCC 1860 AAACATTATA ACTCAGGTGG TRAkTGCTAA TGTATCCTTA AAACGTCTTG AGGAGGTATT 1920 GGCGACAGAA GAAAGAATTC TCTTACCAAA TCCTCCCATT GAACCTGGAG AGCCAGCCAT 1980 CTCAATAAGA AATGGATATT TCTCTTGGGA TTCTAAGGGG GATAGGCCGA CGTTGTCAAA 2040 TATCAACTTG GATGTACCTC TTGGCAGCCT AGTTGCTGTG GTTGGTAGTA CAGGCGAAGG 2100 AAAAACCTCT CTAATATCTG CTATCCTTGG TGAACTTCCT GCAACATCTG ATGCAATAGT 2160 TACTCTCAGA GGATCAGTTG CTTATGTTCC ACAAGTTTCA TGGATCTTTA ATGCAACAGT 2220 ACGCGACAAT ATACTGTTTG GTTCTCCTTT CGACCGTGAA AAGTATGAAA GGGCCATTGA 2280 TGTGACTTCA CTGAAGCATG ACCTAGAGTT ACTGCCTGGT GGTGATCTCA CGGAGATTGG 2340 AGAAAGAGGT GTTAATATCA GTGGAGGACA GAAGCAGAGG GTTTCCATGG CTAGGGCCGT 2400 TTACTCAAAT TCAGATGTGT ACATCTTTGA TGACCCGTTA AGTGCCCTTG ATGCTCATGT 2460 TGGTCAACAG GTTTTTGAAA AATGCATAAA AAGAGAACTG GGGCAGAAAA CGAGAGTTCT 2520 TGTTACAAAC CAGCTCCACT TCCTATCACA AGTGGACAGA ATTGTACTTG TGCATGAAGG 2580 CACAGTGAAA GAGGAAGGAA CATATGAAGA GCTATCCAGT AATGGCCCTT TGTTCCAGAG 2640 GCTAATGGAA AATGCAGGGA AGGTGGAAGA ATATTCAGAA GAAAATGGAG AAGCTGAGGC 2700 AGATCAAACA GCGGAACAAC CAGTTGCGAA TGGGAACACA AATGGTCTTC AAATGGATGG 2760 AAGTGACGAT AAAAAATCCA AAGARGGAAA TAAAAAAGGA GGGAAATCTG TCCTCATCAA 2820 GCAAGAAGAA CGTGAAACCG GAGTTGTAAG TTGGAGAGTC CTGAAGAGGT ACCAGGATGC 2880 ACTTGGAGGG GCATGGGTAG TGATGATGCT CCTTTTATGT TACGTCTTAA CAGAAGTATT 2940 TCGGGTTACT AGCAGCACGT GGTTGAGTGA GTGGACTGAT GCAGGAACTC CAAAGAGTCA 3000 TGGACCCCTT TTCTACAATC TCATATATGC ACTTCTCTCG TTTGGACAGG TTTTGGTGAC 3060 ATTGACCAAT TCATATTGGT TGATTATGTC CAGTCTTTAT GCAGCTAAGA AGTTACACGA 3120 CAATATGCTT CATTCCATAC TGAGGGCCCC GATGTCCTTC TTCCATACCA ATCCGCTAGG 3180 ACGGATAATC AATCGATTCG CAAAAGATCT GGGTGATATT GATCGAACTG TGGCCGTCTT 3240 TGTAAACATG TTTATGGGTC AAGTCTCACA GCTTCTTTCA ACTGTAGTGT TGATTGGCAT 3300 TGTAAGCACT TTGTCCTTGT GGGCCATCAT GCCCCTCCTG GTCTTGTTTT ATGGAGCTTA 3360 TCTTTATTAT CAGAACACAG CCCGTGAGGT TAAGCGTATG GATTCAATTT CAAGATCGCC 3420 TGTTTATGCA CAGTTTGGAG AGGCATTGAA TGGCTTATCA ACTATCCGTG CTTACAAAGC 3480 ATATGATCGT ATGGCTGATA TCAACGGAAG ATCAATGGAT AATAACATCA GATTCACTCT 3540 TGTCAACATG GGTGCCAATC GGTGGCTTGG AATCCGTTTA GAAACTCTGG GTGGTCTTAT 3600 GATATGGCTG ACAGCATCGT TTGCTGTCAT GCAGAATGGA AGAGCGGAGA ACCAACAGGC 3660 ATTTGCATCT ACAATGGGTT TGCTTCTCAG TTATGCCTTA AATATTACTA GCTTGTTAAC 3720 AGGTGTTCTG AGACTTGCGA GTTTGGCTGA GAATAGTCTA AACGCGGTCG AGCGTGTTGG 3780 CAATTATATA GAGATTCCGC CAGAGGCTCC GCCTGTCATT GAGAACAACC GTCCACCTCC 3840 TGGATGGCCA TCATCTGGAT CCATAAAGTT TGAGGATGTT GTTCTCCGTT ACCGCCCTCA 3900 GTTACCGCCT GTGCTTCATG GGGTTTCTTT CTTCATTCAT CCAACAGATA AGGTGGGGAT 3960 TGTTGGAAGG ACTGGTGCTG GAAAGTCAAG CCTGTTGAAT GCATTGTTTA GAATTGTGGA 4020 GGTGGAAGAA GGAAGGATCT TAATCGATGA TTGTGACGTT GGAAAGTTTG GACTGATGGA 4080 CCTACGTAAA GTGCTCGGAA TCATTCCACA GTCACCGGTT CTTTTCTCAG GAACTGTGAG 4140 GTTCAATCTT GATCCATTTG GTGA2CACAA TGATGCTGAT CTTTGGGAAT CTCTAGAGAG 4200 GGCACACTTG AAGGATACCA TCCGCAGAAA TCCTCTTGGT CTTGATGCTG AGGTCTCTGA 4260 GGCAGGAGAG AATTTCAGCG TGGCACAGAG GCAATTGTTG AGTCTTTCAC GTGCGCTGTT 4320 ACGGAGATCT AAGATACTCG TCCTTGATGA AGCAACTGCT GCTGTAGATG TTAGAACCGA 4380 TGCCCTCATT CAGAAGACTA TCCGAGAAGA ATTCAAGTCA TGCACGATGC TCATTATCGC 4440 TCACCGTCTC AATACCATCA TTGACTGTGA CAAAATTCTC GTGCTTGATT CTGGAAGAGT 4500 TCAAGAATTC AGTTCACCGG AGAACCTTCT TTCAAATGAA GGAAGCTCTT TCTCCAAGAT 4560 GGTTCAAAGC ACTGGAGCTG CAAATGCTGA GTACTTGCGT AGTTTAGTAC TCGACAACAA 4620 GCGTGCCAAA GATGACTCAC ACCACTTACA AGGCCAAAGG AAATGGCTGG CTTCTTCTCG 4680 CTGGGCTGCA GCCGCTCAGT TTGCTCTGGC TGCGAGTCTT ACTTCGTCGC ACAACGATCT 4740 TCAAAGCCTT GAAATTGAAG ATGACAGCAG CATTTTGAAG AGAACAAACG ATGCAGTTGT 4800 GACTCTGCGC AGTGTTCTCG AGGGGAAACA CGACAAAGAG ATTGCÆ5AGT CGCTTGAGGA 4860 ACATAATATC TCTAGAGAGG GATGGTTGTC ATCACTCTAT AGAATGGTAG AAGGGCTTGC 4920 AGTGATGAGC AGATTGGCAA GGAACCGAAT GCAACAACCG GATTACAATT TCGAAGGAAA 4980 TACATTTGAC TGGGACAACG TCGAGATGTA GATAAGTTCA TGTTAAACTA GGAATCATTG 5040 TCTCTTCCGT AAGAAACATA TATTTATCTT AACCAAAATT ATTAGTTTGG TTTCCATTTC 5100 ATAAACTTAA TTTTCACCTG CAAAGAkAAT CAAACCCTGT TGTGTTCTTC GTGATAAGTA 5160 GAGAAATTAC TTGAGTATCC TTCTAACTCA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA 5220 AAAAAAAAAA AA 5232 (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9936 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DìiA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: GACTCGATAC CATCTTAAAT GCAGAGTCTT TTCGTGATAA TAAAATTATG GATTCGTTTC 60 AAAGTTTTTT TTTTTTCGTA TGGARAACAC TTGAGCTCTC TCAATCTTGT AGTCTTGACT 120 CTTGATGATT CTTCTATGTT CTCGTTGTGA TTGCTTGTCA CTGTTCTATC TTTATATATG 180 ATTAAATGCA ATTTTGCCCC TTTTTACGCG CGAATGTATT TATTATCTTT CGCACTCTGG 240 GTCCATTTCT TGTCACTTGA GCACATAATG ATTGATTTAT GACTTTTTAA AGTTATGAAA 300 ATTTATTATT TTTGTTGCTA TGGTTTTTTG GAATTAGAAG CTCATTTCAA AGTTGTTGAT 360 TTTCTTTGCA GGGTAGGGAA TTGGTGTGGT AGCTTGTGAT GCACTGTGTT TGAGGGAAAG 420 GAAAGGATAA CGATGGGGTT TGAGTTTATT GAATGGTATT GTAAGCCGGT GCCTAATGGT 480 GTGTGGACTA AAACAAGTGG CTAATGCATT TGGTGCATAC ACGCCTTGTG CTACTGACTC 540 TTTTGTGCTT GGTATCTCTC AACTGGTTCT GTTGGTTCTG TGCCTGTATC GTATATGGCT 600 CGCCTTAAAG GATCACAAGG TGGAGAGGTT CTGTTTGAGG TCGAGATTGT ATAACTATTT 660 CCTGGCTTTG TTGGCTGCGT ATGCTACTGC TGAGCCTTTG TTTAGATTGA TCATGGGGAT 720 TTCAGTTTTA GATTTTGATG GACCTGGACT TCCTCCTTTT GAGGTGCTTT ATTTTCTGTT 780 CCTTATTCTT TATCTTTTAG TTTGTTGTGT ATGTTTTACC TGAAACATGC TATTGTTTGT 840 GTGATTTCTT TGGCAGGCAT TCGGATTGGG TGTCAAAGCT TTTGCTTGGG GCGCTGTAAT 900 GGTCATGATT TTAATGGAAA CTAkAATTTA CATCCGTGAA CTCCGTTGGT ATGTCAGGTT 960 TGCTGTCATA TATGCTCTTG TGGGGGATAT GGTCTTGTTA AATCTTGTTC TCTCAGTCAA 1020 GGAGTACTAT AGCAGGTTGG TACAATTTTG GAGTTACTTT GGTTTATTGA AGTCATTGTT 1080 CTTCTTCTAC AGGGTGAATT CATGTTTTGT TTTCATTGCA GTTATGTTCT GTATCTCTAC 1140 ACAAGCGAAG TGGGAGCTCA GGTTAGCTCA CTTGGACTCC TTTAGAGAGT CCAGAATCCT 1200 AGCATGTGCT ATGATTATAA ATCAGAATCC GATACAGTTT GTTTTCTAAC ATCTTAAGAG 1260 GGTGAATTTT GGTTTTACTT CAGGTTCTGT TTGGAATTCT CTTGTTTATG CATCTTCCCA 1320 ATTTGGATAC TTACCCTGGC TACATGCCAG TGCGGAGTGA AACTGTGGAT GATTATGAGT 1380 ATGAAGAGAT TTCTGATGGA CAP.CAAATAT GCCCTGAGAA GCATCCAAAT ATATTTGACA 1440 GTAAGTCACT CTACATGATT TTCATTTGGT CGCCTGGCTG AAACTTATAA TTAGTAATCA 1500 TAATTTGCAA ACATCGTCTC TGAOTTTTGT TCAGATTGAT CATGGGGATT TAGGTTTTGA 1560 AATTTCACCT GATTTCCTTC TTCCAATTTC CTTGTTTGGT CACAGAA2TC TTCTTCTCAT 1620 GGATGAATCC CTTGATGACT TTGGGATCTA AAAGGCCTCT AACAGAGAAG GATGTGTGGT 1680 ATCTAGACAC TTGGGATCAG ACTGAAACTC TGTTCACGAG GTACTTCTAA CA».TAATTAT 1740 ATCTCTTAAA ATGTATATTA CTGAATTGGC TATTTGATAT TTTCTGTATC CTTTTTAGTT 1800 TCCAGCATTC CTGGGATAAG GAACTACAAA AGCCGCAACC GTGGCTGTTG AGAGCATTGA 1860 ACAATAGCCT GGGAGGAAGG TAGATAGATT TTCTCACCTT ATCGTGCTGT GTTCTCATCT 1920 CTTTTGAGTT TTGAGTATGA TTAGATAGTG CTGGATTTCA CTGTGATGTG CAGATGTTTA 1980 AGTGATCTCT TGAAAGAACC ATCAGGTTTT TAGAATGTGT AGGAAGCAAG ATCAGAATAT 2040 TTCTACTTAT TTAATGTTAG TTGTTTGCTA TAGCAGCTTA ACACATTTCC ATCTTATCAT 2100 AGGCAATCAT GCTTGCTTTC GTACTCTTAT AAATTTAAGA CATAGGGGAT ACAACTTTTA 2160 CTGTAGATTG GTTARATATG TTTTTTTTTC TTGGTTCATA TTGCTTAAGC ATTATTTCGT 2220 TTGTTAACTA CATGTCGTAT GGGGATCTAA TTTTTTGAAT TTTGTAGGTT TTGGTGGGGA 2280 GGATTTTGGA AGGTATTTTC GTCTACCTCT TTCTCTTTTA TTCGTGCTTC CAGAGTCTTT 2340 CCTCTCTTTT ATTCATATGA TCACAGGTTC TGCGTCATGT TGGATAACCT TCTGTCACGT 2400 GGAAGTCATT TATAATTTAC ATGGTGTTAC AGATTATTAG AAGGAt.CTAG TGGGTTCTTA 2460 GTTTTTCTTT ATCAATTCAT TGTACTTGAA CATATTTATT TACATTTGTA TGCACAGATC 2520 GGGAATGATT GCTCACAGTT TGTGGGACCT CTTTTACTGA ATCAACTCTT AAAGGTTTGT 2580 TCTTTTCTTG GCAGATTCGG AAACCTATTA TTGGTTCAAT ATTCTTATCT GACAATATCT 2640 CTCATTTTGG ATGTCAAACT ATATACAGTC AATGCAAGAG GATGCGCCAG CTTGGATGGG 2700 TTACATCTAT GCGTTCTCAA TCTTTGGTGG AGTGGTATGA AATGAAGTCC TCTTTCTCTC 2760 TCTCTCTCTG TCTATTTGGA CTCTCTTCTA TCAACTTGTG AAACTGACAC TTGTTATACT 2820 TCTGTATGTT TGGTCTAAGG TTCTTCTAAA CTGATTATAA TAGCAACACT AGATGTCCCC 2880 TAATGCCACT TTTTGATTTT GTTGCTCTTG GATTTTTTGC GTCTGTTAGA TAGGTTCTGA 2940 CTTTATCTAG TGTAGGGTGA TACTTAAAGC TACAAACTCA TCGAGTGACT GATGTTGATG 3000 ACAACGTTTC TAGGTGTTCG GGGTGCTATG TGAAGCTCAR TATTTCCAGA ATGTCATGCG 3060 TGTTGGTTAC CGACTGAGAT CTGCTCTGGT AAATTTTAAA TTTGCTACCC TGACGTTCTT 3120 CCTTTGCCAT ATGTTTTTGG TGCAGATATG TTTGCTGATA GCATGATTCC CAGTATCTTG 3180 TATAGGAATA AGTATATCAA CATGGTTTCT TTATCCTCTA TATATGATGC ATAAATAAGC 3240 CTTGTGCCAA AAGTTTAGGA ATAAGTTTGT GTTGCTTCAG ATGATTGAGT ATGCTGTTTT 3300 TATTTCTGGA AATTTCCACC ATTTTCAGAT CCTTTCACTA GAGAAATACA AATTTAGCTG 3360 TATTTCCTGA TTCAGTTCAT CGTTTTCTGC GTTTGTAGTG GAGTGAATT AGCTTGTACG 3420 AAATGGAAGA TATTTTGAAC ACAGATGATT TTTAAAATTG GTCTTCCTGT TGATGACTGT 3480 TTTTTTTTTA GATTGCTGCT GTGTTCCGCA AATCGTTGAG GTTAACTAAT GAAGGTCGTA 3540 GAAAGTTTCA AACAGGAAAG ATAACCAACT TAATGACGAC TGATGCCGAA TCTCTTCAGG 3600 TGAGTATCCC TTTCATATTT TCGAATTCAA GTTTGCATGT TTCTCTATAT CATAGTTGCA 3660 GGGCTGTTAA CATCCGGATC TTGAATATTT ATTTTTGTCC GCAGCTGGTA TTGAGTGGGT 3720 TACAGTTACT TTTTATGTTC GGTAATAGAA GTTGGATTTA CTTAGARATG ATTTCCAGCA 3780 TACTGATCTA CTGAATCTGT TTGTTAGGTC TAAGATTGGC TATGAATAGT GATTGCATTT 3840 TCATTTCTAG CTAGCACTTT GTTATCATTG AATTTTTCTT TCTTCTTTTT TATTTTGTTT 3900 CTTATGCCAA CTTAAACTGT GTCTTGTTTA ATGTTTTCGT CTTAACTGTG TCTGGTATCA 3960 ATATTGTTAT CTAATCAACC AGATGTACTT TGTACTRATT TTTCCATTTT CTGTGGCAGC 4020 AAATATGCCA ATCACTTCAT ACCATGTGGT CGGCTCCATT TCGTATAATT ATAGCACTGA 4080 TTCTCCTCTA TCAGCAATTG GGTGTTGCCT CGCTCATTGG TGCATTGTTG TTGGTCCTTA 4140 TGTTCCCTTT ACAGGTACAT GACTTCTAAA TTTCCTCATT TTTTTTCCTT TGTAGCTTAT 4200 TTTTCTCTAT ACTGTTCGCT TGTTCATTCG TACTCCTAAA GGCTACTTCT TCTTCGTCTC 4260 CTGAACTTGT TCTCTGTTTT CTTALAACAG ACTGTTATTA TAAGCAARAT GCAGAAGCTG 4320 ACAAAGGAAG GTCTGCAGCG TACTGACAAG AGAATTGGCC TTATGAATGA AGTTTTAGCT 4380 GCAATGGATA CAGTAAAGTA AGARATTCTA GAACCAATTT TGTTAACATA GTTATTAATT 4440 TGCAGGAAAC TTGTACTAAA CCALAATGCT ACAGGTGTTA TGCTTGGGAA AACAGTTTCC 4500 AGTCCAAGGT CCAAACTGTC GTGATGATGA ATTATCTTGG TTCCGGAAAT CACAGCTCCT 4560 GGGAGCGGTA TGACTACAGC GTAGTTACTT TTGTTTTTCC TCTAATTATT GTATATTTCT 4620 AACTCTTGCT TGGTCTTGTC TTGTTTTGCA GTTGAkTATG TTCATACTGA ATAGCATTCC 4680 TGTTCTTGTG ACTATTGTTT CATTTGGTGT GTTCACATTA CTTGGAGGAG ACCTGACCCC 4740 TGCAAGAGCA TTTACGTCAC TCTCTCTCTT TGCTGTGCTT CGTTTCCCTC TCTTCATGCT 4800 TCCAAACATT ATAACTCAGG TGATTTCTTA AATATGTTGT TGCAATGCAT GTGTATTAAG 4860 TAGAACTGTT AGTGCTTGTA GTAACTGTCG TTTGGTTATC AAATCCATGA CTTATATTTC 4920 GAATTTACAT GCTGGAGGGT ATCCTTGCTG GTGCCAGAAA CAGATGCCGA TGCTGACTAG 4980 TTTTCACTTG TAGGTGGTAA ATGCTAATGT ATCCTTAAAA CGTCTTGAGG AGGTATTGGC 5040 GACAGAAGAA AGAATTCTCT TACCAAATCC TCCCATTGAA CCTGGAGAGC CAGCCATCTC 5100 AATAAGAAAT GGATATTTCT CTTGGGATTC TAAGGTGTCG CTTGGCTATT CTATACCATG 5160 TTCCTTCTTT CGCTTCTCTC ATTACCTTTA TCCATAGAAA GTACAAAAAT CGAGCTAACC 5220 CTATGTATCT ACAGGGGGAT AGGCCGACGT TGTCAAATAT CAACTTGGAT GTACCTCTTG 5280 GCAGCCTAGT TGCTGTGGTT GGTAGTACAG GCGAAGGALk AACCTCTCTA ATATCTGCTA 5340 TCCTTGGTGA ACTTCCTGCA ACATCTGATG CARTAGTTAC TCTCAGAGGA TCAGTTGCTT 5400 ATGTTCCACA AGTTTCATGG ATCTTTAATG CAACAGTATG TTCTTCTTTT CTTTGACTTT 5460 TAAGTTGGGC TGACGTTGCA AATTTTTCTG TTGTACATAA TGTTAAATGT ATTTTCTGTC 5520 TTTTATAGTA GAACAATATG TGTTCTCARA TGCGTCAGTT ACTTCACCAA CTTAGTGGAA 5580 ACCTTCTTCA ATATTTGATT CTCTAAGCTA TTTTGAACAG AAGACTGATA TGCATTTTCT 5640 TATAAAAATT TGTAGGTACG CGACAATATA CTGTTTGGTT CTCCTTTCGA CCGTGAAAAG 5700 TATGAAAGGG CCATTGATGT GACTTCACTG AAGCATGACC TAGAGTTACT GCCTGTAAGT 5760 TTTGAGGAGA GCTTCGTGGA GTTGATRACA AGGATTTGTC TTGCCTGTTC TCGTGTTGCT 5820 AAGTTTGTTT CAACCTCTTT CTCTTGCTTA ATAGGGTGGT GATCTCACGG AGATTGGAGA 5880 AAGAGGTGTT AATATCAGTG GAGGACAGAA GCAGAGGGTT TCCATGGCTA GGGCCGTTTA 5940 CTCAAATTCA GATGTGTACA TCTTTGATGA CCCGTTAAGT GCCCTTGATG CTCATGTTGG 6000 TCAACAGGTA CTAACTCATT GATTCTCTTT GATAAGGCTA GTCTATTTCA TTTTTGAATT 6060 TATCTAACAT TTTTGTGTCT GGTCATTATG GGAATACTGT CAGTCTGATT TCTAGGAATA 6120 TTGTTTCAGG TTTTTGAAAA ATGCATAAAA AGAGAACTGG GGCAGAAAAC GAGAGTTCTT 6180 GTTACAAACC AGCTCCACTT CCTATCACAA GTGGACAGAA TTGTACTTGT GCATGAAGGC 6240 ACAGTGAAAG AGGAAGGAAC ATATGAAGAG CTATCCAGTA ATGGGCCTTT GTTCCAGAGG 6300 GTAATGGAAA ATGCAGGGAA GGTGGAAGAA TATTCAGAAG AAAATGGAGA AGCTGAGGCA 6360 GACCAAACAG CGGAACAACC AGTTGCGAAT GGGAACACAA ATGGTCTTCA AATGGATGGA 6420 AGTGACGATA AAAAATCCAA AGAAGGAAkT AAAAAAGGAG GGAAATCTGT CCTCATCAAG 6480 CAAGAAGAAC GTGAAACCGG AGTTGTARGT TGGAGAGTCC TGAAGAGGTA ACTTGAACAT 6540 TTGGCTTTTG CAATCTTACT ATTTGTTTGC AACTTTCCCC ATACTCGATC CAAGAGGTCC 6600 ATTCATTTGT GGTGTTTCAC AACAAACTAG CATGTTCCTT ATGTTTTTAG GCTGAACTAT 6660 ACCTTTGCGG GATATCAGAA TGACTTTTCC AGGCTTTCAA TGTTTTCAGG TACCAGGATG 6720 CACTTGGAGG GGCATGGGTA GTGATGATGC TCCTTTTATG TTACGTCTTA ACAGAAGTAT 6780 TTCGGGTTAC TAGCAGCACG TGGTTGAGTG AGTGGACTGA TGCAGGAACT CCAAAGAGTC 6840 ATGGACCCCT TTTCTACAAT CTCATATATG CACTTCTCTC GTTTGGACAG GTATGAGTTA 6900 TGTTTGCTTG ATGGATGAGT GAAGATTTGA TATAATCTTG ACCTCATGAT ATARCATATA 6960 TAGCTGAAAC CTGACCAGCT TAGAAAGATC TTATATAATT CTACTTTTGT GATTTTACTT 7020 TGAGAATCCA AAGGTGGAGG TAGAAAAGGT TAGTAAAGAA TTGATTTTTT TGCTGAGACT 7080 CTTTCTTCTT GCTTACAGGT TTTGGTGACA TTGACCAATT CATATTGGTT GATTATGTCC 7140 AGTCTTTATG CAGCTAAGAA GTTACACGAC AATATGCTTC ATTCCATACT GAGGGCCCCG 7200 ATGTCCTTCT TCCATACCAA TCCGCTAGGA CGGATAATCA ATCGATTCGC AAAAGATCTG 7260 GGTGATATTG ATCGAACTGT GGCCGTCTTT GTAAACATGT TTATGGGTCA AGTCTCACAG 7320 CTTCTTTCAA CTGTAGTGTT GATTGGCATT GTAAGCACTT TGTCCTTGTG GGCCATCATG 7380 CCCCTCCTGG TCTTGTTTTA TGGAGCTTAT CTTTATTATC AGGTAATGTA CCTTCTGACC 7440 GCAGCATTTA AATAACTGAG ATTAAGTGAC AGAAAGAGAA AAGGACACAG ATGATGGATG 7500 TTACACATAC TTTTTTAGCC TCATTTGTCA TGTCTGAGTT CGTTTGGTGC TTAAGCTATC 7560 TACACTCATC TGTCACCAAA AATCATGCTG TATATGTTGT GTGTTAAATA TTTTTCTTAT 7620 TGCAGAACAC AGCCCGTGAG GTTAAGCGTA TGGATTCAAT TTCARGATCG CCTGTTTATG 7680 CACAGTTTGG AGAGGCATTG AATGGCTTAT CAACTATCCG TGCTTACAAA GCATATGATC 7740 GTATGGCTGA TATCAACGGA AGATCAATGG ATAATAACAT CAGATTCACT CTTGTCAACA 7800 TGGGTGCCAA TCGGTGGCTT GGAATCCGTT TAGAAACTCT GGGTGGTCTT ATGATATGGC 7860 TGACAGCATC GTTTGCTGTC ATGCAGAATG GAAGAGCGGA GAACCAACAG GCATTTGCAT 7920 CTACAATGGG TTTGCTTCTC AGTTATGCCT TAAATATTAC TAGCTTGTT ACAGGTGTTC 7980 TGAGACTTGC GAGTTTGGCT GAGAATAGTC TAAACGCGGT CGAGTGTTGG CAATTATATA 8040 GAGATTCCGC CAGAGGTCCG CCTGTCATTG AGAACAACCG TCCACCTCCT GGATGGCCAT 8100 CATCTGGATC CATAAAGTTT GAGGATGTTG TTCTCCGTTA CCGCCCTCAG TTACCGCCTG 8160 TGCTTCATGG GGTTTCTTTC TTCATTCATC CAACAGATAA GGTGGGGATT GTTGGAAGGA 8220 CTGGTGCTGG AAAGTCAAGC CTGTTGAATG CATTGTTTAG AATTGTGGAG GTGGAAAAAG 8280 GAAGGATCTT AATCGATGAT TGTGACGTTG GAAAGTTTGG ACTGATGGA CTACGTAAAG 8340 TGCTCGGAAT CATTCCACAG TCACCGGTTC TTTTCTCAGG AACTGTGAGG TTCAATCTTG 8400 ATCCATTTGG TGAACACAAT GATGCTGATC TTTGGGAATC TCTAGAGAGG GCACACTTGA 8460 AGGATACCAT CCGCAGAAAT CCTCTTGGTC TTGATGCTGA GGTATTCAGT TGCTGCCTAT 8520 ATTGATATGA AGTCTCATTT TTTAAGTGGT AATAACTGAT TTTCAATCTT TGTTCAGGTC 8580 TCTGAGGCAG GAGAGAATTT CAGCGTGGGA CAGAGGCAAT TGTTGAGTCT TTCACGTGCG 8640 CTGTTACGGA GATCTAAGAT ACTCGTCCTT GATGAAGCAA CTGCTGCTGT AGATGTTAGA 8700 ACCGATGCCC TCATTCAGAA GACTATCCGA GAAGAATTCA AGTCATGCAC GATGCTCATT 8760 ATCGCTCACC GTCTCAATAC CATCATTGAC TGTGACAAAA TTCTCGTGCT TGATTCTGGA 8820 AGAGTATGAT TTTAAACACT CTCTCTCTTT CAATCTCACA CTCTCCTTGT TTCTCAGCTA 8880 ACCTGTTCTA TTCCAATTTG TTAACTCAGG TTCAAGARTT CAGTTCACCG GAGAACCTTC 8940 TTTCAAATGA AGGAAGCTCT TTCTCCAAGA TGGTTCAAG CACTGGAGCT GCAAATGCTG 9000 AGTACTTGCG TAGTTTAGTA CTCGACAACA AGCGTGCCAA AGATGACTCA CACCACTTAC 9060 AAGGCCAAAG GAAATGGCTG GCTTCTTCTC GCTGGGCTGC AGCCGCTCAG TTTGCTCTGG 9120 CTGCGAGTCT TACTTCGTCG CACAACGATC TTCAAAGCCT TGAAATTGAA GATGACAGCA 9180 GCATTTTGAA GAGAACAAAC GATGCAGTTG TGACTCTGCG CAGTGTTCTC GAGGGGAAAC 9240 ACGACAAAGA GATTGCAGAG TCGCTTGAGG AACATAATAT CTCTAGAGAG GGATGGTTGT 9300 CATCACTCTA TAGAATGGTA GAAGGTAAAC CAAATATGCA TCTCTACAAA TGCTTATGCA 9360 AAATCTTAAT CACCACACTG AAACATTAAA GTCAAATCGT GCTCTTATAT TGCAAGCCTG 9420 CTTTCCGCTG TCTACGTTTC AGGGCTTGCA GTGATGAGCA GATTGGCAAG GAACCGAATG 9480 CAACAACCGG ATTACAATTT CGAAGGAAAT ACATTTGACT GGGACAACGT CGAGATGTAG 9540 ATAAGTTCAT GTTAAACTAG GAATCATTGT CTCTTCCGTA AGAAACATAT ATTTATCTTA 9600 ACCAAAATTA TTAGTTTGGT TTCCATTTCA TAAACTTAAT TTTCACCTGC AAAGAAAATC 9660 AAACCCTGTT GTGTTCTTCG TGATAAGTAG AGAAATTACT TGAGTATCCT TCTAACTCAT 9720 AAATGGGATC TCATGATTCA TGAACAAGCA GCAACACAAT AATACCCTTT TCAGATTTTG 9780 GAGCTGGACA AAGTTGTTAA GTTGAGTTTC TCTTACAGTC ATTCATATAC AAAAACCTCT 9840 TCGACTGAAG CACCAAGAAA GAAACAAACA TCARAAGGGA ATGAGGTCTT TTCTTAGGGC 9900 TGAGATCATC GGAATGTGGG AGTGCGGAAC ACGACC 9936 (2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1621 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: Met Gly Phe Glu Phe Ile Glu Trp Tyr Cys Lys Pro Val Pro Asn Gly 1 5 10 15 Val Trp Thr Lys Thr Val Ala Asn Ala Phe Gly Ala Tyr Thr Pro Cys 20 25 30 Ala Thr Asp Ser Phe Val Leu Gly Ile Ser Gln Leu Val Leu Leu Val 35 40 45 Leu Cys Leu Tyr Arg Ile Trp Leu Ala Leu Lys Asp His Lys Val Glu 50 55 60 Arg Phe Cys Leu Arg Ser Arg Leu Tyr Asn Tyr Phe Leu Ala Leu Leu 65 70 75 80 Ala Ala Tyr Ala Thr Ala Glu Pro Leu Phe Arg Leu Ile Met Gly Ile 85 90 95 Ser Val Leu Asp Phe Asp Gly Pro Gly Leu Pro Pro Phe Glu Ala Phe 100 105 110 Gly Leu Gly Val Lys Ala Phe Ala Trp Gly Ala Val Met Val Met Ile 115 120 125 Leu Met Glu Thr Lys Ile Tyr Ile Arg Glu Leu Arg Trp Tyr Val Arg 130 135 140 Phe Ala Val Ile Tyr Ala Leu Val Gly Asp Met Val Leu Leu Asn Leu 145 150 155 160 Val Leu Ser Val Lys Glu Tyr Tyr Ser Ser Tyr Val Leu Tyr Leu Tyr 165 170 175 Thr Ser Glu Val Gly Ala Gln Val Leu Phe Gly Ile Leu Leu Phe Met 180 185 190 His Leu Pro Asn Leu Asp Thr Tyr Pro Gly Tyr Met Pro Val Arg Ser 195 200 205 Glu Thr Val Asp Asp Tyr Glu Tyr Glu Glu Ile Ser Asp Gly Gln Gln 210 215 220 Ile Cys Pro Glu Lys His Pro Asn Ile Phe Asp Lys Ile Phe Phe Ser 225 230 235 240 Trp Met Asn Pro Leu Met Thr Leu Gly Ser Lys Arg Pro Leu Thr Glu 245 250 255 Lys Asp Val Trp Tyr Leu Asp Thr Trp Asp Gln Thr Glu Thr Leu Phe 260 265 270 Thr Ser Phe Gln His Ser Trp Asp Lys Glu Leu Gln Lys Pro Gln Pro 275 280 285 Trp Leu Leu Arg Ala Leu Asn Asn Ser Leu Gly Gly Arg Phe Trp Trp 290 295 300 Gly Gly Phe Trp Lys Ile Gly Asn Asp Cys Ser Gln Phe Val Gly Pro 305 310 315 320 Leu Leu Leu Asn Gln Leu Leu Lys Ser Met Gln Glu Asp Ala Pro Ala 325 330 335 Trp Met Gly Tyr Ile Tyr Ala Phe Ser Ile Phe Gly Gly Val Val Phe 340 345 350 Gly Val Leu Cys Glu Ala Gln Tyr Phe Gln Asn Val Met Arg Val Gly 355 360 365 Tyr Arg Leu Arg Ser Ala Leu Ile Ala Ala Val Phe Arg Lys Ser Leu 370 375 380 Arg Leu Thr Asn Glu Gly Arg Arg Lys Phe Gln Thr Gly Lys Ile Thr 385 390 395 400 Asn Leu Met Thr Thr Asp Ala Glu Ser Leu Gln Gln Ile Cys Gln Ser 405 410 415 Leu His Thr Met Trp Ser Ala Pro Phe Arg Ile Ile Ile Ala Leu Ile 420 425 430 Leu Leu Tyr Gln Gln Leu Gly Val Ala Ser Leu Ile Gly Ala Leu Leu 435 440 445 Leu Val Leu Met Phe Pro Leu Gln Thr Val Ile Ile Ser Lys Met Gln 450 455 460 Lys Leu Thr Lys Glu Gly Leu Gln Arg Thr Asp Lys Arg Ile Gly Leu 465 470 475 480 Met Asn Glu Val Leu Ala Ala Met Asp Thr Val Lys Cys Tyr Ala Trp 485 490 495 Glu Asn Ser Phe Gln Ser Lys Val Gln Thr Val Arg Asp Asp Glu Leu 500 505 510 Ser Trp Phe Arg Lys Ser Gln Leu Leu Gly Ala Leu Asn Met Phe Ile 515 520 525 Leu Asn Ser Ile Pro Val Leu Val Thr Ile Val Ser Phe Gly Val Phe 530 535 540 Thr Leu Leu Gly Gly Asp Leu Thr Pro Ala Arg Ala Phe Thr Ser Leu 545 550 555 560 Ser Leu Phe Ala Val Leu Arg Phe Pro Leu Phe Met Leu Pro Asn Ile 565 570 575 Ile Thr Gln Val Val Asn Ala Asn Val Ser Leu Lys Arg Leu Glu Glu 580 585 590 Val Leu Ala Thr Glu Glu Arg Ile Leu Leu Pro Asn Pro Pro Ile Glu 595 600 605 Pro Gly Glu Pro Ala Ile Ser Ile Arg Asn Gly Tyr Phe Ser Trp Asp 610 615 620 Ser Lys Gly Asp Arg Pro Thr Leu Ser Asn Ile Asn Leu Asp Val Pro 625 630 635 640 Leu Gly Ser Leu Val Ala Val Val Gly Ser Thr Gly Glu Gly Lys Thr 645 650 655 Ser Leu Ile Ser Ala Ile Leu Gly Glu Leu Pro Ala Thr Ser Asp Ala 660 665 670 Ile Val Thr Leu Arg Gly Ser Val Ala Tyr Val Pro Gln Val Ser Trp 675 680 685 Ile Phe Asn Ala Thr Val Arg Asp Asn Ile Leu Phe Gly Ser Pro Phe 690 695 700 Asp Arg Glu Lys Tyr Glu Arg Ala Ile Asp Val Thr Ser Leu Lys His 705 710 715 720 Asp Leu Glu Leu Leu Pro Gly Gly Asp Leu Thr Glu Ile Gly Glu Arg 725 730 735 Gly Val Asn Ile Ser Gly Gly Gln Lys Gln Arg Val Ser Met Ala Arg 740 745 750 Ala Val Tyr Ser Asn Ser Asp Val Tyr Ile Phe Asp Asp Pro Leu Ser 755 760 765 Ala Leu Asp Ala His Val Gly Gln Gln Val Phe Glu Lys Cys Ile Lys 770 775 780 Arg Glu Leu Gly Gln Lys Thr Arg Val Leu Val Thr Asn Gln Leu His 785 790 795 800 Phe Leu Ser Gln Val Asp Arg Ile Val Leu Val His Glu Gly Thr Val 805 810 815 Lys Glu Glu Gly Thr Tyr Glu Glu Leu Ser Ser Asn Gly Pro Leu Phe 820 825 830 Gln Arg Leu Met Glu Asn Ala Gly Lys Val Glu Glu Tyr Ser Glu Glu 835 840 845 Asn Gly Glu Ala Glu Ala Asp Gln Thr Ala Glu Gln Pro Val Ala Asn 850 855 860 Gly Asn Thr Asn Gly Leu Gln Met Asp Gly Ser Asp Asp Lys Lys Ser 865 870 875 880 Lys Glu Gly Asn Lys Lys Gly Gly Lys Ser Val Leu Ile Lys Gln Glu 885 890 895 Glu Arg Glu Thr Gly Val Val Ser Trp Arg Val Leu Lys Arg Tyr Gln 900 905 910 Asp Ala Leu Gly Gly Ala Trp Val Val Met Met Leu Leu Leu Cys Tyr 915 920 925 Val Leu Thr Glu Val Phe Arg Val Thr Ser Ser Thr Trp Leu Ser Glu 930 935 940 Trp Thr Asp Ala Gly Thr Pro Lys Ser His Gly Pro Leu Phe Tyr Asn 945 950 955 960 Leu Ile Tyr Ala Leu Leu Ser Phe Gly Gln Val Leu Val Thr Leu Thr 965 970 975 Asn Ser Tyr Trp Leu Ile Met Ser Ser Leu Tyr Ala Ala Lys Lys Leu 980 985 990 His Asp Asn Met Leu His Ser Ile Leu Arg Ala Pro Met Ser Phe Phe 995 1000 1005 His Thr Asn Pro Leu Gly Arg Ile Ile Asn Arg Phe Ala Lys Asp Leu 1010 1015 1020 Gly Asp Ile Asp Arg Thr Val Ala Val Phe Val Asn Met Phe Met Gly 1025 1030 1035 1040 Gln Val Ser Gln Leu Leu Ser Thr Val Val Leu Ile Gly Ile Val Ser 1045 1050 1055 Thr Leu Ser Leu Trp Ala Ile Met Pro Leu Leu Val Leu Phe Tyr Gly 1060 1065 1070 Ala Tyr Leu Tyr Tyr Gln Asn Thr Ala Arg Glu Val Lys Arg Met Asp 1075 1080 1085 Ser Ile Ser Arg Ser Pro Val Tyr Ala Gln Phe Gly Glu Ala Leu Asn 1090 1095 1100 Gly Leu Ser Thr Ile Arg Ala Tyr Lys Ala Tyr Asp Arg Met Ala Asp 1105 1110 1115 1120 Ile Asn Gly Arg Ser Met Asp Asn Asn Ile Arg Phe Thr Leu Val Asn 1125 1130 1135 Met Gly Ala Asn Arg Trp Leu Gly Ile Arg Leu Glu Thr Leu Gly Gly 1140 1145 1150 Leu Met Ile Trp Leu Thr Ala Ser Phe Ala Val Met Gln Asn Gly Arg 1155 1160 1165 Ala Glu Asn Gln Gln Ala Phe Ala Ser Thr Met Gly Leu Leu Leu Ser 1170 1175 1180 Tyr Ala Leu Asn Ile Thr Ser Leu Leu Thr Gly Val Leu Arg Leu Ala 1185 1190 1195 1200 Ser Leu Ala Glu Asn Ser Leu Asn Ala Val Glu Arg Val Gly Asn Tyr 1205 1210 1215 Ile Glu Ile Pro Pro Glu Ala Pro Pro Val Ile Glu Asn Asn Arg Pro 1220 1225 1230 Pro Pro Gly Trp Pro Ser Ser Gly Ser Ile Lys Phe Glu Asp Val Val 1235 1240 1245 Leu Arg Tyr Arg Pro Gln Leu Pro Pro Val Leu His Gly Val Ser Phe 1250 1255 1260 Phe Ile His Pro Thr Asp Lys Val Gly Ile Val Gly Arg Thr Gly Ala 1265 1270 1275 1280 Gly Lys Ser Ser Leu Leu Asn Ala Leu Phe Arg Ile Val Glu Val Glu 1285 1290 1295 Glu Gly Arg Ile Leu Ile Asp Asp Cys Asp Val Gly Lys Phe Gly Leu 1300 1305 1310 Met Asp Leu Arg Lys Val Leu Gly Ile Ile Pro Gln Ser Pro Val Leu 1315 1320 1325 Phe Ser Gly Thr Val Arg Phe Asn Leu Asp Pro Phe Gly Glu His Asn 1330 1335 1340 Asp Ala Asp Leu Trp Glu Ser Leu Glu Arg Ala His Leu Lys Asp Thr 1345 1350 1355 1360 Ile Arg Arg Asn Pro Leu Gly Leu Asp Ala Glu Val Ser Glu Ala Gly 1365 1370 1375 Glu Asn Phe Ser Val Gly Gln Arg Gln Leu Leu Ser Leu Ser Arg Ala 1380 1385 1390 Leu Leu Arg Arg Ser Lys Ile Leu Val Leu Asp Glu Ala Thr Ala Ala 1395 1400 1405 Val Asp Val Arg Thr Asp Ala Leu Ile Gln Lys Thr Ile Arg Glu Glu 1410 1415 1420 Phe Lys Ser Cys Thr Met Leu Ile Ile Ala His Arg Leu Asn Thr Ile 1425 1430 1435 1440 Ile Asp Cys Asp Lys Ile Leu Val Leu Asp Ser Gly Arg Val Gln Glu 1445 1450 1455 Phe Ser Ser Pro Glu Asn Leu Leu Ser Asn Glu Gly Ser Ser Phe Ser 1460 1465 1470 Lys Met Val Gln Ser Thr Gly Ala Ala Asn Ala Glu Tyr Leu Arg Ser 1475 1480 1485 Leu Val Leu Asp Asn Lys Arg Ala Lys Asp Asp Ser His His Leu Gln 1490 1495 1500 Gly Gln Arg Lys Trp Ala Ser Ser Arg Trp Ala Ala Ala Ala Gln Phe 1505 1510 1515 1520 Ala Leu Ala Ala Ser Leu Thr Ser Ser His Asn Asp Leu Gln Ser Leu 1525 1530 1535 Glu Ile Glu Asp Asp Ser Ser Ile Leu Lys Arg Thr Asn Asp Ala Val 1540 1545 1550 Val Thr Leu Arg Ser Val Leu Glu Gly Lys His Asp Lys Glu Ala Glu 1555 1560 1565 Ser Leu Glu Glu His Asn Ile Ser Arg Glu Gly Trp Leu Ser Ser Leu 1570 1575 1580 Tyr Arg Met Val Glu Gly Leu Ala Val Met Ser Arg Leu Ala Arg Asn 1585 1590 1595 1600 Arg Met Gln Gln Pro Asp Tyr Asn Phe Glu Gly Asn Thr Phe Asp Trp 1605 1610 1615 Asp Asn Val Glu Met 1620 (2) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 5175 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: GAATTCGCGG CCGCCGGCGA ATTTGCACTC TTTACCTCTC TTTGACTCCG TGAGATTCGA 60 GGATTGTTAG TTTCTTGTGA TGTGTAGTCT TTGAAGCAGG GGATTTTTAT TGTATTGAGG 120 AAGAAGATGG GGTTTGAGCC GTTGGATTGG TATTGCAAGC CGGTGCCGAA TGGTGTGTGG 180 ACTAAAACTG TGGATTATGC GTTTGGTGCA TACACGCCTT GTGCTATTGA CTCTTTTGTG 240 CTTGGTATCT CTCATCTGGT TCTGTTGATT CTGTGTCTTT ATCGCTTGTG GCTCATCACG 300 AAGGATCACA AAGTGGATAA GTTCTGCTTG AGGTCTAAAT GGTTTAGCTA TTTTCTGGCT 360 CTTTTGGCTG CTTATGCTAC TGCGGAGCCT TTGTTTAGAT TGGTCATGAG GATCTCTGTT 420 TTGGATTTGG ATGGAGCTGG GTTTCCTCCC TATGAGGCGT TTATGTTGGT CCTTGAGGCT 480 TTTGCTTGGG GTTCTGCTTT GGTCATGACT GTTGTGGAAA CTAAAACGTA TATCCATGAA 540 CTCCGTTGGT ATGTCAGATT CGCTGTCATT TATGCTCTTG TGGGAGACAT GGTGTTGTTA 600 AATCTTGTTC TCTCTGTTAA GGAGTACTAT GGCAGTTTTA AACTGTATCT TTACATAAGC 660 GAGGTGGCAG TTCAGGTTGC ATTTGGAACC CTCTTGTTTG TGTATTTCCC TAATTTGGAC 720 CCTTACCCTG GTTACACACC AGTTGGGACT GAAAATTCCG AGGATTACGA GTATGAAGAG 780 CTTCCTGGAG GAGAAAATAT ATGTCCTGAG AGGCATGCAA ATTTATTTGA CAGTATCTTC 840 TTCTCATGGT TGAACCCATT GATGACTCTG GGATCAAAAC GACCTCTCAC CGAGAAGGAT 900 GTATGGCATC TGGACACTTG GGATAAAACT GAAACTCTTA TGAGGAGCTT CCAGAAGTCC 960 TGGGATAAGG AACTAGAAAA GCCCAAACCG TGGCTTTTGA GAGCACTGAA CAACAGCCTT 1020 GGGGGAAGGT TTTGGTGGGG TGGCTTTTGG AAGATTGGGA ATGACTGTTC ACAGTTCGTG 1080 GGGCCTCTTC TACTGAATGA GCTCTTAAAG TCAATGCAAC TTAATGAACC AGCGTGGATA 1140 GGTTACATCT ATGCAATCTC AATCTTTGTT GGAGTGGTAT TGGGGGTTTT ATGTGAAGCT 1200 CAGTATTTCC AAAATGTGAT GCGTGTTGGT TACCGGCTTA GGTCTGCACT GATTGCTGCT 1260 GTGTTCCGAA AATCTTTGAG GCTAACTAAT GAGGGGCGGA AGAAGTTTCA AACAGGAAA 1320 ATAACAAACT TAATGACTAC TGATGCTGAG TCGCTGCAGC AAATCTGCCA ATCACTTCAT 1380 ACCATGTGGT CGGCGCCATT TCGTATAATT GTAGCACTGG TTCTCCTCTA TCAACAATTG 1440 GGTGTTGCCT CGATCATTGG TGCATTGTTT CTTGTCCTTA TGTTCCCCAT ACAGACTGTT 1500 ATTATAAGCA AAACGCAGAA GTTAACAAAA GAAGGGTTGC AGCGTACTGA CAAGAGAATT 1560 GGCCTAATGA ATGAGGTTTT AGCGGCAATG GATACAGTGA AGTGTTACGC TTGGGAAAAC 1620 AGTTTTCAGT CCAAGGTTCA AACTGTACGT GATGATGAAT TATCTTGGTT CCGGAAAGCA 1680 CAACTCCTGT CAGCGTTCAA TATGTTCATA CTAAACAGCA TCCCTGTCCT CGTGACTGTT 1740 GTTTCATTTG GTGTGTTCTC ATTGCTTGGA GGAGATCTGA CACCTGCAAG AGCGTTTACG 1800 TCACTCTCTC TATTTTCTGT GCTTCGCTTC CCTTTATTCA TGCTTCCAAA CATTATAACT 1860 CAGATGGTAA ATGCTAATGT ATCCTARACC GTTTGGAGGA GGTACTGTCA ACCGAAGAGA 1920 GAGTTCTCTT ACCGAATCCT CCCATTGAAC CTGGACAGCC AGCTATCTCA ATAAGAAATG 1980 GATACTTCTC CTGGGATTCA AAGGCGGATA GGCCAACATT GTCAAACATC AACCTGGACA 2040 TACCTCTTGG CAGCCTAGTT GCGGTAGTTG GCAGCACAGG AGAAGGAAAFI ACCTCCCTGA 2100 TATCTGCTAT GCTTGGGGAA CTTCCTGCAA GATCTGATGC GACTGTTACT CTTAGAGGAT 2160 CAGTCGCTTA TGTTCCACAA GTTTCATGGA TCTTTAACGC AACAGTACGT GACAATATAT 2220 TGTTTGGGGC TCCTTTTGAC CAAGAARAAT ATGAAAGGGT GATTGATGTG ACAGCACTCC 2280 AGCATGACCT TGAGTTACTG CCTGGAGGTG ACCTCACGGA GATCGGAGAA AGGGGTGTTA 2340 ACATCAGTGG GGGACAAAAG CAGAGGGTTT CTATGGCTAG GGCCGTTTAC TCAAATTCAG 2400 ACGTGTGCAT CTTAGATGAA CCATTGAGTG CCCTTGATGC GCATGTTGGT CAGCAGGTTT 2460 TTGAAAAATG CATAAAAAGG GAACTAGGGC AGACAACGAG AGTACTTGTT ACAAATCAGC 2520 TCCACTTCCT ATCACAAGTG GATAAAATCC TACTTGTCCA TGAGGGAACA GTAAAAGAGG 2580 AAGGAACATA TGAAGAATTA TGCCATAGTG GCCCGTTGTT CCCGAGGTTA ATGGAAAATG 2640 CAGGGAAGGT TGAAGATTAT TCCGAAGAAA ATGGAGAAGC TGAAGTACAT CAAACATCTG 2700 TAAAACCAGT TGAAAATGGG AACGCTAATA ATCTGCAGAA GGATGGAATC GAGACAAAGA 2760 ATTCCAAAGA AGGAAACTCT GTTCTTGTCA AACGAGAAGA ACGTGAAACT GGAGTTGTGA 2820 GTTGGAAAGT CCTGGAGAGG TACCAGAATG CACTTGGAGG TGCATGGGTA GTGATGATGC 2880 TCGTTATATG CTACGTCTTG ACTCAAGTAT TTCGGGTTTC AAGCATCACT TGGTTGAGTG 2940 AGTGGACTGA TTCAGGAACC CCAAAGACTC ATGGACCCCT ATTCTATAAT ATTGTCTATG 3000 CGCTTCTTTC GTTTGGACAG GTCTCTGTGA CATTGATCAA TTCATATTGG TTGATTATGT 3060 CCAGTCTATA TGCAGCTAAA AAGATGCATG ATGCTATGCT TGGTTCCATA CTAAGGGCTC 3120 CAATGGTGTT CTTTCAAACC AATCCATTAG GACGGATAAT CAATCGATTT GCAAAAGATA 3180 TGGGAGATAT TGATCGAACT GTGGCAGTCT TTGTAAACAT GTTTATGGGT TCAATCGCAC 3240 AGCTTCTTTC AACTGTTATC TTGATTGGCA TTGTCAGCAC TCTGTCCCTG TGGGCCATCA 3300 TGCCCCTGTT GGTCGTGTTC TATGGAGCTT ATCTGTATTA CCAGAACACA TCTCGGGAAA 3360 TTAAACGTAT GGATTCCACT ACAAGATCGC CAGTTTATGC TCAATTTGGT GAGGCATTGA 3420 ATGGACTATC TAGTATCCGT GCTTATAAAG CATATGACAG GATGGCTGAL ATTAATGGAA 3480 GGTCAATGGA CAATAACATC AGATTCACAC TTGTAAACAT GGCTGCAAAT CGGTGGCTGG 3540 GAATCCGTTT GGAAGTTTTG GGAGGTCTCA TGGTTTGGTG GACTGCTTCA TTAGCCGTCA 3600 TGCAGAACGG AAAGGCAGCG AACCAACAAG CATATGCATC TACGATGGGT TTGCTTCTCA 3660 GTTATGCGTT AAGCATTACC AGCTCTTTAA CAGCTGTACT GAGACTCGCG AGTCTAGCTG 3720 AGAATAGTTT AAACTCGGTT GAGCGTGTTG GAAATTATAT CGAGATACCA TCAGAGGCTC 3780 CATTGGTCAT TGAAAACAAC CGTCCACCTC CCGGATGGCC ATCATCTGG TCCATAAAAT 3840 TTGAGGATGT TGTTCTTCGT TACCGCCCTG AGTTACCTCC TGTTCTTCAT GGAGTTTCGT 3900 TCTTGATTTC TCCAATGGAT AAGGTGGGAk TTGTTGGGAG GACAGGCGCT GGGAAATCAA 3960 GCCTCTTAAA TGCCTTATTC AGGATTGTGG AGCTGGAAAA AGGAAGGATT TTAATTGATG 4020 AATGCGACAT TGGAAGATTT GGACTGATGG ACCTACGTAA AGTGGTCGGA ATTATACCGC 4080 AAGCGCCAGT TCTTTTCTCA GGTACCGTGA GATTCAATCT TGACCCATTT AGTGAACACA 4140 ACGACGCCGA TCTCTGGGAA TCTCTTGAGA GGGCACACTT GARAGATACT ATCCGCAGAA 4200 ATCCTCTTGG TCTTGATGCT GAGGTAACTG AGGCAGGAGA GAATTTCAGT GTTGGACAGA 4260 GACAGTTGTT GAGTCTTGCA CGTGCATTGT TACGAAGATC TAAGATACTT GTTCTTGATG 4320 AAGCAACTGC TGCAGTTGAC GTAAGAACTG ATGTTCTCAT CCARAAGACC ATCCGAGAAG 4380 AATTCAAGTC ATGCACAATG CTAATCATCG CTCATCGTCT CAATACTATC ATCGACTGTG 4440 ACAAAGTTCT TGTCCTTGAT TCTGGAAAAG TTCAGGAATT CAGTTCACCG GAGAATCTTC 4500 TTTCAAATGG AGAAAGTTCT TTCTCGAAGA TGGTTCARAG TACAGGAACT GCAAACGCGG 4560 AGTACTTACG TAGTATAACA CTAGAGAACA ARCGTACCAG AGAAGCTAAC GGTGATGATT 4620 CACAACCTTT AGAAGGTCAA AGGAAATGGC AAGCTTCTTC TCGTTGGGCT GCAGCTGCTC 4680 AATTTGCATT GGCTGTGAGC CTCACTTCAT CTCACAACGA CCTCCAAAGC CTTGAAATCG 4740 AAGATGATAA CAGTATTTTG AAGAAAACAA AGGACGCCGT CGTCACTTTA CGCAGTGTCC 4800 TTGAAGGGAA ACATGATAAA GAGATTGAAG ACTCTCTAAA CCAAAGTGAC ATCTCTAGAG 4860 AGCGTTGGTG GCCATCTCTT TACAAAATGG TCGAAGGGCT TGCCGTGATG AGCAGATTGG 4920 CGAGGAACAG AATGCAACAC CCGGATTACA ATTTAGAAGG GARATCGTTT GACTGGGACA 4980 ATGTCGAGAT GTAAACGATG AAAGGCTTAC ACTAATAGAC CTAAAACTCC CATTTTGATG 5040 GAACTTTTAT TTGTATTGCT TGGGATACAC GTAACAAAAT GCCCATTAAT CGTGGTGTAA 5100 CTATATAGGC TATGCTTCTT TTGGGAAAAA GAGAGTTTGA TTACAGAGGA TGTGATGATA 5160 ACACAATTGG AATTC 5175 (2) INFORMATION FOR SEQ ID NO:5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 10342 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: GGGAGGTTTG GTTTTTTCCC TATCAATCGA ATTCCATTTC GTGCTCGTAA CGTGGATTTT 60 GGTAGATTTT TTTTAGGGGG ATGGAAACTT GTTTATTATC TATAGATGAT GATTTTGTTT 120 TCTCCATGAG AATGTATGCT TTTAAACTTT TTTTTTTTTG TTTTTTGCCT TCGGAGCTAA 180 CTTTGGGGGC TGGTCTCGGT CTCTGTTTTC TCTCCACTAA AARGATAAAA AGCTTTTGCC 240 ATCTTTTTTT TTTTCTCAAT ARTCTATCAC ATCGTTTTTT TTCTTTGTTT TTTTCTCCAT 300 TTGTCTTCAT TGAGTTCATA GCCACATAAT TATTGATTTC TTTTTCTTTT AGTGTTTCTG 360 TTACTGATGC GTTTCATTAT TTATACTTCT CACTTGCAGA TTCGAGGATT GTTAGTTTCT 420 TGTGATGTGT AGTCTTTGAA GCAGGGGATT TTTATTGTAT TGAGGAAGAA GATGGGGTTT 480 GAGCCGTTGG ATTGGTATTG CAAGCCGGTG CCGAATGGTG TGTGGACTAA AACTGTGGAT 540 TATGCGTTTG GTGCATACAC GCCTTGTGCT ATTGACTCTT TTGTGCTTGG TATCTCTCAT 600 CTGGTTCTGT TGATTCTGTG TCTTTATCGC TTGTGGCTCA TCACGAAGGA TCACAAAGTG 660 GATAAGTTCT GCTTGAGGTC TAARTGGTTT AGCTATTTTC TGGCTCTTTT GGCTGCTTAT 720 GCTACTGCGG AGCCTTTGTT TAGATTGGTC ATGAGGATCT CTGTTTTGGA TTTGGATGGA 780 GCTGGGTTTC CTCCCTATGA GGTGTGTTAT CACTTTGCTG TTTTGTTGAT GTTGTTCTCC 840 TTCTGTATGT TTTTTCCTGA GAGATGCTGT TGTTTTGTGC TTTATTTGGC AGGCGTTTAT 900 GTTGGTCCTT GAGGCTTTTG CTTGGGGTTC TGCTTTGGTC ATGACTGTTG TGGAAACTAA 960 AACGTATATC CATGAACTCC GTTGGTATGT CAGATTCGCT GTCATTTATG CTCTTGTGGG 1020 AGACATGGTG TTGTTAAATC TTGTTCTCTC TGTTAAGGAG TACTATGGCA GGTTGGTAAA 1080 TTTGCAGTCT GTATGGTTTA TGCAATTTTG TTTCCCTGGT CTGGCACGAT GAACTTATAT 1140 GCGTCATTTT TTTTTTGTTT TTGGCAGTTT TAAACTGTAT CTTTACATAz. GCGAGGTGGC 1200 AGTTCAGGTT TGCACTTTAA AACTCCTTTT TGCATTCTCC AAACTACTCT TTACCATGTG 1260 CTGTATCTAA GTCACACTGT AAATGATACA ACTTTGTTTT TATAATGACG TTAAGGATGG 1320 TTTTTGGATC CAGGTTGCAT TTGGAACCCT CTTGTTTGTG TATTTCCCTA ATTTGGACCC 1380 TTACCCTGGT TACACACCAG TTGGGACTGA AAATTCCGAG GATTACGAGT ATGAAGAGCT 1440 TCCTGGAGGA GAAAATATAT GTCCTGAGAG GCATGCAAAT TTATTTGACA GTATGTCACT 1500 CTACACTTCT CATTCCCTAC TTTGTTTTTA TAGGTGCATT TTCTATTTTA ATTGTGAGAA 1560 TTGCCACCGC ATCTTTTATC ACTTTTCTGC ACTTACTACC TATCTAAGTT GGTTATTTAT 1620 GCAGAGCTTA AATATTTCCC TGGAATTGTA AATTTTCTTA TGGAGTGCTA ATACGTAGTA 1680 GGTCATTAAA ATTGTTTCCG CAGAGAGTAG TCTATAGTCT CTTCAAAATT TTTTTTTGAC 1740 TTATCCTCCC GTTCTCCCTA GAAATGAACT TATGATTTGT GACTGTGCCG AGGTTTTTGC 1800 TTAGTGATCA TCACTTCGAC TAAGCTGCAA CATTTTATAT AGTATATTCG TCAACATTTG 1860 TCAAACTTTG ACTATTATGT TCCTTCTTAC CCTTGTCTTT CAACCCACAG GTATCTTCTT 1920 CTCATGGTTG AACCCATTGA TGACTCTGGG ATCAAAACGA CCTCTCACCG AGAAGGATGT 1980 ATGGCATCTG GACACTTGGG ATAAAACTGA AACTCTTATG AGGAGGTATA TTTTAATAAA 2040 TAACAACTGT TCTCATACTG TCTATGACTG GCATGGTTGC GTGACATATT TTTATCTCAT 2100 TTTTTAGCTT CCAGAAGTCC TGGGATAAGG AACTAGAAAA GCCCAAACCG TGGCTTTTGA 2160 GAGCACTGAA CAACAGCCTT GGGGGAAGGT AAACARAAAC TTCTTCACAG TCATGTGTTT 2220 TCATCTTTTT GGGCTTTGAC ATGATGTGTG ATTTGTAAAA GGAAGCATTT GGTTGTAATA 2280 ATAAATGCAT TATGAATAAC TAGAAGCTGA GAAATCTGTT ATGGCTGTGA CTTCAAGTAT 2340 GTTTTGATGC GTGTCGAGTT GAATAAGAAA TGTGTTACTT TTCTGGTTAT AATCTGCCAT 2400 AGATACTTTC CATCCTTATG GACTGTCTGT TTCTGCATTT TGTAGGTTTT GGTGGGGTGG 2460 CTTTTGGAAG GTACTTTTGT ACTCTTTATT GTGTTTTATT CTTTATTCTG AAACAGTCTT 2520 TTCCTTGTCT ATTTGATAAT ATTGATGGCT TCTGAGGTCT TAGTTTTCCT AAATGGTGTG 2580 TTTTGTAACT GTTTAATCTT GACATTTCAA TCTAAATTGT ATCATAGATT GGGAATGACT 2640 GTTCACAGTT CGTGGGGCCT CTTCTACTGA ATGAGCTCTT AAAGGTTTGT TCCTTTACTT 2700 CTTTTTACCC CGTGCACATT GTGCTTGAAC CTATTTAACA C;ATGCTTTG TAATTTTTCC 2760 ATTCACATGG ATCTTTGAGA TGGATTCATA TTCCTACTGG CTCGAATAAG TGTTTAAACG 2820 TTCTTGATAG ATTCAAAATC CTATCATCCT TTGAATATTA TGTTCTGACG ATATCTCACA 2880 ATGTCTCCTT TAACTTTCCG CAGTCAATGC AACTTAATGA ACCAGCGTGG ATAGGTTACA 2940 TCTATGCAAT CTCAATCTTT GTTGGAGTGG TATGCAACAA ATTCTCTTTT TCTTCGCTGC 3000 CTTTATTATT CTCTTGCATG GACTGCAAAG GATATGAAAC ARE2ACTCTA CTTTCCTTGG 3060 ATTCTTTTCT TTCTTGCTAG GACTTCATGG TATTTTTGGT CIAGAGTAG2 TGCTACGAAT 3120 TGTAGGACCA GTTTAATTTT CTTAAGCTGA AAGTAATCTC TGTGCGATTC GATTGTATTA 3180 GAAAATAGCC TGATTCTACT CTTAGAGTTA GTTTTTTTTG TTTGTTAATA CATTTGCATG 3240 TTGAAAAGGT TTTGTTTAAT GTAGGTCAAG GTGACACTTG ACCAATGGAC TCCTTGATCG 3300 CTTGATGTTG ATGTTGACAT TTTCAGGTAT TGGGGGTTTT ATGTGAAGCT CAGTATTTCC 3360 AAAATGTGAT GCGTGTTGGT TACCGGCTTA GGTCTGCACT GGTAAGAAAA AGTTTCACAT 3420 GAATTATCTT TTGCTACTTA GTTTTTCTTT TTGCTCTGCT TCTCATGTTT TGATGCAATA 3480 CCTGTACTGT TATGTCTGTT GAAAGCTATA GCAGATGCTT ATAGATTGCT TCATTCTGCT 3540 GATGAATTCT CCCTTAATAG ATTGCTGCTG TGTTCCGAAA ATCTTTGAGG CTAACTAATG 3600 AGGGGCGGAA GAAGTTTCAA ACAGGAAkAA TAACAAACTT AATGACTACT GATGCTGAGT 3660 CGCTGCAGGT GTATCTTTGT TACCTTTACT CTCTTTAGCC TTGTCTGTTT CTTGATATAA 3720 ATTTACACTG CATAGTTGTA TATCTACCTC AAAATATGAG TCTTAGATGC AATTTACCAA 3780 GATAGTCTTT TTCCTGCAAC TGACGACTGA ATCTGAAGCT TATTCTAAGA TTCTAGAAAT 3840 CCTAAGAGTT GTGATTACAT TTTCAACACC CTTGTTCTTT TGTTGCCGTT GTAGGATTTG 3900 ATTTTCCTTT ATTAGCCAAT AAACCTTTAA TTCGCTTGAT TTGTAGAAAA AAGTTACCTT 3960 TGAACAGTGC TTTTATCTAA GCTCTTGCTT GAAATCAAAG TGTTTATCTA GCTGATAGCT 4020 GTTCTTTTTC CCTAACGTTT CTCTTGTGTG TGACAGCAAA TCTGCCAATC ACTTCATACC 4080 ATGTGGTCGG CGCCATTTCG TATAATTGTA GCACTGGTTC TCCTCTATCA ACAATTGGGT 4140 GTTGCCTCGA TCATTGGTGC ATTGTTTCTT GTCCTTATGT TCCCCATACA GGTTCGTATA 4200 TCTTAATAAT TCCCCATTCT CTTTGCGCTG TCGGTTTTTT TTTCCTTTTG ATTGCTTATT 4260 TCTCATTTGC TTTTCACACC AATGAAAATG ATTCATTTCC TCCGTTTATT TGGTTGAAAC 4320 AGACTGTTAT TATAAGCAAA ACGCAGAAGT TAACAAAAGA AGGGTTGCAG CGTACTGACA 4380 AGAGAATTGG. CCTAATGAAT GAGGTTTTAG CGGCAATGGA TACAGTGAAG TACGATACTT 4440 TGGAAGCCTG AAACCTAATA TTTATTTTCT TGCATAGTTG G;AGTTTGTG GCAGTGTTTA 4500 ACTATCTCAC TAAACCAAAA TACTGTAGGT GTTACGCTTG GGAAAACAGT TTTCAGTCCA 4560 AGGTTCAAAC TGTACGTGAT GATGAATTAT CTTGGTTCCG GA2AGCACAA CTCCTGTCAG 4620 CGGTATGGCT TGAGTGCAGT GACTGTTATA TTAATTGATT TTATAGACCG TATGCATGAT 4680 GTGCATAGTT GTCTTGGTCA TTTACTTGTC GCTCTCCTAA CGGTATGATT GTATACAAGG 4740 ACAAATCCAA GTTGCTCGTC TTTTTAAATG CCTTTGACCA TTTTGAGAAT GGTATCCATC 4800 AATATGTGTT TAGGCATTTT CTGTACTATT TTCTAGTTCA TTGAACATTG ATTCAGTTGT 4860 TTCGGGCATG TGTAGCAGCA TTCATGCATG ATCTTTAACA TATATTGCAT TAATGTTTCT 4920 GACTCATTCT TGGTCTTCTA TTTGCTCTGC AGTTCAATAT GTTCATACTA AACAGCATCC 4980 CTGTCCTCGT GACTGTTGTT TCATTTGGTG TGTTCTCATT GCTTGGAGGA GATCTGACAC 5040 CTGCAAGAGC GTTTACGTCA CTCTCTCTAT TTTCTGTGCT TCGCTTCCCT TTATTCATGC 5100 TTCCAAACAT TATAACTCAG GTGATTTCCT TAAAATGTTT CTTGAACCAT GTTTTCATGT 5160 CCAGTACTGA ATAATGTGGC ATCATAGTAA TGATTGCTTC TGATTGCTCT TTTAATTTTC 5220 CATCTCTACC TCTTTTTCTA GACCAGTCGT TGTCATAATG TTTTTGCAGA TGCTGACCAG 5280 GCTTTACTTT TGTAGATGGT AAATGCTAAT GTATCCTTAA ACCGTTTGGA GGAGGTACTG 5340 TCAACCGAAG AGAGAGTTCT CTTACCGAAT CCTCCCATTG AACCTGGACA GCCAGCTATC 5400 TCAATAAGAA ATGGATACTT CTCCTGGGAT TCAAAGGTCT TCTTTGTCTA TTTTATCACA 5460 TGTTCTTACT TCTATTAGTT TCTATCATTA CATATTGTCA ATGAAGTACA AAAAGTGAGC 5520 TAGAAGTATA CATATGCAGG CGGATAGGCC AACATTGTCA AACATCAACC TGGACATACC 5580 TCTTGGCAGC CTAGTTGCGG TAGTTGGCAG CACAGGAGAA GGAAAAACCT CCCTGATATC 5640 TGCTATGCTT GGGGAACTTC CTGCAAGATC TGATGCGACT GTTACTCTTA GAGGATCAGT 5700 CGCTTATGTT CCACAAGTTT CATGGATCTT TAACGCAACA GTAAGTTTAT ATATGCTACT 5760 CAGTTTATAG TATGGTTCTC AATGCGARAA TGTCAAATTC TCCTCTTGGA TTGTTACTTA 5820 TTTTGTATGT ATTTTATGTT TTGTATATGA TGATGTGTGC TTTTAGATAC GTCCACATGC 5880 TGATGGTTGT AATTAACATC GCGTAGGTAC GTGACAATAT ATTGTTTGGG GCTCCTTTTG 5940 ACCAAGAAAA ATATGAAAGG GTGATTGATG TGACAGCACT CCAGCATGAC CTTGAGTTAC 6000 TGCCTGTAAG TTTTGTGGAG AGTTACTTAG CCATGTGCAT TGAAAATTTC CTGAGGTGAA 6060 ACGAACCTTG AAATCTGTTG GTGCGATGTA AATCGAAAAA ACTGAATTGC ATCAGTTCTG 6120 TTGATAGCAT GTACTTCTAT TTTCTAGTGC TCAGGTATCT AAGCTTGTTT CCTCTTCTTT 6180 CTCTTGATTG ATAGGGAGGT GACCTCACGG AGATCGGAGA AAGGGGTGTT AACATCAGTG 6240 GGGGACAAAA GCAGAGGGTT TCTATGGCTA GGGCCGTTTA CTCAAATTCA GACGTGTGCA 6300 TCTTAGATGA ACCATTGAGT GCCCTTGATG CGCATGTTGG TCAGCAGGTA AACTAGCCAT 6360 AGGCTCTTTT GGATAGAACA ATACTTTGTT TTTCTTTCAA TTTTGCAAAT CGTGAACTCT 6420 ATAACGTTTT GTTTTTCAAT CTGCATGGAT ATTCTACCTTC TTGTTTGCCA CGGATCTCTG 6480 CCATATACTA CTTTTAAGCA AACATTGTTA TCTGATGTTC GA2ACTGGCT GTTATATATA 6540 GGTTTTTGAA AAATGCATAA AAAGGGAACT AGGGCAGACA ACGAGAGTAC TTGTTACAAA 6600 TCAGCTCCAC TTCCTATCAC AAGTGGATAA AATCCTACTT GTCCATGAGG GAACAGTAAA 6660 AGAGGAAGGA ACATATGAAG AATTATGCCA TAGTGGCCCG TTGTTCCCGA GGTTAATGGA 6720 AAATGCAGGG AAGGTTGAAG ATTATTCCGA AGAkAkTGGA GA2GCTGAAG TACATCAAAC 6780 ATCTGTAAAA CGAGTTGAAA ATGGGAACGC TAATAATCTG CAGAAGGATG GAATCGAGAC 6840 AAAGAATTCC AAAGAAGGAA ACTCTGTTCT TGTCAAACGA GAAGAACGTG AAACTGGAGT 6900 TGTGAGTTGG AAAGTCCTGG AGAGGTAAGT TGGCATTCGG ATTTTTGCTC TTTCTTGTTG 6960 TGTTGTTGCA GTATTCCTTT CTATCGACAG TGGAAATATC CGTAAATAAG ACATATTCTT 7020 TGGTTTAGAG CAATATGTCA ATTTATCTGT GGTGTTTCTT TACTACAAA TGGATATATA 7080 TTGTTTGACT CGCTCTATTC ATATTCATAC AAAATGTATA TATATTTTCC GTATTAAGGT 7140 TCGTATTGTA AAGCCATTGT AATAACTTGT GAGGTGT0AC CATGTTCCAG GTACCAGAAT 7200 GCACTTGGAG GTGCATGGGT AGTGATGATG CTCGTTATAT GCTACGTCTT GACTCAAGTA 7260 TTTCGGGTTT CAAGCATCAC TTGGTTGAGT GAGTGGACTG ATTCAGGAAC CCCAAAGACT 7320 CATGGACCCC TATTCTATAA TATTGTCTAT GCGCTTCTTT CGTTTGGACA GGTATGAGTT 7380 GCATTTGGCA AATGTTTGAG TCGGTATCTT CATGATCGGA TAACAATATA TAACTGAACA 7440 TTAAAGGCTG ATCAGTTAAG AATATACACC ATGTTTCTTC TGCGCCAAAG TATCGAGCAA 7500 ACAAAATGGA AAATAAAAGG ATACAGAGAG CAAAACGTTT ATTGCTAACA CGTATTTCTG 7560 CGGGGGTTTG TCAGGTCTCT GTGACATTGA TCAATTCATA TTGGTTGATT ATGTCCAGTC 7620 TATATGCAGC TAAAAAGATG CATGATGCTA TGCTTGGTTC CATACTAAGG GCTCCAATGG 7680 TGTTCTTTCA AACCAATCCA TTAGGACGGA TAATCAATCG ATTTGCAAAA GATATGGGAG 7740 ATATTGATCG AACTGTGGCA GTCTTTGTAA ACATGTTTAT GGGTTCAATC GCACAGCTTC 7800 TTTCAACTGT TATCTTGATT GGCATTGTCA GCACTCTGTC CCTGTGGGCC ATCATGCCCC 7860 TGTTGGTCGT GTTCTATGGA GCTTATCTGT ATTACCAGTG TAACCTACAT ACTTTTTAAA 7920 CGCAATGCTA TCTACATTCA TGACTACAGA TCGAGACATG GAAAACTGAG ACCAAAAGGA 7980 ACACTGATTG TGTCATATCT GTTGTGTCAT AACCTGATTT TTCCTTATTG TAGAACACAT 8040 CTCGGGAAAT TAAACGTATG GATTCCACTA CAAGATCGCC AGTTTATGCT CAATTTGGTG 8100 AGGCATTGAA TGGACTATCT AGTATCCGTG CTTATAPAGC ATATGACAGG ATGGCTGAAA 8160 TTAATGGAAG GTCAATGGAC AATAACATCA GATTCACACT TGTAAACATG GCTGCAAATC 8220 GGTGGCTGGG AATCCGTTTG GAAGTTTTGG GAGGTCTCAT GGTTTGGTGG ACTGCTTCAT 8280 TAGCCGTCAT GCAGAACGGA AAGGCAGCGA ACCAACA2GC ATATGCATCT ACGATGGGTT 8340 TGCTTCTCAG TTATGCGTTA AGCATTACCA GCTCTTTAAC AGCTGTACTG AGACTCGCGA 8400 GTCTAGCTGA GAATAGTTTA AACTCGGTTG AGCGTGTTGG AAATTATATC GAGATACCAT 8460 CAGAGGCTCC ATTGGTCATT GAAAACAACC GTCCACCTCC CGGATGGCC TCATCTGGAT 8520 CCATAAAATT TGAGGATGTT GTTCTTCGTT ACCGCCCTGA GTTACCTCCT GTTCTTCATG 8580 GAGTTTCGTT CTTGATTTCT CCAATGGATA AGGTGGGAAT TGTTGGGAGG ACAGGCGCTG 8640 GGAAATCAAG CCTCTTAAAT GCCTTATTCA GGATTGTGGA GCTGGALARS GGAAGGATTT 8700 TAATTGATGA ATGCGACATT GGAAGATTTG GACTGATGGA CCTACGTAAA GTGGTCGGAA 8760 TTATACCGCA AGCGCCAGTT CTTTTCTCAG GTACCGTGAG ATTCAATCTT GACCCATTTA 8820 GTGAACACAA CGACGCCGAT CTCTGGGAAT CTCTTGAGAG GGCACACTTG AAAGATACTA 8880 TCCGCAGAAA TCCTCTTGGT CTTGATGCTG AGGTACTTAA TTAAATATTT CCATTTGGGA 8940 AAGTCTCATG TATTCAGTAA TAATAACTCA GTCTTTTTGG TCAGGTAACT GAGGCAGGAG 9000 AGAATTTCAG TGTTGGACAG AGACAGTTGT TGAGTCTTGC ACGTGCATTG TTACGAAGAT 9060 CTAAGATACT TGTTCTTGAT GAAGCAACTG CTGCAGTTGA CGTAAGAACT GATGTTCTCA 9120 TCCAAAAGAC CATCCGAGAA GAATTCAAGT CATGCACAAT GCTAATCATC GCTCATCGTC 9180 TCAATACTAT CATCGACTGT GACAAAGTTC TTGTCCTTGA TTCTGGAAAA GTACGTATAC 9240 AAAATATTCG ACCACTACTT GCATCAATTT AATCACTTTT GAGCTAACAT ATATTGAGAT 9300 TCCCAACACC TCAGGTTCAG GAATTCAGTT CACCGGAGAA TCTTCTTTCA AATGGAGAAA 9360 GTTCTTTCTC GAAGATGGTT CA2AGTACAG GAACTGCAAA CGCGGAGTAC TTACGTAGTA 9420 TAACACTAGA GAACAAACGT ACCAGAGAAG CTAACGGTGA TGATTCACAl CCTTTAGAAG 9480 GTCAAAGGAA ATGGCAAGCT TCTTCTCGTT GGGCTGCAGC TGCTCAATTT GCATTGGCTG 9540 TGAGCCTCAC TTCATCTCAC AACGACCTCC AAAGCCTTGA AATCGAAGAT GATAACAGTA 9600 TTTTGAAGAA AACAAAGGAC GCCGTCGTCA CTTTACGCAG TGTCCTTGAA GGGAAACATG 9660 ATAAAGAGAT TGAAGACTCT CTA2ACCAaA GTGACATCTC TAGAGAGCGT TGGTGGCCAT 9720 CTCTTTACAA AATGGTCGAA GGTAACGTTA TTCTTAAGAT TTCTGATACG AGTATACGAC 9780 ATAAAGAATT GTTGAAGTTT CTTGATCTAk TAATTTGTGT ATATACTCTC AGGGCTTGCC 9840 GTGATGAGCA GATTGGCGAG GAACAGAATG CAACACCCGG ATTACAATTT AGAAGGGAAA 9900 TCGTTTGACT GGGACAATGT CGAGATGTAA ACGATGAAAG GCTTACACTA ATAGACCTAA 9960 AACTCCCATT TTGATGGAAC TTTTATTTGT ATTGCTTGGG ATACACGTA2. CAAAATGCCC 10020 ATTAATCGTG GTGTAACTAT ATAGGCTATG CTTCTTTTGG GAAAAAGAGA GTTTGATTAC 10080 AGAGGATGTG ATGATAACAC AATTGGAATT CAAATTTGCA GCAAAATTTG GGAGAAAAAA 10140 AAAAGTCAAT GAGTGCAACA TGCCAACATG GTTTCAACTT CTGGACATGG ACAACCATTG 10200 GACATAATTT CTCTCACAGG ACCATGTTTT GTCATTGACA TTTTGCACAA AAATGTTCTA 10260 TTAAACATAT ATCTATAAAG AATTTGAACA ATTGTTAAAA AAACACTTAA AATATAAATT 10320 GCAATACAAA TTTCCTTTTT TT 10342 (2) INFORMATION FOR SEQ ID NO:6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1622 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: Met Gly Phe Glu Pro Leu Asp Trp Tyr Cys Lys Pro Val Pro Asn Gly 1 5 10 15 Val Trp Thr Lys Thr Val Asp Tyr Ala Phe Gly Ala Tyr Thr Pro Cys 20 25 30 Ala Ile Asp Ser Phe Val Leu Gly Ile Ser His Leu Val Leu Leu Ile 35 40 45 Leu Cys Leu Tyr Arg Leu Trp Leu Ile Thr Lys Asp His Lys Val Asp 50 55 60 Lys Phe Cys Leu Arg Ser Lys Trp Phe Ser Tyr Phe Leu Ala Leu Leu 65 70 75 80 Ala Ala Tyr Ala Thr Ala Glu Pro Leu Phe Arg Leu Val Met Arg Ile 85 90 95 Ser Val Leu Asp Leu Asp Gly Ala Gly Phe Pro Pro Tyr Glu Ala Phe 100 105 110 Met Leu Val Leu Glu Ala Phe Ala Trp Gly Ser Ala Leu Val Met Thr 115 120 125 Val Val Glu Thr Lys Thr Tyr Ile His Glu Leu Arg Trp Tyr Val Arg 130 135 140 Phe Ala Val Ile Tyr Ala Leu Val Gly Asp Met Val Leu Leu Asn Leu 145 150 155 160 Val Leu Ser Val Lys Glu Tyr Tyr Gly Ser Phe Lys Leu Tyr Leu Tyr 165 170 175 Ile Ser Glu Val Ala Val Gln Val Ala Phe Gly Thr Leu Leu Phe Val 180 185 190 Tyr Phe Pro Asn Leu Asp Pro Tyr Pro Gly Tyr Thr Pro Val Gly Thr 195 200 205 Glu Asn Ser Glu Asp Tyr Glu Tyr Glu Glu Leu Pro Gly Gly Glu Asn 210 215 220 Ile Cys Pro Glu Arg His Ala Asn Leu Phe Asp Ser Ile Phe Phe Ser 225 230 235 240 Trp Leu Asn Pro Leu Met Thr Leu Gly Ser Lys Arg Pro Leu Thr Glu 245 250 255 Lys Asp Val Trp His Leu Asp Thr Trp Asp Lys Thr Glu Thr Leu Met 260 265 270 Arg Ser Phe Gln Lys Ser Trp Asp Lys Glu Leu Glu Lys Pro Lys Pro 275 280 285 Trp Leu Leu Arg Ala Leu Asn Asn Ser Leu Gly Gly Arg Phe Trp Trp 290 295 300 Gly Gly Phe Trp Lys Ile Gly Asn Asp Cys Ser Gln Phe Val Gly Pro 305 310 315 320 Leu Leu Leu Asn Glu Leu Leu Lys Ser Met Gln Leu Asn Glu Pro Ala 325 330 335 Trp Ile Gly Tyr Ile Tyr Ala Ile Ser Ile Phe Val Gly Val Val Leu 340 345 350 Gly Val Leu Cys Glu Ala Gln Tyr Phe Gln Asn Val Met Arg Val Gly 355 360 365 Tyr Arg Leu Arg Ser Ala Leu Ile Ala Ala Val Phe Arg Lys Ser Leu 370 375 380 Arg Leu Thr Asn Glu Gly Arg Lys Lys Phe Gln Thr Gly Lys Ile Thr 385 390 395 400 Asn Leu Met Thr Thr Asp Ala Glu Ser Leu Gln Gln Ile Cys Gln Ser 405 410 415 Leu His Thr Met Trp Ser Ala Pro Phe Arg Ile Ile Val Ala Leu Val 420 425 430 Leu Leu Tyr Gln Gln Leu Gly Val Ala Ser Ile Ile Gly Ala Leu Phe 435 440 445 Leu Val Leu Met Phe Pro Ile Gln Thr Val Ile Ile Ser Lys Thr Gln 450 455 460 Lys Leu Thr Lys Glu Gly Leu Gln Arg Thr Asp Lys Arg Ile Gly Leu 465 470 475 480 Met Asn Glu Val Leu Ala Ala Met Asp Thr Val Lys Cys Tyr Ala Trp 485 490 495 Glu Asn Ser Phe Gln Ser Lys Val Gln Thr Val Arg Asp Asp Glu Leu 500 505 510 Ser Trp Phe Arg Lys Ala Gln Leu Leu Ser Ala Phe Asn Met Phe Ile 515 520 525 Leu Asn Ser Ile Pro Val Leu Val Thr Val Val Ser Phe Gly Val Phe 530 535 540 Ser Leu Leu Gly Gly Asp Leu Thr Pro Ala Arg Ala Phe Thr Ser Leu 545 550 555 560 Ser Leu Phe Ser Val Leu Arg Phe Pro Leu Phe Met Leu Pro Asn Ile 565 570 575 Ile Thr Gln Met Val Asn Ala Asn Val Ser Leu Asn Arg Leu Glu Glu 580 585 590 Val Leu Ser Thr Glu Glu Arg Val Leu Leu Pro Asn Pro Pro Ile Glu 595 600 605 Pro Gly Gln Pro Ala Ile Ser Ile Arg Asn Gly Tyr Phe Ser Trp Asp 610 615 620 Ser Lys Ala Asp Arg Pro Thr Leu Ser Asn Ile Asn Leu Asp Ile Pro 625 630 635 640 Leu Gly Ser Leu Val Ala Val Val Gly Ser Thr Gly Glu Gly Lys Thr 645 650 655 Ser Leu Ile Ser Ala Met Leu Gly Glu Leu Pro Ala Arg Ser Asp Ala 660 665 670 Thr Val Thr Leu Arg Gly Ser Val Ala Tyr Val Pro Gln Val Ser Trp 675 680 685 Ile Phe Asn Ala Thr Val Arg Asp Asn Ile Leu Phe Gly Ala Pro Phe 690 695 700 Asp Gln Glu Lys Tyr Glu Arg Val Ile Asp Val Thr Ala Leu Gln His 705 710 715 720 Asp Leu Glu Leu Leu Pro Gly Gly Asp Leu Thr Glu Ile Gly Glu Arg 725 730 735 Gly Val Asn Ile Ser Gly Gly Gln Lys Gln Arg Val Ser Met Ala Arg 740 745 750 Ala Val Tyr Ser Asn Ser Asp Val Cys Ile Leu Asp Glu Pro Leu Ser 755 760 765 Ala Leu Asp Ala His Val Gly Gln Gln Val Phe Glu Lys Cys Ile Lys 770 775 780 Arg Glu Leu Gly Gln Thr Thr Arc Val Leu Val Thr Asn Gln Leu His 785 790 795 800 Phe Leu Ser Gln Val Asp Lys Ile Leu Leu Val His Glu Gly Thr Val 805 810 815 Lys Glu Glu Gly Thr Tyr Glu Glu Leu Cys His Ser Gly Pro Leu Phe 820 825 830 Pro Arg Leu Met Glu Asn Ala Gly Lys Val Glu Asp Tyr Ser Glu Glu 835 840 845 Asn Gly Glu Ala Glu Val His Gln Thr Ser Val Lys Pro Val Glu Asn 850 855 860 Gly Asn Ala Asn Asn Leu Gln Lys Asp Gly Ile Glu Thr Lys Asn Ser 865 870 875 880 Lys Glu Gly Asn Ser Val Leu Val Lys Arg Glu Glu Arg Glu Thr Gly 885 890 895 Val Val Ser Trp Lys Val Leu Glu Arg Tyr Gln Asn Ala Leu Gly Gly 900 905 910 Ala Trp Val Val Met Met Leu Val Ile Cys Tyr Val Leu Thr Gln Val 915 920 925 Phe Arg Val Ser Ser Ile Thr Trp Leu Ser Glu Trp Thr Asp Ser Gly 930 935 940 Thr Pro Lys Thr His Gly Pro Leu Phe Tyr Asn Ile Val Tyr Ala Leu 945 950 955 960 Leu Ser Phe Gly Gln Val Ser Val Thr Leu Ile Asn Ser Tyr Trp Leu 965 970 975 Ile Met Ser Ser Leu Tyr Ala Ala Lys Lys Met His Asp Ala Met Leu 980 985 990 Gly Ser Ile Leu Arg Ala Pro Met Val Phe Phe Gln Thr Asn Pro Leu 995 1000 1005 Gly Arg Ile Ile Asn Arg Phe Ala Lys Asp Met Gly Asp Ile Asp Arg 1010 1015 1020 Thr Val Ala Val Phe Val Asn Met Phe Met Gly Ser Ile Ala Gln Leu 1025 1030 1035 1040 Leu Ser Thr Val Ile Leu Ile Gly Ile Val Ser Thr Leu Ser Leu Trp 1045 1050 1055 Ala Ile Met Pro Leu Leu Val Val Phe Tyr Gly Ala Tl'r Leu Tyr Tyr 1060 1065 1070 Gln Asn Thr Ser Arg Glu Ile Lys Arg Met Asp Ser Thr Thr Arg Ser 1075 1080 1085 Pro Val Tyr Ala Gln Phe Gly Glu Ala Leu Asn Gly Leu Ser Ser Ile 1090 1095 1100 Arg Ala Tyr Lys Ala Tyr Asp Arg Met Ala Glu Ile Asn Gly Arg Ser 1105 1110 1115 1120 Met Asp Asn Asn Ile Arg Phe Thr Leu Val Asn Met Ala Ala Asn Arg 1125 1130 1135 Trp Leu Gly Ile Arg Leu Glu Val Leu Gly Gly Leu Met Val Trp Trp 1140 1145 1150 Thr Ala Ser Leu Ala Val Met Gln Asn Gly Lys Ala Ala Asn Gln Gln 1155 1160 1165 Ala Tyr Ala Ser Thr Met Gly Leu Leu Leu Ser Tyr Aia Leu Ser Ile 1170 1175 1180 Thr Ser Ser Leu Thr Ala Val Leu Arg Leu Ala Ser Leu Ala Glu Asn 1185 1190 1195 1200 Ser Leu Asn Ser Val Glu Arg Val Gly Asn Tyr Ile Glu Ile Pro Ser 1205 1210 1215 Glu Ala Pro Leu Val Ile Glu Asn Asn Arg Pro Pro Pro Gly Trp Pro 1220 1225 1230 Ser Ser Gly Ser Ile Lys Phe Glu Asp Val Val Leu Arg Tyr Arg Pro 1235 1240 1245 Glu Leu Pro Pro Val Leu His Gly Val Ser Phe Leu Ile Ser Pro Met 1250 1255 1260 Asp Lys Val Gly Ile Val Gly Arg Thr Gly Ala Gly Lys Ser Ser Leu 1265 1270 1275 1280 Leu Asn Ala Leu Phe Arg Ile Val Glu Leu Glu Lys Gly Arg Ile Leu 1285 1290 1295 Ile Asp Glu Cys Asp Ile Gly Arg Phe Gly Leu Met Asp Leu Arg Lys 1300 1305 1310 Val Val Gly Ile Ile Pro Gln Ala Pro Val Leu Phe Ser Gly Thr Val 1315 1320 1325 Arg Phe Asn Leu Asp Pro Phe Ser Glu His Asn Asp Ala Asp Leu Trp 1330 1335 1340 Glu Ser Leu Glu Arg Ala His Leu Lys Asp Thr Ile Arg Arg Asn Pro 1345 1350 1355 1360 Leu Gly Leu Asp Ala Glu Val Thr Glu Ala Gly Glu Asn Phe Ser Val 1365 1370 1375 Gly Gln Arg Gln Leu Leu Ser Leu Ala Arg Ala Leu Leu Arg Arg Ser 1380 1385 1390 Lys Ile Leu Val Leu Asp Glu Ala Thr Ala Ala Val Asp Val Arg Thr 1395 1400 1405 Asp Val Leu Ile Gln Lys Thr Ile Arg Glu Glu Phe Lys Ser Cys Thr 1410 1415 1420 Met Leu Ile Ile Ala His Arg Leu Asn Thr Ile Ile Asp Cys Asp Lys 1425 1430 1435 1440 Val Leu Val Leu Asp Ser Gly Lys Val Gln Glu Phe Ser Ser Pro Glu 1445 1450 1455 Asn Leu Leu Ser Asn Gly Glu Ser Ser Phe Ser Lys Met Val Gln Ser 1460 1465 1470 Thr Gly Thr Ala Asn Ala Glu Tyr Leu Arg Ser Ile Thr Leu Glu Asn 1475 1480 1485 Lys Arg Thr Arg Glu Ala Asn Gly Asp Asp Ser Gln Pro Leu Glu Gly 1490 1495 1500 Gln Arg Lys Trp Gln Ala Ser Ser Arg Trp Ala Ala Ala Ala Gln Phe 1505 1510 1515 1520 Ala Leu Ala Val Ser Leu Thr Ser Ser His Asn Asp Leu Gln Ser Leu 1525 1530 1535 Glu Ile Glu Asp Asp Asn Ser. Ile Leu Lys Lys Thr Lys Asp Ala Val 1540 1545 1550 Val Thr Leu Arg Ser Val Leu Glu Gly Lys His Asp Lys Glu Ile Glu 1555 1560 1565 Asp Ser Leu Asn Gln Ser Asp Ile Ser Arg Glu Arg Trp Trp Pro Ser 1570 1575 1580 Leu Tyr Lys Met Val Glu Gly Leu Ala Val Met Ser Ag Leu Ala Arg 1585 1590 1595 1600 Asn Arg Met Gln His Pro Asp Tyr Asn Leu Glu Gly L's Ser Phe Asp 1605 1610 1615 Trp Asp Asn Val Glu Met 1620 (2) INFORMATION FOR SEQ ID NO:7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1251 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: TTCACTTTTG TCCTTTTTTT CTTAACATCT ACTTTTGTCA TCAGCAAATT ATCTGTAAAT 60 AAGATAGGGT TTATGCTTAT TGCTACAATG AACCTAATCC TATGATGTGT ATTGCAATTT 120 GCAACCATGC GAGTTTAATT ATTTGTTTAC TGCTATAGTG ATCATTTTAT GATGTGTTTT 180 TATTAATTAC AAAACAGAGC ATCAAAAATC AAAAGAACAT ATCGCATAAT CGAACTATGC 240 TAATACCTCT CCTCAATCTT TGTTGTTGTT ATATTCAAGT AGCTTATTCT TTTGTTTTAT 300 TTTACGATTA GATTTCTCTA GAATTTAATT TATATTATTT AATCATACTT GATCAAGGTT 360 TGTAGCTTAA TCAATATCGT TATCGTGTCA TCCTGCAGAT TCAAATGATC AAGTCTAATA 420 ATCTACTTAT ATGTATTATA TATATTAGAT ACCACCAACG AAACAAAATC ATATTTCTAT 480 AACATTTGTT TGGTTAAATA TATTTAAAGA TTTGTAACAG TTGTTCGGGT TCAAAACTAT 540 CACTTTGTAG TTGTAGGATG AGGAAAAGTC GTGATATGAT CATCTACTAA AATCATGTGT 600 TTTTTAAAGA ACATGATTTT CATTGGATAG TTTAATAAAT GTTAAAAAAA TACTAAGTGT 660 CAAAGAAGAG ATTTGAACCA TATGTAGAAT ACTTGATTCG AATTTTTCCT GACGAATAAT 720 CTAATATCCT TTTCTCAAAA GAAAAAAATG TTTGTTAACT TGGACACGAT ATTATTATCC 780 AACTTCCTTT CTAGATATTC ATTTTTAAAT TACCTATATA TTTTTATTTT CTCAAAATAT 840 ACTAAAAATT GGATAGAGCT ATTAAATAAA AAAGATAGAA TTTAGAGAGA AATAGCAACA 900 TAATGAATTA TAATATAAAT ATTTTGTAAA GAAATAACAA ACTTTATAGT TAGTTTGCCT 960 AATATAGAAA AAAGATACAG TTATTTACCC ATTTGTTTGT GTGTAAAAAA AGGAGTAAAA 1020 TAAACAGAGA AAAGAGCTTC TTGTTTTTAC TTGTGAACGT TATTGACTTT TCGGCCTCTC 1080 TCTCTTCTCT ATACAAkTAT ATGGATCTTC ATTTCTTCGT ATAGTGTAAG CAGTGACGCA 1140 TCCATTTATC ATCATCTCCT TAT.thATCTC GAATCTGCCA CAGAGAGAGC GTGTGACAAA 1200 ATGAGTTCAT AAGATTCCGT TATCGTCTTC CTGATTCCTC CAAATCTCCG G 1251 (2) INFORMATION FOR SEQ ID NO:8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1368 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: AAACAATTGG TGTATTTTGA ATTTTTCATG CAACGCACGT GAACAGCTTA ATTGCTTGAT 60 TGGAAACAAA CCTTTTTAGA ATTCATTAAT CAGTTTTAGG TGTTTTGGAA AATTAACGAA 120 CTATAGTGGA GATTmkTTAA TTTTATATTA GTCTTTTTTA GTACACAAAT CGAAGTTTCC 180 TAGATTTTTT CAAAGTTGAA AATAATATTG ATAATATTTA TCAACAATGA ATCTACAAAA 240 ACATAATTTT TTTGCCAAAC AAATAACACC GAAACAAGAT TCATTCACTA TTTTTGGTTT 300 AAAAAAAAAA ATCAAAATTA CACTATTATG AAGCCAATTT TTGTATGCAA AAAACCTGTA 360 TGTATCAATT TGTTTGTATT ALRAAGTAAG CATTTATGTC TTTTTTTTAT AAATAATAGA 420 AACACTTACT AGATGAATAG ATTTTTTGGT TTTAGAACAG AATACTATAA TTGTATTTAT 480 ATAGCTTTTT TATATTATTC GATATAGAAA AGTGTTATAA TAGGAAAAAT GTACCATATA 540 CTGTCAATAA CATATTTGAT TCTAAATATA ARTAGAATTG TTTTAAAGAA ATATGATCGT 600 TTATAATTAA ATGGTTTTTA ATGTCTTTTC TTGGGGCAAA AAACAAAGCT TGTCTTTCGT 660 CCATATATTT GCATCGTL.G GGGTGACGTA TCACTCTCTC TTTCTCTCAA ATATTATTCT 720 TCAATCTCTT TTTGGGGAAT CTTCGAGCAA ATTAGTGAGA GAACCCACCC ACTTTCTTTC 780 TCATATGAGT ACATAkGATC CCTTTTGAGT TTTCGTGTTT TGCCAAAATC TCCAGGTAAA 840 GCTTCTCCCT TTTTCTCTGT TTTCTCTGTT TTGTTATTCT CCCTTTTCTC CATTGTAGCT 900 TTTTCCTGTA AAGTGGGATT GATAGTTTTG TTTCATGGAT TTCAAATTTG TGTTATTTGA 960 CTCGATACCA TCTTAAATGC AGAGTCTTTT CGTGATAATA AAATTATGGA TTCGTTTCAA 1020 AGTTTTTTTT TTTTCGTATG GAAAACACTT GAGCTCTCTC AATCTTGTAG TCTTGACTCT 1080 TGATGATTCT TCTATGTTCT CGTTGTGATT GCTTGTCACT GTTCTATCTT TATATATGAT 1140 TAAATGCAAT TTTGCCCCTT TTTACGCGCG AATGTATTTA TTATCTTTCG CACTCTGGGT 1200 CCATTTCTTG TCACTTGAGC ACATAATGAT TGATTTATGA CTTTTTAAAG TTATGAAAAT 1260 TTATTATTTT TGTTGCTATG GTTTTTTGGA ATTAGAAGCT CATTTCAAAG TTGTTGATTT 1320 TCTTTGCAGG GTAGGGAATT GGTGTGGTAG CTTGTGATGC ACTGTGTT 1368 (2) INFORMATION FOR SEQ ID NO:9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 6 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: Ala Cys Asp Glu Phe Gly 1 5 (2) INFORMATION FOR SEQ ID NO:10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 6 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: Ala Cys Asp His Ile Lys 1 5 (2) INFORMATION FOR SEQ ID NO:ll: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 12 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:ll: GTGACAATAT GC 12 (2) INFORMATION FOR SEQ ID NO:12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 54 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: GTTTCACAGT TTALAGCGTA GTCTGGGACG TCGTATGGGT AATTTTCATT GACC 54 (2) INFORMATION FOR SEQ ID NO:13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: AAACTGCAGA TGGCTGGTAA TCTTGTTTC 29 (2) INFORMATION FOR SEQ ID NO:14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 50 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: GCCTCTAGAT CAAGCGTAGT CTGGGACGTC GTATGGGTAA TTTTCATTGA 50 (2) INFORMATION FOR SEQ ID NO:15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: AGATTAAGCC ATGCATGTCT 20 (2) INFORMATION FOR SEQ ID NO:16: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: TGCTGGTACC AGACTTGCCC TCC 23 (2) INFORMATION FOR SEQ ID NO:17: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: GAAAGTGGAT GTGGGACGGG C 21 (2) INFORMATION FOR SEQ ID NO:18: (i) SEQUENCE z NCE CHARACTERISTICS: (A) LENGTH: 17 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: TCCATATGTT TACTGGC 17 (2) INFORMATION FOR SEQ ID NO:19: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: AAACCGGTGC GGCCGCCATG GGGTTTGAGC CGT 33 (2) INFORMATION FOR SEQ ID NO:20: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: AATTAACCCT CACTAAAGGG 20