GENES ENCODING A FAMILY OF POTASSIUM CHANNELS

Abstract: This invention relates generally to the potassium channel gene family. More particularly, the present invention relates to the cloning and characterization of potassium channel genes from Drosophila melanogaster and Caenorhabditis elegans. Other aspects of the present invention include methods of assaying substances to determine effects on cell growth. Also presented are methods of controlling nematode and insect pests by inhibiting potassium channels substantially homologous to those encoded by nucleotide sequences as described herein.

Description: 32 , 421-01 GENES ENCODING A FAMILY OF POTASSIUM CHANNELS Field of Invention This invention relates generally to the potassium channel gene family. More particularly, the present invention relates to the cloning and characterization of potassium channel genes from Drosophila melanogaster and Caenorhabditis elegans . Background of the Invention Synthetic organic insecticides are primarily nerve poisons acting on the cholinergic system (organophosphorus compounds and methylcarbamates) , the voltage-gated sodium channel (pyrethroids and DDT) , and the GABA-gated chloride channel (cyclodienes and other polychlorocycloalkanes) . Potassium channels comprise a large and diverse group of integral membrane proteins that determine the level of excitability and repolarization properties of neurons and muscle fibers [B. Hille, Ionic Channels of Excitable Membranes, Sinauer, Sunderland, MA (1984)]. The multiple essential functions encoded by the potassium channels make them excellent targets for new pesticides and animal and human therapeutics. Potassium channel diversity in the fruitfly Drosophila melanogaster results from an extended gene family coding for homologous proteins. Six genes encoding potassium channels have been cloned from Drosophila melanogaster which account for a large part of the diversity of potassium currents observed in insect nervous tissue [A. Wei, M. Covarrubias, A. Butler, K. Baker, M. Pak, L. Salkoff, Science 248, 599-603 (1990) , N.S. Atkinson, G.A. Robertson, B. Ganetzky, Science 253,551-555, (1991) , J. War ke, R. Drysdale, B. Ganetzky, Science 252, 1560-1564 (1991) , A. Bruggemann, L.A. Pardo, W. Stuhmer, 0. Pongs, Nature 365, 445-448 (1993)] . ShaJcer and Shal encode voltage- gated potassium channels with rapid current activation and inactivating properties. Shab and Shaw encode delayed rectifier channels, with slow inactivating (Shah) and non-inactivating (Shav) properties. Slo encodes a calcium-activated potassium channel and eag encodes a voltage-gated channel permeable to both potassium and calcium which is modulated by cyclic AMP. Modulation of cardiac action potential by compounds that effect the behavior of potassium channels may be a useful treatment for serious heart conditions. In this regard, each of the potassium channels cloned from insects have corresponding versions in mammalian species, including, specifically, a delayed rectifier potassium channel homolog, RAK, cloned from rat cardiac tissue [M. Paulmichl, P. Νasmith, R. Hellmiss, K. Reed, W.A. Boyle, J.M. Νerbonne, E.G. Peralta, D.E. Clapham, Proc . Natl . Acad. Sci USA 88, 7892-7895 (1991)] . Thus, the RAK channel represents an important target of new drugs for the control of heart failure. The delayed rectifier potassium current in heart cells regulates the duration of the plateau of the cardiac action potential by countering the depolarizing, inward calcium current. Delayed rectifier potassium currents characteristically are activated upon depolarization from rest, display a sigmoidal or delayed onset, and have a nonlinear, or rectifying, current-voltage relation. Several types of delayed potassium conductances have been identified in cardiac cells based on measured single-channel conductances. Heart rate and contractility are regulated by second messenger modification of delayed rectifier potassium conductances, and species differences in the shape of the plateau may be influenced by the type and level of channel expression. On the basis of predicted membrane spanning topology, potassium channels may be subdivided into two distinct classes: voltage-gated, calcium- activated, and cyclic nucleotide-g ted potassium channels that are composed of six membrane spanning domains (S1-S6) and a single pore forming domain (H5) , and inward rectifying potassium channels that pass through the membrane twice and also contain a single pore forming region [Y. Kubo, E. Reuveny, P.A. Slesinger, Y.N. Jan, L.Y. Jan Nature 364, 802-806 (1993); Y. Kubo, T.J. Baldwin, Y.Ν. Jan, L.Y. Jan Nature 362, 127-133 (1993)]. Here, we report the cloning and functional expression in yeast of a novel Drosophila melanogaster potassium channel. Further, we identify a Caenorhabditis elegans homolog that constitutes the second member of a new family of potassium channels exhibiting a topological configuration unique among the known classes of potassium channels. The yeast Saccharomyces cerevisiae is utilized as a model eukaryotic organism for the purpose of studying potassium transport mechanisms. Due to the ease with which one can manipulate the genetic constitution of the yeast Saccharomyces cerevisiae, researchers have developed a detailed understanding of many complex biological pathways, including potassium transport. In yeast, high affinity potassium uptake is performed by the product of the BK1 gene [R.F. Gaber, CA. Styles, G.R. Fink Mol . Cell . Biol . 8, 2848-2859 (1988)]. Mutant yeast strains lacking trkl function are incapable of growing in medium lacking high concentrations of potassium. Since potassium transport mechanisms are present in organisms as divergent as yeast and man, one could predict that expression of heterologous potassium channels in mutant cells might replace trkl function, and support growth on medium containing low potassium concentration. In this regard, plant potassium channels were shown to function in yeast and represent important targets for new herbicides [J.A Anderson, S.S. Huprikar, L.V. Kochian, W.J. Lucas, R.F. Gaber, Proc. Natl . Acad. Sci USA 89, 3736-3740 (1992); H. Sentenac, N. Bonnaud, M. Minet, F. Lacroute, J.-M. Salmon, F. Gaynard, C. Grignon, Science 256, 663-665 (1992); D.P. Schachtman and J.I. Schroeder, Nature 370, 655-658]. Thus, we have employed this yeast expression system for cloning and expression of potassium channels from heterologous species, making it useful for discovery of new pesticides, and animal and human therapeutics. Discovery of such compounds will necessarily require screening assays of high specificity and throughput. For example, new pesticides directed at potassium channels require high selectivity for insect channels and low activity against non-insect species. Screening assays utilizing yeast strains genetically modified to accommodate functional expression of heterologous potassium channels offer significant advantages in this area. Summary of the Invention A first aspect of the present invention is the discovery of a new subclass of potassium channel genes and proteins encoded thereby. Potassium channels belonging to this new subclass comprise four hydrophobic domains capable of forming transmembrane helices, wherein a first pore-forming domain is interposed between the first and second transmembrane helices and a second pore-forming domain is interposed between the third and fourth transmembrane helices, and wherein each pore-forming domain contains a potassium selective peptide motif. In preferred embodiments, the peptide motif is selected from the group consisting of a Y/F-G dipeptide motif. In certain preferred embodiments, the isolation and characterization of invertebrate (i.e. insect and nematode) potassium channel genes belonging to this new subclass is presented. In more preferred embodiments, the present invention provides for the isolation of complementary DMA fragments from Drosophila melanogaster and Caenorhabditis elegans which encode conserved amino acid sequence elements unique to this potassium channel gene family. A yeast expression technology is employed to clone cDNAs from Drosophila melanogaster and C. elegans and a hybridization approach is utilized to isolate additional cDMAs from Caenorhabditis elegans . A second aspect of the present invention is a method of assaying substances to determine effects on cell growth. Yeast cells of the kind described above are cultured in appropriate growth medium to cause expression of heterologous proteins, embedded in agar growth medium, and exposed to chemical compounds applied to the surface of the agar plates. Effects on the growth of embedded cells are found around compounds that have effects on the heterologous potassium channel. A third aspect of the present invention is a method of controlling nematode and insect pests by inhibiting potassium channels substantially homologous to those encoded by nucleotide sequences as presented herein. Brief Description of the Drawings FIGURE 1. Growth of CY162 cells bearing pDmORFl. CY162 cells transformed with plasmids isolated from survivors of a primary library screen for plasmids that support the growth of CY162 on medium contain low potassium concentration. Six individual transformants of each plasmid-bearing strain are cultured in patches on the indicated medium. CY162 cells bearing pDmORFl are found in the upper left-hand corner of each plate while pKATl containing cells are found in the lower right hand corner. FIGURE 2A and 2B. DNA sequence and deduced amino acid sequence of Dm ORFl [SEQ ID NOS: 1 and 2] . The nucleotide sequence of the 2.4 kb cDNA revealed a single long open reading frame proximal to the GAL1 promoter. Segments corresponding to putative transmembrane (M1-M4) and pore-forming H5 domains in the predicted polypeptide are underlined. The single amino-terminal asparagine linked glycosylation site is indicated by a G. FIGURE 3A and 3B. DNA sequence and deduced amino acid sequence of the F22b7.7 segment of the Caenorhabditis elegans genome [SEQ ID NO:3] . Segments corresponding to putative transmembrane (M1-M4) and pore-forming H5 domains in the predicted polypeptide are underlined. FIGURE 4. Alignment of DmORFl and F22b7.7 sequences. Protein-coding regions of DmORFl [SEQ ID NO: 37] and F22b7.7 [SEQ ID NO: 38] (designated as CeORF-1 in this FIGURE) are compared using the protein sequence alignment algorithm in Genework DNA sequence analysis software. Identical amino acids are boxed. FIGURE 5A. Comparison of the pore-forming domains of DmORFl and F22b7.7. Amino acid sequences from the six cloned Drosophila melanogaster potassium channels and three inward rectifier channels [SEQ ID NOS:7 through 21] are compared to DmORFl and F22b7.7 within the pore-forming H5 regions. Amino acid identities are indicated by a vertical line and conserved substitutions indicated by a dot. Amino acid substitutions deemed acceptable are indicated. FIGURE 5B. Hydropathy plot analysis of the DmORFl and F22b7.7 polypeptide sequence. The Kyte-Doolittle hydropathy algorithm in the Geneworks DNA analysis software is used to predict the topology of DmORFl and F22b7.7. The position of predicted membrane spanning domains (M1-M4) and pore-forming domains are indicated. FIGURE 6. Predicted membrane spanning topology of DmORFl . FIGURE 7. Heterologous potassium channel-dependent growth of plasmid bearing CY162 ( trklΔ) strains. CY162 bearing pYES2, pKATl, pDmORFl, and pRATRAK are cultured at 30°C for four days on arginine phosphate agar medium containing 0 mM, 0.2 mM, or 100 mM added KC1. FIGURE 8. Inhibition of growth of yeast cells containing heterologous potassium channels. CY162 cells (10 5 ) bearing the indicated plasmids are plated in arginine phosphate agar medium containing 0.2 mM potassium chloride. Sterile filter disks were placed on the surface of the agar and saturated with 20 ml of a 1 M solution of potassium channel blocking compound. Clockwise from upper left-hand corner is BaCl 2 , CsCl, TEA, and RbCl. KCl is applied to the center disk. FIGURE 9A and 9B. DNA sequence and deduced amino acid sequence of CORK [SEQ ID NO: 36] . The nucleotide sequence of the 1.4 kb cDNA revealed a single long open reading frame proximal to the GAL1 promoter. Segments corresponding to pore-forming H5 domains in the predicted polypeptide are underlined. Asparagine- linked glycosylation sites are indicated by a G. Detailed Description of the Invention Nucleotide bases are abbreviated herein as follows : Ade;A-Adenine G-Guanine Ura; U-Uracil C-Cytosine T-Thymine Amino acid residues are abbreviated herein to either three letters or a single letter as follows: Ala A-Alanine Leu;L-Leucine Arg R-Arginine Lys;K-Lysine Asn N-Asparagine Met;M-Methionine Asp D-Aspartic acid Phe;F-Phenylalanine Cys C-Cysteine Pro;P-Proline Gin Q-Glutamine Ser;S-Serine Glu E-Glutamic acid Thr;T-Threonine Gly G-Glycine Trp;W-Tryptophan His H-Histidine Tyr;Y-Tyrosine He I-Isoleucine Val;V-Valine The term "mammalian" as used herein refers to any mammalian species (e.g., human, mouse, rat, and monkey). The term "heterologous" as used herein refers to DNA sequences, proteins, and other materials originating from organisms other than the organism used in the expression of the potassium channels or portions thereof, or described herein (e.g., mammalian, avian, amphibian, insect, plant), or combinations thereof not naturally found in yeast. The terms "upstream" and "downstream" are used herein to refer to the direction of transcription and translation, with a sequence being transcribed or translated prior to another sequence being referred to as "upstream" of the latter. The potassium channels of the present invention possess properties in common with known potassium channels including, voltage-gated channels, calcium activated channels, cyclic nucleotide gated channels, inward rectifier channels, and the like, and especially with regard to electrophysiological properties. Certain preferred channels exhibit inward and outward currents that are affected by potassium concentration, particularly characteristic of voltage-gated channels. The term "channel" and the nucleotide sequences encoding same, is intended to encompass subtypes of the aforementioned classes of channels, and mutants, derivatives and homologs thereof. The nucleotide sequences encoding the potassium channels or parts thereof may be expressed recombinantly, and utilized for a variety of reasons, the most notable of which is for screening of substances that modulate the activity of the potassium ion channels. Such substances, especially inhibitors of the activity of the potassium channels of the present invention, may be utilized as insecticides, antihelmenthics, drugs suitable for the control of heart failure, and the like. Heterologous DNA sequences are typically expressed in a host by means of an expression vector. An expression vector is a replicable DNA construct in which a DNA sequence encoding the heterologous DNA sequence is operably linked to suitable control sequences capable of affecting the expression of a protein or protein subunit coded for by the heterologous DNA sequence in the intended host. Generally, control sequences include a transcriptional promoter, an optional operator sequence to control transcription, a sequence encoding suitable mRNA ribosomal binding sites, and (optionally) sequences which control the termination of transcription and translation. Vectors useful for practicing the present invention include plasmids, viruses (including bacteriophage) , and integratable DNA fragments (i.e., fragments integratable into the host genome by genetic recombination) . The vector may replicate and function independently of the host genome, as in the case of a plasmid, or may integrate into the genome itself, as in the case of an integratable DNA fragment. Suitable vectors will contain replicon and control sequences which are derived from species compatible with the intended expression host. For example, a promoter operable in a host cell is one which binds the RNA polymerase of that cell, and a ribosomal binding site operable in a host cell is one which binds the endogenous ribosomes of that cell. DNA regions are operably associated when they are functionally related to each other. For example, a promoter is operably linked to a coding sequence if it controls the transcription of the sequence; a ribosome binding site is operably linked to a coding sequence if it is positioned so as to permit translation. Generally, operably linked means contiguous and, in the case of leader sequences, contiguous and in reading phase. Transformed host cells of the present invention are cells which have been transformed or transfected with the vectors constructed using recombinant DNA techniques and express the protein or protein subunit coded for by the heterologous DNA sequences. In preferred embodiments, the transformed host cells are yeast. A variety of yeast cultures, and suitable expression vectors for transforming yeast cells, are known. See e.g., U.S. Patent No. 4,745,057; U.S. Patent No. 4,797,359; U.S. Patent No. 4,615,974; U.S. Patent No. 4,880,734; U.S. Patent No. 4,711,844; and U.S. Patent No. 4,865,989. Saccharomyces cerevisiae is the most commonly used among the yeasts, although a number of other yeast species are commonly available. See. e.g., U.S. Patent No. 4,806,472 (Kluveromyces lactis and expression vectors therefore) ; 4,855,231 (Pichia pastoris and expression vectors therefore) . A heterologous potassium channel may permit a yeast strain unable to grow in medium containing low potassium concentration to survive [CY162, for example, see J.A Anderson, S.S. Huprikar, L.V. Kochian, W.J. Lucas, R.F. Gaber, Proc. Natl . Acad. Sci USA 89, 3736-3740 (1992)]. Yeast vectors may contain an origin of replication from the endogenous 2 micron (2μ) yeast plasmid or an autonomously replicating sequence (ARS) which confer on the plasmid the ability to replicate at high copy number in the yeast cell, centromeric (CEN) sequences which limit the ability of the plasmid to replicate at only low copy number in the yeast cell, a promoter, DNA encoding the heterologous DNA sequences, sequences for poly-adenylation and transcription termination, and a selectable marker gene. An exemplary plasmid is YRp7, (Stinchcomb et al., (1979) Nature 282, 39; Kingsman et al. , (1979) Gene 7, 141; Tschemper et al., (1980) Gene 10, 157] . This plasmid contains the TRP1 gene, which provides a selectable marker for a mutant strain of yeast lacking the ability to grow in the absence tryptophan, for example ATCC No. 44076. The presence of the trpl lesion in the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan. Suitable promoting sequences in yeast vectors include the promoters for metallothionein (YEp52) , 3- phosphoglycerate kinase [pPGKH, Hitzeman et al. , (1980) J. Biol . Chem. 255, 2073] or other glycolytic enzymes [pYSK153, Hess et al. , (1968) J. Adv. Enzyme Reg. 7, 149] ; and Holland et al. , (1978) Biochemistry 17, 4900], such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phospho-fructokinase, glucose-6- phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, trioseposphate isomerase, phosphoglucose isomerase, and glucokinase. Suitable vectors and promoters for use in yeast expression are further described in R. Hitzeman et al . , EPO Publn. No. 73,657. Other promoters, which have the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2 (pAD4M) , isocytochrome C, acid phosphates, degradative enzymes associated with nitrogen metabolism, and the aforementioned metallothionein and glyceraldehyde-3- phosphate dehydrogenase, as well as enzymes responsible for maltose and galactose (pYES2) utilization. Finally, in constructing suitable expression plasmids, the termination sequences associated with these genes may also be ligated into the expression vector 3' of the heterologous coding sequences to provide polyadenylation and termination of the mRNA. In one embodiment of the present invention, a yeast expression system is described, wherein yeast cells bear heterologous potassium channels. In preferred embodiments, these channels are DmORF-1, CORK, or RAK. As noted above, transformed host cells of the present invention express the proteins or proteins subunit coded for by the heterologous DNA sequences. When expressed, the potassium channel is located in the host cell membrane (i.e., physically positioned therein in proper orientation for both the stereoselective binding of ligands and passage of potassium ions) . In certain preferred screening embodiments of the present invention, a transformed yeast cell is presented, containing a heterologous DNA sequence which codes for a rat cardiac delayed rectifier potassium channel, RAK, cloned into a suitable expression vector. RAK is capable of complementing the potassium-dependent phenotvpe of Saccharomyces cerevisiae strain CY162 on medium containing low potassium concentration. The potassium channel subclass of the present invention is characterized in that the potassium channels have four hydrophobic domains capable of forming transmembrane helices. These channels are further characterized in that they comprise two pore-forming domains, one of which is interposed between said first helix and said second helix, and the other of which is interposed between said third helix and said fourth helix. The pore-forming domains further contain a potassium selective motif which serves to confer upon the channel the ability to pass potassium ions to the exclusion of other ions, such as sodium, calcium, and the like. In certain preferred embodiments, this motif contains the peptide Y/G, and particularly in either a dipeptide or tripeptide motif, and frequently with Y/F-G bonding. In most preferred embodiments, the motif is selected from the group consisting of G-V-G, G-L-G, G-Y-G, G-F-G, and G-I-G. In certain embodiments of the present invention, the potassium channel is positioned within a cell membrane in such a manner as to allow it to function as a modulator of the flow of potassium ions into and out of the cell. To best regulate this activity, at least one pore-forming domain may be positioned proximal to a exterior portion of the cell membrane. In other embodiments, the potassium channels of the present invention further comprise an amino-terminal glycosylation site, and especially wherein that site is asparagine-linked. Potassium channels belonging to the subclass as presented herein may be derived from a wide variety of animal species, both vertebrate and invertebrate. Using the yeast expression technology and other teachings as set forth herein, the present inventors have isolated a single 2463 base pair cDNA fragment from an invertebrate source, designated Dm ORFl [SEQ ID NO: 1] , by complementation of the potassium-dependent phenotype of Saccharomyces cerevisiae strain CY162 ( trklΔ) on medium containing low potassium concentration [J.A Anderson, S.S. Huprikar, L.V. Kochian, W.J. Lucas, R.F. Gaber, Proc . Natl . Acad. Sci USA 89, 3736- 3740 (1992) ] . Dm ORFl contains a single long open reading frame encoding a protein of 618 amino acids [SEQ ID NO:2] that exhibits substantial amino acid identity to the pore- forming regions of other potassium channels. The DmORFl contains structural features that distinguish it from other classes of potassium channels, including four hydrophobic domains capable of forming transmembrane helices (M1-M4) and two putative pore forming H5 domains found between transmembrane helices Ml and M2, and M3 and M4. Each pore forming H5 domain contains the Y/F-G dipeptide motif required for potassium selectivity [L. Heginbotham, T. Abramson, R. MacKinnon, Science 258, 1152-1155, (1992)]. This work was expanded to clone a construct derived from C. elegans having a single open reading frame sufficient to encode a protein of 434 amino acids, designated pCORK. A search of the GENBANK database for DNA and protein sequences similar to DmORFl revealed several cloned potassium channel sequences including a putative protein coding DNA sequence, F22b7.7, reported in the Caenorhabditis elegans genome sequencing project [R. Wilson, R. Ainscough, K Anderson, et al. Nature 368, 32-38 (1994)]. The DΝA sequence contained a single long open reading frame sufficient to encode a protein of 336 amino acids (predicted MW 38.5 kDa) with substantial homoiogy to known potassium channel sequences. Using the hybridization approach, a cDΝA sequence designated CeORFl [SEQ ID NO: 38] was isolated by probing a Caenorhabditis elegans cDNA library with oligonucleotides designed using F22b7.7 DNA sequences [T.N. Davis and J. Thorner Meth . Enzymol . 139, 246-262 (1987)]. CeORFl contains a single long open reading frame encoding a protein that exhibits substantial amino acid identity to pore- forming regions of other potassium channels. CeORFl and pCORK each contain structural features similar to DmORFl, including two putative pore forming H5 domains. Each pore forming H5 domain contains the Y/F-G dipeptide motif required for potassium selectivity [L. Heginbotham, T. Abramson, R. MacKinnon, Science 258, 1152- 1155, (1992)]. These features form the basis of the designation of a new sub-family of potassium channels comprising DmORFl, CORK, and CeORFl. Other aspects of the present invention relate to methods of modulating potassium channel activity, by affecting the ability of such channel to allow the flow of ions into, through, or out of a cellular membrane, and particularly when these ions are potassium ions. Certain substances whether biological or chemical in nature, may be applied to cell membranes having as an integral part of their structure, one or more potassium channels comprising the amino acid sequences of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 36, or RAK, in an amount and for a time sufficient to affect the ability of the potassium channel to so regulate the flow of ions. Substances that are potassium channel blockers will inhibit the ability of the channel to regulate the flow of such ions. Substances that enhance such ability may be considered potassium channel "activators." Substances that modulate the activity of RAK may do so by modulation of cardiac action potential, upward or downward. Application of such substances may take the form of in vitro, ex vivo, or in vivo application, each in a formulation suitable to deliver the substance to the cell membrane and to sustain such delivery for a time sufficient to allow the substance to interact with the membrane. Appropriate formulations, concentrations of substances, application time, and other relevant parameters may be established by utilizing, inter alia , known assays for measuring ion channel current flow. Another suitable endpoint one skilled in the art may utilize in optimizing these parameters, especially in the case of potassium channel blockers, is "cell death". Such assays may be performed in vitro and extrapolated to in vivo conditions, or in some cases may be easily established directly in vivo , as for example, by applying the substance directly to a test sample comprising the target insect pest (whole organism) and noting the appropriate parameters at which an acceptable per cent of insect death is attained. In certain preferred embodiments, methods of selectively inhibiting insect pests are presented by applying to such insect pests a substance capable of selectively inhibiting the activity of a potassium channel contained in the cells of such insect, and comprising the amino acid sequence of SEQ ID NO:2, or a potassium channel substantially homologous thereto. In the most preferred embodiments, the inhibitor will inhibit the activity of the aforementioned potassium channel without inhibition of other, non-homologous potassium channels that may be present in species other than the targeted insect pest. It is envisioned that such other species may also be present at the site of application of the inhibitor, such as in a garden, crop, or other site wherein it is desired to control insect pests. In other preferred embodiments, methods of selectively inhibiting nematode pests are presented much in the same manner as discussed for control of insect pests, by applying to such pests a substance capable of selectively inhibiting the activity of a potassium channel contained in the cells of such pest, and comprising the the amino acid sequence of SEQ ID NO:4 or SEQ ID NO: 36, or potassium channels substantially homologous thereto. The following Examples are provided to further illustrate various aspects of the present invention. They are not to be construed as limiting the invention. Example 1 Recombinant expression library screening. Saccharomyces cerevisiae strain CY162 is described in Anderson, J.A. et al. (1992) Proc. Natl . Adad. Sci . USA 89, 3736-3740] . Growth of bacterial strains and plasmid manipulations are performed by standard methods (Maniatis T. , Molecular Cloning. Cold Spring Harbor Laboratory Press, 1982) . Media conditions for growth of yeast, isolation of plasmid DNA from yeast, and DNA-mediated transformation of yeast strains are as described (Rose M. D. , Methods in yeast genetics, Cold Spring Harbor Laboratory Press, 1990) . A multifunctional expression library constructed in pYES2 and containing cDNA made from 3rd instar male Drosophila melanogaster mRNA is used as described [S.J. Elledge, J.T. Mulligan, S.W. Ramer, M. Spottswood, R.W. Davis Proc. Natl . Acad. Sci USA 88, 1731-1735 (1991)]. A multifunctional expression library constructed in pYES2 and containing cDNA made from mRNA obtained from all life stages of Caenorhabditis elegans is custom-made by Invitrogen Corporation. Isolation of expression plasmids encoding heterologous potassium channels. CY162 cells are transformed with plasmid DNA from each library to give 3 x 10 6 transformants from each library on SCD-ura (synthetic complete dextrose (2 %) medium containing all necessary nutritional supplements except uracil) containing 0.1 M KC1 agar medium. Transformants are replica-plated to SCG-ura (synthetic complete galactose (2 %) medium containing all necessary nutritional supplements except uracil) agar medium. Colonies that grow on this selective agar medium are transferred to SCG-ura agar medium to obtain single colonies clones and while reassaying suppression of the potassium- dependent phenotype. Plasmid DNA is isolated from surviving colonies and used to transform CY162. Six individual transformant strains containing one plasmid, pDmORFl , hat confers the potassium independent phenotype is cultured on SCD-ura and SCG-ura medium along with CY162 strains bearing pKATl, which encodes a plant inward rectifier potassium channel that supports the growth of CY162 on selective medium (FIGURE 1) . The plasmid bearing strains exhibit potassium-independent growth on both dextrose and galactose containing medium. Growth on dextrose is likely due to basal level of transcription leading to sufficient potassium channel expression to support growth. Example 2 DNA sequence analysis of DmORFl. Plasmids that confer suppression of the potassium-dependent phenotype are subjected to automated DNA sequence analysis performed by high temperature cycle sequencing (Applied Biosystems) . Geneworks DNA sequence analysis software (Intelligenetics) is used to align raw DNA sequence information and to identify open reading frames. The DNA sequence of the 2.4 kb insert in pDmORFl is displayed in FIGURE 2A and 2B [SEQ ID NO:l]. The 5' untranslated sequences of the cDNA contain long poly A and poly T tracts not likely to be found in protein coding regions. The first ATG proximal to the 5' end is present in a consensus Drosophila melanogaster translational initiation site [D.R. Cavener Nucleic Acids Res . , 15, 1353-1361 (1987)], consistent with the designation of this site as the translational start site. A single long open reading frame sufficient to encode a protein of 618 amino acids (predicted MW 68 kDa) is encoded in pDmORFl. A consensus polyadenylation site, AATCAA, occurs at position 2093-2098 in 3' untranslated sequences. The DmORFl contains structural features that distinguish it from other classes of potassium channels, including four hydrophobic domains capable of forming transmembrane helices (M1-M4) and two pore forming H5 domains found between transmembrane helices Ml and M2, and M3 and M4. Each pore forming H5 domain contains the Y/F-G dipeptide motif required for potassium selectivity [L. Heginbotham, T. Abramson, R. MacKinnon, Science 258, 1152-1155, (1992)]. Example 3 Identification of Caenorhabditis elegans sequences homologous to DmORFl. A search of the GENBANK database protein sequences similar to DmORFl reveals significant matches with several known potassium channel sequences. The closest match is to a putative protein coding DNA sequence, F22b7.7, reported in the Caenorhabditis elegans genome sequencing project [R. Wilson, R. Ainscough, K. Anderson, et al., Nature 368, 32-38 (1994)]. The DΝA sequence and predicted amino acid sequence assembled from putative exons recognized by a GEΝBAΝK exon identification algorithm is displayed in FIGURE 3A and 3B [SEQ ID ΝOS:3 and 4]. The DΝA sequence contains a single long open reading frame sufficient to encode a protein of 336 amino acids (predicted MW 38.5 kDa) with substantial homoiogy to known potassium channel sequences. The F22b7.7 sequence contains structural features that distinguish it from other classes of potassium channels, including three of four hydrophobic domains capable of forming transmembrane helices (M1-M4) identified in DmORFl and two pore forming H5 domains found between transmembrane helices a predicted Ml and M2, and M3 and M4. Each pore forming H5 domain contains the Y/F-G dipeptide motif required for potassium selectivity [L. Heginbotham, T. Abramson, R. MacKinnon, Science 258, 1152-1155, (1992)]. The lack of an amino terminal transmembrane domain homologous to DmORFl Ml in the F22b7.7 sequence may be due to failure of the search algorithm to identify exon(s) encoding the amino terminus. Alternatively, an amino terminal coding sequence may be added by trans-splicing, which occurs frequently in Caenorhabditis elegans . Example 4 Cloning and DNA sequence analysis of CeORFl. Oligonucleotides corresponding to DNA sequences encoding the two pore forming domains of F22b7.7 are synthesized using an Applied Biosystems DNA synthesizer. F22b7.7-H2-1: 5'TCCATTTTCTTTGCCGTAACCGTCGTCACTACCATCGGATACGGTAATCCA [SEQ ID NO:5]. F22b7.7-H2-2: 5'TCATTCTACTGGTCCTTCATTACAATGACTACTGTCGGGTTTGGCGACTTG [SEQ ID NO:6]. The oligos were labelled at their 5' ends with 32 P using a 5'-end labelling kit according to manufacturers instructions (New England Nuclear) . The labelled oligos are pooled and used to screen 6 x 10 5 plaques from a λZAP- Caenorhabditis elegans cDNA library (obtained from Clontech) by published methods [T.N. Davis and J. Thorner Meth . Enzymol . 139, 246-262 (1987)]. Hybridization is at 42°C for 16 hours. Positive clones are plaque-purified by twice repeating the hybridization screening process. Plasmid DNAs, excised from phage DNA according to manufacturers instructions, are subjected to automated DNA sequence analysis performed by high temperature cycle sequencing (Applied Biosystems). Geneworks DNA sequence analysis software (Intelligenetics) is used to align raw DNA sequence data and to identify open reading frames. Example 5 Comparison of the putative proteins encoded by DmORFl and F22b7.7. Predicted amino acid sequences of DmORFl and F22b7.7 are aligned and displayed in FIGURE 4 [SEQ ID NOS:37 and 38] . Only limited overall amino acid homoiogy is exhibited by these two proteins with regions of greatest homoiogy existing in the pore forming H2-1 and H2-2 domains. FIGURE 5A shows a comparison of the pore forming domains of DmORFl and F22b7.7 with those of the known Drosophila melanogaster potassium channel and inward rectifier sequences [SEQ ID NOS:7 through 21]. Amino acid identities greater than 50 % are observed with all potassium channel sequences. FIGURE 5B shows hydropathy plot analysis of DmORFl and F22b7.7. The two proteins, which show remarkable topological similiarity through their length, are predicted to be composed of four membrane-spanning hydrophobic domains (M1-M4) , and two pore forming H2 domains. These data suggest the predicted topology shown in FIGURE 6. Both proteins are predicted to span the membrane four times with amino and carboxyl termini residing within the cell. This topology places the single amino-terminal asparagine-linked glycosylation site and H2 domains on the cell exterior permitting permeation of the membrane by the pore forming domains from the outside, an absolute requirement for the formation of a functional potassium channel. Example 6 Functional expression of a rat atrial delayed rectifier potassium channel in yeast. CY162 transformants containing plasmids pKATl, which encodes a plant inward rectifier potassium channel, pRATRAK, which encodes a rat atrial delayed rectifier potassium channel, pDmORFl, and control plasmid pYES are cultured on arginine-phosphate-dextrose agar medium lacking ura medium [A. Rodriguez-Navarro and J. Ramos, J. Bacteriol . 159, 940-945, (1984)] containing various KC1 concentrations (FIGURE 7) . Strains containing pKATl, pRATRAK, and pDmORFl all support the growth of CY162 on medium containing a low concentration of potassium, while pYES2 containing CY162 cells only grow on medium containing a high potassium concentration, indicating that heterologous potassium channels of several different types function to provide high affinity potassium uptake. pRATRAK is constructed by modifying the protein-coding sequences of RATRAK to add 5' Hindlll and 3' Xbal sites using PCR. In addition, four A residues are added to the sequences immediately 5' proximal to the initiator ATG to provide a good yeast translational initiation site. The modified fragment is cloned into the Hindlll and Xbal sites in the yeast expression vector pYES2 (Invitrogen) , forming pRATRAK. Example 7 Bioassay of functional expression of heterologous potassium channels Yeast strains dependent on heterologous potassium channels for growth should be sensitive to non-specific potassium channel blocking compounds. To test the potassium channel blocking properties of several compounds, a convenient agar plate bioassay is employed. Strains containing pKATl, pRATRAK, pDmORFl, and pYES2 are plated in arginine-phosphate-dextrose agar medium lacking ura and containing various amounts of potassium chloride. Arginine- phosphate-dextrose medium is used to avoid interference from potassium and ammonium ions present in standard synthetic yeast culture medium. Sterile filter disks were placed on the surface of the agar and saturated with potassium channel blocking ions CsCl, BaCl 2 , and TEA. The growth of heterologous potassium channel containing strains is inhibited by potassium channel blocking ions, in a channel dependent manner. DmORFl-dependent growth is blocked by BaCl 2 but not by CsCl or TEA. KAT-dependent growth is blocked by BaCl 2 , CsCl and TEA. RATRAK-dependent growth is blocked by BaCl 2 , CsCl and TEA to a much greater extent than pKATl, reflecting in part a slower growth rate of pRATRAK- containing cells. These observations confirm that these channels support the growth of the mutant yeast cells and demonstrate the efficacy of the yeast bioassay for screening for compounds that block potassium channel function. The control pYES-containing strain grows only around applied KCl and RbCl, a congener of KCl. Example 8 Identification of compounds that alter potassium channel activity Yeast strains made capable of growing on medium containing low potassium concentration by expression of heterologous potassium channels are used to screen libraries of chemical compounds of diverse structure for those that interfere with channel function. CY162 cells containing pKATl, pRATRAK, pDmORFl, pCeORFl, and pYES2-_PRKl (lOVml) are plated in 200 ml of arginine-phosphate-dextrose agar medium lacking ura and containing 0.2 mM potassium chloride in 500 cm 2 plates. The CY162 cells bearing pYES2- RK1 are included in the assay as a control to identify compounds that have non-specific effects on the yeast strain and are therefore not specifically active against the heterologous potassium channels. Samples of chemical compounds of diverse structure (2 μl of 10 mg/ml solution in DMSO) are applied to the surface of the hardened agar medium in a 24 x 24 array. The plates are incubated for 2 days at 30°C during which time the applied compounds radially diffuse into the agar medium. The effects of applied compounds on strains bearing heterologous potassium channel genes are compared to the pYES2-_PR 2 bearing strain. Compounds that cause a zone of growth inhibition around the point of application that is larger on plates containing cells bearing the heterologous potassium channels than that observed around the pYES2-_P l bearing strains are considered selective potassium channel blockers. Compounds that induce a zone of enhanced growth around the point of application that is larger on plates containing cells bearing the heterologous potassium channels than that observed around the pYES2-_TRKl bearing strains are considered selective potassium channel openers. Example 9 DmORFl-induced currents in X. laevis oocytes assayed by two- electrode voltage clamp DNA sequence analysis of the pDmORF insert strongly suggest that the protein encoded by the single long ORF possesses properties in common with known potassium channels. To test this hypothesis, the electrophysiological properties of the putative potassium channel encoded by DmORF was examined by expression in X. laevis oocytes. Currents were measured by two-electrode whole-cell voltage clamp. DNA sequences encoding the open reading frame of DmORFl were amplified by polymerase chain reaction (PCR) using the following oligonucleotides: MP023: ATAAAGCTTAAAAATGTCGCCGAATCGATGGAT [SEQ ID NO:22] MP024: AGCTCTAGACCTCCATCTGGAAGCCCATGT [SEQ ID NO:23] The full length PCR product was cloned into corresponding sites in pSP64 poly A (Promega), forming pMPl47. Template DNA was linearized with EcoRI and RNA transcribed using the Message Machine (Ambion) in vitro transcription kit according to manufacturers instructions. A sample of the RNA was resolved in a MOPS-acetate-formaldehyde agarose gel and RNA content was estimated by ethidium bromide staining. The remainder was stored on dry ice. X. laevis oocytes were isolated and injected with 50 nl of sterile TE containing 5- 20 ng transcript according to published procedures. After three days, whole oocyte currents were recorded using a two- electrode voltage clamp. Electrodes contained 3M KCl and had resistances of 0.3-1.0 MQ. Recordings were performed with constant perfusion at room temperature in the presence of either low (10 mM) or high (90 mM) potassium chloride. Two electrode voltage clamp analysis of the DmORFl gene product expressed in X. laevis oocytes demonstrates properties of a voltage- and potassium-dependent potassium channel. At low potassium concentrations, DmORFl exhibited outward current at depolarizing potentials. At high potassium concentration, DmORFl exhibits both inward and outward currents. The DmORFl channel displays a high preference for potassium and shows cation selectivity in the rank order K>Rb>NH 4 >Cs>Na>Li. Potassium currents were greatly attenuated by BaCl 2 . Example 10 Developmental regulation of DmORFl expression in D. melanogaster determined by northern blotting analysis Isolation of pDmORFl from a D. melanogaster expression library strongly suggests that the insert contained within originated in mRNA from that species. Detailed understanding of the developmental regulation of DmORFl expression should aid in determining strategies for use of DmORFl as a target for novel insecticides. To characterize DmORFl expression, northern blotting analysis of poly A RNA from various stages of the D. melanogaster life cycle was carried out. D. melanogaster poly A + RNA from embryo, larvae and adult forms (Invitrogen, 5 μg) was resolved in a MOPS- acetate-formaldehyde agarose gel according to standard procedures. The gel was stained with ethidium bromide and photographed to mark the positions of 18 S and 28 S ribosomal RNAs used as molecular weight markers. RNA was transferred by capillary action to nitrocellulose with 10 x SSPE. The blot was air-dried, baked for one hour at 80°C, and prehybridized in 4x SSPE, 1% SDS, 2x Denhardt's, 0.1 % single stranded DNA at 68 °C for 2 hours. A 2.4 kb Xhol fragment of DmORFl was isolated from pDmORFl and labeled with o- 32 P dCTP using the Ready-to-Go kit (Pharmacia) according to manufacturers instructions. The probe was denatured by heating to 100°C for 5 minutes followed by quenching in an ice water bath. The probe was added to the prehybridization solution and hybridization continued for 24 hours at 68 °C. The blot was washed briefly with 2x SSPE, 0.1% SDS at room temperature followed by 0.5 x SSPE, 0.1 % SDS at 65 °C for 2 hours. The blot was air-dried and exposed to Reflection X-ray film (NEN) using an intensifying screen at -70 °C for 48 hours. Northern blotting analysis indicates that the DmORFl probe hybridizes to an mRNA species of approximately 2.8 kb isolated from D. melanogaster embryo, larvae, and adult forms. The length of the DmORFl mRNA corresponds well with the length of the predicted ORF. Thus, the DmORF is expressed at all developmental stages in the life cycle of D. melanogaster. Example 11 Expression of the DmORFl gene product in vitro. DNA sequence analysis of the pDmORFl insert reveals a single long ORF with conserved amino acid sequence domains in common with known potassium channels. The DNA sequence predicts an ORF sufficient to encode a protein of 618 amino acid in length. The DmORFl polypeptide contains four segments of at least 20 hydrophobic amino acids in length suggesting that the segments span the plasma membrane. In addition, the DmORFl protein sequence contains a putative N- linked glycosylation site (Asn-Thr-Thr) at amino acids 58- 60. To confirm that a protein of the predicted size of DmORF is expressed from the insert in pDmORFl and to test the proposition that DmORFl is glycosylated, pDmORFl was used as template to drive coupled in vitro transcription/translation. Plasmid pMP147 was used as template to produce 35 S- labeled DmORF gene product in vitro using a TnT coupled transcription-translation kit (Promega) according to manufacturers instructions. Glycosylation of the nascent DmORFl polypeptide was accomplished by addition of canine pancreatic microsomes (Promega) to the transcription- translation reaction. Samples of glycosylated DmORF protein were treated with endoglycosidase H to remove added carbohydrate moieties. Aliquots were precipitated with TCA and collected on GF/C filters, washed with ethanol, dried and counted. Equivalent cpm's were resolved by SDS-PAGE. The gel was impregnated with soluble fluor Amplify (Amersham) and dried onto Whatman 3MM paper. The dried gel was exposed to Reflection X-ray film at room temperature. Translation of the DmORFl gene product in vitro produced a polypeptide of 68 kDa, consistent with the predicted molecular weight of the ORF. Translation of DmORFl in the presence of canine pancreatic microsomes results in synthesis of a protein with reduced electrophoretic mobility, consistent with glycosylation of the nascent polypeptide. Treatment of glycosylated DmORF with EndoH increased its relative mobility as expected upon removal of carbohydrate moieties. Thus, the pDmORFl insert is capable of directing the expression of a glycoprotein with the expected molecular weight. EndoH treatment removes carbohydrate residues consistent with the sugar added through N-linked glycosylation. Example 12 High-affinity K* uptake and selectivity of DmORFl expressed in yeast. Expression of DmORF permits CY162 cells to grow on medium containing a low concentration of potassium, implying that DmORFl supplies high affinity potassium uptake capacity. To characterize the potassium uptake properties of CY162 cells containing DmORFl, 86 Rb uptake studies were performed. Examination of the uptake of this potassium congener revealed important aspects of potassium uptake by DmORFl. Yeast strains containing heterologous potassium- expression plasmids CYl62-DmORFl, CY162-pKAT and the control strain CY162-pYES2 (Clontech) were cultured overnight in SC Gal-ura containing 0.1 M KCl. The cells were harvested, washed with sterile doubled distilled water and starved for K + for 6 hours in Ca-MES buffer. Cells were washed again and distributed to culture tubes (10 β cells/tube) containing 86 RbCl in Ca-MES buffer. The tubes were incubated at room temperature, samples filtered at various time intervals and counted. 86 Rb uptake into cells was displayed. For Double Reciprocal analysis, 86 Rb was held constant and barium ions varied to determine Ki values. The high-affinity potassium uptake capacity encoded by DmORFl permits high-affinity uptake of the potassium congener, 86 Rb, as well. Barium inhibited B6 Rb uptake with a Ki of μM as demonstrated in Double Reciprocal analysis. No high affinity ββ Rb uptake is observed in control CY162-pYES2 cells and 86 Rb uptake into CYl62-pKAT cells is consistent with its published properties. Example 13 Expression of Drosophila melanogaster potassium channels in yeast. Voltage-gated potassium channel diversity in the fruitfly Drosophila melanogaster is encoded in large part by six genes, Shaker, Shab, Shal, Shaw, Eag, and Slo. Expression of these potassium channels in yeast will permit their introduction into screening assays for novel insecticidal compounds and facilitate characterization of their ion channel properties and sensitivity to compounds with activating and inhibitory properties. DNA sequences encoding Drosophila melanogaster potassium channels were amplified by PCR using synthetic oligonucleotides that add 5' Hindlll or Kpn I, sites and 3' Xbal, SphI, or Xhol sites: Shaker 5' :AAAAAGCTTAAAATGGCACACATCACG [SEQ ID NO:24] Shaker 3' :AAACTCGAGTCATACCTGTGGACT [SEQ ID NO:25] Shab 5' :AAAAAGCTTAAAATGGTCGGGCAATTG [SEQ ID NO:26] Shab 3' :AAAAGCATGCTCATCTGGATGGGCA [SEQ ID NO:27] Shal 5' :AAAAAGCTTAAAATGGCCTCGGTCGCC [SEQ ID NO:28] Shal 3' :TTTTCTAGACTACATCGTTGTCTT [SEQ ID NO:29] Shaw 5' :AAAAAGCTTAAAATGAATCTGATCAAC [SEQ ID NO:30] Shaw 3' :AAATCTAGATTAGTCGAAACTGAA [SEQ ID NO:31] Eag 5' :AAAAAGCTTAAAATGCCTGGCGGA [SEQ ID NO:32] Eag 3' :AAATCTAGAGGCTACAGGAAGTCC [SEQ ID NO:33] Slo 5' :GGGGGTACCAAAATGTCGGGGTGTGAT [SEQ ID NO:34] Slo 3* :TTTTTCTAGATCAAGAGTTATCATC [SEQ ID NO:35] Plasmids used as templates for the PCR reactions were: pBSc-DShakerH37, pBSc-dShabll, ρBSc-dShal2+(A) 36 , pBScMXT- dShaw [A. Wei, M. Covarrubias, A. Butler, K. Baker, M. Pak, L. Salkoff, Science 248, 599-603 (1990), provided by L. Salkoff] , pBScMXT-slo,v4 [N.S. Atkinson, G.A. Robertson, B. Ganetzky, Science 253,551-555, (1991), provided by L. Salkoff], and pBIMCH20 Eag [CH20] [J. Warmke, R. Drysdale, B. Ganetzky, Science 252, 1560-1564 (1991), A. Bruggemann, L.A. Pardo, W. Stuhmer, 0. Pongs, Nature 365, 445-448 (1993) , provided by B. Ganetzky] . Amplified fragments were digested with the appropriate restriction endonucleases, purified using GeneClean (Bio 101) , and ligated into corresponding sites in pYES2 (Invitrogen) . CY162 cells were transformed with assembled Drosophila melanogaster potassium channel expression plasmids by the LiCl method and plated on SCD-ura containing 0.1M KCl agar medium. Selected transformants were tested for growth on arginine-phosphate-galactose (2 %)/sucrose (0.2 %)-ura agar medium containing 1-5 mM KCl. CY162 cells containing pKATl or pDmORFl were cultured as positive controls and CY162 cells containing pYES2 were grown to provide a negative control. CY162 cells bearing Drosophila melanogaster potassium channel expression plasmids survive under conditions in which growth is dependent on functional potassium channel expression. At potassium ion concentrations between 1-3 mM, negative control CY162 cells containing pYES2 grow poorly. Expression of the Drosophila melanogaster potassium channels Shal, Shaw and Eag substantially improve growth of CY162. These results are consistent with the Drosophila melanogaster potassium channels providing high-affinity potassium uptake capacity. This capacity is apparently sufficient to replace the native high-affinity potassium transport capacity encoded by TRKl which is lacking in CY162 ( trkl trk2) cells. Example 14 Cloning of a novel C. elegans sequence with homoiogy to potassium channels. In order to expand the applicability of this technology to discover compounds with novel anhelmenthic activity, CY162 cells were transformed with a pYES2-based yeast expression library constructed using cDNA synthesized from C. elegans mRNA (Invitrogen) . Plasmid DNA isolated from yeast cells that survived the selection scheme described in EXAMPLE 1 were subjected to automated DNA sequence analysis performed by high temperature cycle sequencing (Applied Biosystems) . Geneworks DNA sequence analysis software (Intelligenetics) is used to align raw DNA sequence information and to identify open reading frames. The DNA sequence of the 1.4 kb insert in pCORK is displayed in FIGURE 9A and 9B. The 5' untranslated sequences of the cDNA are present in this construct. A single long open reading frame sufficient to encode a protein of 434 amino acids (predicted MW 48 kDa) is predicted in pCORK [SEQ ID NO:38]. A consensus polyadenylation site, AATAAA, occurs at position 1359-1364 in 3' untranslated sequences and is followed by a tract of 15 consecutive A residues. The CORK ORF contains structural features that resemble pore forming H5 domains found in potassium channels. Two putative pore forming H5 domains (residues 76-39 and 150-162) contain the G-Y/F-G tripeptide motif required for potassium selectivity [L. Heginbotham, T. Abramson, R. MacKinnon, Science 258, 1152- 1155, (1992)]. SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: American Cyanamid Company (ii) TITLE OF INVENTION: Genes Encoding a Novel Family of Potassium Channels (iii) NUMBER OF SEQUENCES: 38 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: American Cyanamid Company (B) STREET: One Cyanamid Plaza (C) CITY: Wayne (D) STATE: New Jersey (E) COUNTRY: USA (F) ZIP: 07470-8426 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentin Release #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE: (C) CLASSIFICATION: (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Matthews, Gale F. (B) REGISTRATION NUMBER: 32.369 (C) REFERENCE/DOCKET NUMBER: 32,421-01 PCT (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 201-660-6329 (B) TELEFAX: 201-660-7160 (2) INFORMATION FOR SEQ ID NO:l: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2441 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 190..2043 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: ACGCGATCGC CGCGAGTGTA TATTTTTTTT TTAGCTCAGT CTTCAGTGTT TCGCGATTCT 60 CTTTAAAAGA AAAAAAAAAT AATAAGTCAA AACTACAAAC CACACAGCGA AAGGCGAAAG 120 CAACGGTTCC TGCGAGTGTT TATTTTTTTT TTCAACAATT TTTGATCGTA GTGCGACAAT 180 CCGTCGAGC ATG TCG CCG AAT CGA TGG ATC CTG CTG CTC ATC TTC TAC 228 Met Ser Pro Asn Arg Trp lie Leu Leu Leu lie Phe Tyr 1 5 10 ATA TCC TAC CTG ATG TTC GGG GCG GCA ATC TAT TAC CAT ATT GAG CAC 276 lie Ser Tyr Leu Met Phe Gly Ala Ala lie Tyr Tyr His He Glu His 15 20 2 5 GGC GAG GAG AAG ATA TCG CGC GCC GAA CAG CGC AAG GCG CAA ATT GC 324 Gly Glu Glu Lys He Ser Arg Ala Glu Gin Arg Lys Ala Gin He A la 30 35 40 45 ATC A A C G AA TAT CTG CTG G AG GAG CTG GGC GAC AAG A A T A CG ACC ACA 372 He Asn Glu Tyr Leu Leu Glu Glu Leu Gly Asp Lys Asn Thr Thr Thr 50 55 CAG GAT GAG ATT CTT CAA CGG ATC TCG GAT TAC TOT GAC AA A CCG GTT 420 Gin Asp Glu He Leu Gin A rg He Ser Asp Tyr Cys Asp Lys Pro Val 65 70 7S ACA TTG CCG CCG ACA TAT GAT GAT ACG CCC TAC ACG TGG ACC TTC T 468 Thr Leu Pro Pro Thr Tyr Asp Asp Thr Pro Tyr Thr Trp Thr Phe Tyr 80 85 90 CAT GCC TTC TTC TTC GCC TTC ACC GTT TGC TCC ACG GTG GGA TAT GGG 516 His Ala Phe Phe Phe Ala Phe Thr Val Cys Ser Thr V al Gly Tyr Gly 95 100 105 AAT ATA TCG CCA ACC ACC TTC GCC GGA CGG ATG ATC ATG ATC GCG T 564 A β n He Ser Pro Thr Thr Phe Ala Gly Arg Met He Met He Ala Tyr 110 115 120 125 612 TCG G TG ATT GGC ATC CCC GTC AAT GGT ATC CTC TTT <« « « <« Ser Val He Gly He Pro Val Aβn Gly He Leu Phe Ala Gly Leu Gly 130 13S GAA TAC TTT GGA CGT ACG TTT GAA GCG ATC TAC AGA CGC TAC AA AAG 660 Glu Tyr Phe Gly A rg Thr Phe Glu Ala He Tyr Arg Arg Tyr Lys Lys 145 150 155 708 TAC AAG ATG TCC ACG GAT ATG CAC TAT GTC CCG CCG CM CTG GGA TTG Tyr Lys Met Ser Thr Asp Met His Tyr Val Pro Pro Gin Leu Gly Leu 160 165 ■**■ '" 756 ATC ACC ACG GTG GTG ATT GCC CTG ATT CCG GGA ATA GC - CTC! TTC^CTG lie Thr Thr Val Val He Ala L.u He Pro Gly He Ala Leu Phe Leu 175 180 185 804 GTG CTG CCC TGC GTG GGT GTT CAC CTA CTT CGA GAA TO MC CΛ TCI Val Leu Pro Cys Val Gly Val His Leu Leu Arg Glu Leu Gly Leu Ser 190 195 200 205 852 TCC ATC TCG CTG TAC TAC AGC TAT GTG ACC ACC ACA ACA ATT ∞A ™ Ser He Ser Leu Tyr Tyr Ser Tyr Val Thr Thr Thr Thr He Gly Phe 210 215 -" - 900 GGT GAC TAT GTG CCC ACA TTT GGA GCC AAC CAG CCC AAGi GAG TTC GGC Gly Asp Tyr Val Pro Thr Phe Gly Ala Aβn Gin Pro Lye Glu Phe G l y 225 230 948 GGC TGG TTC GTG GTC TAT CAG ATC TTT GTG ATC GTG TGG TTC ATC TTC Gly Trp Phe Val Val Tyr Gin He Phe Val He Val Trp Phe He Phe 24C 245 250 996 TCG CTG GGA TAT CTT GTG ATG ATC ATG ACA TTT ATC ACT CGG GGC CTC Ser Leu Gly Tyr Leu Val Met He Met Thr Phe He Thr Arg Gly Leu 255 260 265 1044 CAG AGC AAG AAG CTG GCA TAC CTG GAG CAG CAG ™ TCC *CC * ^ Gin Ser Lye Lye Leu Ala Tyr Leu Glu Gin Gin Leu Ser Ser Asn Leu 270 275 280 1092 AAG GCC ACA CAG AAT CGC ATC TGG TCT GGC GTC ACC AAG GAT GTG GGC Lye Ala Thr Gin Asn Arg He Trp Ser Gly Val Thr Lye A β p Val Gl y 290 295 TAC CTC CGG CGA ATG CTC AAC GAG CTG TAC ATC CTC AAA GTG AAG CCT 1140 Tyr Leu Arg Arg Met Leu Asn Glu Leu Tyr He Leu Lys Val Lys Pro 305 310 315 GTG TAC ACC GAT GTA GAT ATC GCC TAC ACA CTG CCA CGT TCC AAT TCG 1188 Val Tyr Thr Asp Val Asp He Ala Tyr Thr Leu Pro Arg Ser Asn Ser 320 325 330 TGT CCG GAT CTG AGC ATG TAC CGC GTG GAG CCG GCT CCC ATT CCC AGC 1236 Cys Pro Asp Leu Ser Met Tyr Arg Val Glu Pro Ala Pro He Pro Ser 335 340 345 CGG AAG AGG GCA TTC TCC GTG TGC GCC GAC ATG GTT GGC GCC CAA AGG 1284 Arg Lys Arg Ala Phe Ser Val Cys Ala Asp Met Val Gly Ala Gin Arg 350 355 360 365 GAG GCG GGC ATG GTA CAC GCC AAT TCC GAT ACG GAT CTA ACC AAA CTG 1332 Glu Ala Gly Met Val His Ala Asn Ser Asp Thr Asp Leu Thr Lys Leu 370 375 380 GAT CGC GAG AAG ACA TTC GAG ACG GCG GAG GCG TAC CAC CAG ACC ACC 1380 Aβp Arg Glu Lys Thr Phe Glu Thr Ala Glu Ala Tyr His Gin Thr Thr 385 390 395 GAT TTG CTG GCC AAG GTG GTC AAC GCA CTG GCC ACG GTG AAG CCA CCG 1428 Asp Leu Leu Ala Lys Val Val Asn Ala Leu Ala Thr Val Lys Pro Pro 400 405 410 CCG GCG GAA CAG GAA GAT GCG GCT CTC TAT GGT GGC TAT CAT GGC TTC 1476 Pro Ala Glu Gin Glu Aβp Ala Ala Leu Tyr Gly Gly Tyr His Gly Phe 415 420 425 TCC GAC TCC CAG ATC CTG GCC AGC GAA TGG TCG TTC TCG ACG GTC AAC 1524 Ser Aβp Ser Gin He Leu Ala Ser Glu Trp Ser Phe Ser Thr Val Aβn 430 435 440 445 GAG TTC ACA TCA CCG CGA CGT CCA AGA GCA CGT GCC TGC TCC GAT TTC 1572 Glu Phe Thr Ser Pro Arg Arg Pro Arg Ala Arg Ala Cyβ Ser Aβp Phe 450 455 460 AAT CTG GAG GCA CCT CGC TGG CAG AGC GAG AGG CCA CTG CGT TCG AGC 1620 λβn Leu Glu Ala Pro Arg Trp Gin Ser Glu Arg Pro Leu Arg Ser Ser 465 470 475 CAC AAC GAA TGG ACA TGG AGC GGC GAC AAC CAG CAG ATC CAG GAG GCA 1668 Hie Aβn Glu Trp Thr Trp Ser Gly Asp Aβn Gin Gin He Gin Glu Ala 480 485 490 TTC AAC CAG CGC TAC AAG GGA CAG CAG CGT GCC AAC GGA GCA GCC AAC 1716 Phe Aβn Gin Arg Tyr Lye Gly Gin Gin Arg Ala Aβn Gly Ala Ala Aβn 495 500 505 TCG ACC ATG GTC CAT CTG GAG CCG GAT GCT TTG GAG GAG CAG CTG AGA 1764 Ser Thr Met Val Hie Leu Glu Pro Aβp Ala Leu Glu Glu Gin Leu Arg 510 515 520 525 AAC AAT CAC CGG GTG CCG GTC GCG TCA AGA AGT TCT CCA TGC CGG ATG 1812 Aβn Aβn Hie Arg Val Pro Val Ala Ser Arg Ser Ser Pro Cyβ Arg Met 530 535 540 GTC TGC GAC GTC TGT TTC CCT TCC AGA AGA AGC ACC CCT CGC AGG ATC 1860 Val Cyβ Aβp Val Cys Phe Pro Ser Arg Arg Ser Thr Pro Arg Arg He 545 550 555 TGG AGC GCA AGT TGT CCG TGG TCT CGG TAC CCG AGG GTG TCA TCT CGC 1908 Trp Ser Ala Ser Cys Pro Trp Ser Arg Tyr Pro Arg Val Ser Ser Arg 560 565 570 AGG AAG CCA GAT CCC CGC TGG ACT ACT ACA TCA ACA CGG TCA CGG CGG 1956 Arg Lys Pro Asp Pro Arg Trp Thr Thr Thr Ser Thr Arg Ser Arg Arg 575 580 585 CCT CCA GTC AAT CCT ATT TGC GCA ACG GAC GCG GTC CGC CAC CGC CCT 2004 Pro Pro Val Asn Pro He Cys Ala Thr Asp Ala Val Arg His Arg Pro 590 595 600 605 TCG AAT CGA ATG GCA GCT TGG CCA GCG GCG GCG GCG GGC TAACGAACAT 2053 Ser Aβn Arg Met Ala Ala Trp Pro Ala Ala Ala Ala Gly 610 615 GGGCTTCCAG ATGGAGGATG GAGCAACCCC GCCATCGGCA TTGGGCGGTG GAGCCTATCA 2113 ACGCAAGGCG GCTGCTGGCA AGCGCCGACG CGAGAGCATC TACACCCAGA ATCAAGCCCC 2173 ATCCGCTCGC CGGGGCAGCA TGTATCCGCC GACCGCGCAC GCCTTGGCCC AGATGCAGAT 2233 GCGACGCGGC AGCTTGGCAA CCAGTGGCTC TGGATCGGCG GCCATGGCGG CAGTGGCCGC 2293 GCGTCGTGGC AGCCTCTTCC CAGCTACAGC ATCGGCATCA TCGCTGACCT CTGCTCCGCG 2353 CCGAAGCAGC ATATTCTCGG TTACCTCCGA AAAGGATATG AATGTGCTGG AGCAGACGAC 2413 CATTGCGGAT CTGATTCGTG CGCTCGAG 2441 (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 618 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Met Ser Pro Aβn Arg Trp He Leu Leu Leu He Phe Tyr He Ser Tyr 1 5 10 15 Leu Met Phe Gly Ala Ala He Tyr Tyr Hie He Glu Hie Gly Glu Glu 20 25 30 Lye He Ser Arg Ala Glu Gin Arg Lye Ala Gin He Ala He A β n Glu 35 40 45 Tyr Leu Leu Glu Glu Leu Gly Aβp Lys Aβn Thr Thr Thr Gin Aβp Glu 50 55 60 He Leu Gin Arg He Ser Aβp Tyr Cyβ Aβp Lye Pro Val Thr Leu Pro 65 70 75 80 Pro Thr Tyr Aep Aβp Thr Pro Tyr Thr Trp Thr Phe Tyr Hie Ala Phe 85 90 95 Phe Phe Ala Phe Thr Val Cys Ser Thr Val Gly Tyr Gly Aβn He Ser 100 105 110 Pro Thr Thr Phe Ala Gly Arg Met He Met He Ala Tyr Ser Val He 115 120 125 Gly He Pro Val Aβn Gly He Leu Phe Ala Gly Leu Gly Glu Tyr Phe 130 135 140 Gly Arg Thr Phe Glu Ala He Tyr Arg Arg Tyr Lye Lys Tyr Lye Met 145 150 155 160 Ser Thr Aβp Met His Tyr Val Pro Pro Gin Leu Gly Leu He Thr Thr 165 170 175 Val Val He Ala Leu He Pro Gly He Ala Leu Phe Leu Val Leu Pro 180 185 190 Cys Val Gly Val His Leu Leu Arg Glu Leu Gly Leu Ser Ser He Ser 195 200 205 Leu Tyr Tyr Ser Tyr Val Thr Thr Thr Thr He Gly Phe Gly Asp Tyr 210 215 220 Val Pro Thr Phe Gly Ala Asn Gin Pro Lys Glu Phe Gly Gly Trp Phe 225 230 235 240 Val Val Tyr Gin He Phe Val He Val Trp Phe He Phe Ser Leu Gly 245 250 255 Tyr Leu Val Met He Met Thr Phe He Thr Arg Gly Leu Gin Ser Lys 260 265 270 Lys Leu Ala Tyr Leu Glu Gin Gin Leu Ser Ser Asn Leu Lys Ala Thr 275 280 285 Gin Aβn Arg He Trp Ser Gly Val Thr Lys Aβp Val Gly Tyr Leu Arg 290 295 300 Arg Met Leu Aβn Glu Leu Tyr He Leu Lys Val Lye Pro Val Tyr Thr 305 310 315 320 Aβp Val Aβp He Ala Tyr Thr Leu Pro Arg Ser Asn Ser Cys Pro Asp 325 330 335 Leu Ser Met Tyr Arg Val Glu Pro Ala Pro He Pro Ser Arg Lys Arg 340 345 350 Ala Phe Ser Val Cys Ala Aβp Met Val Gly Ala Gin Arg Glu Ala Gly 355 360 365 Met Val Hie Ala Aβn Ser λβp Thr Aβp Leu Thr Lye Leu A β p Arg Glu 370 375 380 Lye Thr Phe Glu Thr Ala Glu Ala Tyr His Gin Thr Thr Asp Leu Leu 385 390 395 400 Ala Lye Val Val λβn Ala Leu Ala Thr Val Lye Pro Pro Pro Ala Glu 405 410 415 Gin Glu Aβp Ala Ala Leu Tyr Gly Gly Tyr Hie Gly Phe Ser A β p Ser 420 425 430 Gin He Leu Ala Ser Glu Trp Ser Phe Ser Thr Val Aβn Glu Phe Thr 435 440 445 Ser Pro Arg Arg Pro Arg Ala Arg Ala Cyβ Ser Aβp Phe A β n Leu Glu 450 455 460 Ala Pro Arg Trp Gin Ser Glu Arg Pro Leu Arg Ser Ser His A β n Glu 465 470 475 480 Trp Thr Trp Ser Gly Aβp Aβn Gin Gin He Gin Glu Ala Phe A β n Gin 485 490 495 Arg Tyr Lye Gly Gin Gin Arg Ala Aβn Gly Ala Ala Asn Ser Thr Met 500 505 510 Val His Leu Glu Pro Asp Ala Leu Glu Glu Gin Leu Arg Asn A β n Hie 515 520 525 Arg Val Pro Val Ala Ser Arg Ser Ser Pro Cyβ Arg Met Val Cy β A β p 530 535 540 Val Cys Phe Pro Ser Arg Arg Ser Thr Pro Arg Arg He Trp Ser Ala 545 550 555 560 Ser Cys Pro Trp Ser Arg Tyr Pro Arg Val Ser Ser Arg Arg Lys Pro 565 570 575 Asp Pro Arg Trp Thr Thr Thr Ser Thr Arg Ser Arg Arg Pro Pro Val 580 585 590 Asn Pro He Cys Ala Thr Asp Ala Val Arg His Arg Pro Ser Asn Arg 595 600 605 Met Ala Ala Trp Pro Ala Ala Ala Ala Gly 610 615 (2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1011 baβe pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..1008 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: ATG TCC GAT CAG CTG TTT GTC GCA TTT GAG AAG TAT TTC TTG ACG AGT 48 Met Ser Aβp Gin Leu Phe Val Ala Phe Glu Lye Tyr Phe Leu Thr Ser 1 5 10 15 AAC GAG GTC AAG AAG AAT GCA GCA ACG GAG ACA TGG ACA TTT TCA TCG 96 Aβn Glu Val Lye Lye Aβn Ala Ala Thr Glu Thr Trp Thr Phe Ser Ser 20 25 30 TCC ATT TTC TTT GCC GTA ACC GTC GTC ACT ACC ATC GGA TAC GGT AAT 144 Ser He Phe Phe Ala Val Thr Val Val Thr Thr He Gly Tyr Gly A β n 35 40 45 CCA GTT CCA GTG ACA AAC ATT GGA CGG ATA TGG TGT ATA TTG TTC TCC 192 Pro Val Pro Val Thr Aβn He Gly Arg He Trp Cyβ He Leu Phe Ser 50 55 60 TTG CTT GGA ATA CCT CTA ACA CTG GTT ACC ATC GCT GAC TTG GCA GGT 240 Leu Leu Gly He Pro Leu Thr Leu Val Thr He Ala Aβp Leu Ala Gly 65 70 75 80 AAA TTC CTA TCT GAA CAT CTT GTT TGG TTG TAT GGA AAC TAT TTG AAA 288 Lys Phe Leu Ser Glu His Leu Val Trp Leu Tyr Gly Aβn Tyr Leu Lye 85 90 95 TTA AAA TAT CTC ATA TTG TCA CGA CAT CGA AAA GAA CGG AGA GAG CAC 336 Leu Lye Tyr Leu He Leu Ser Arg Hie Arg Lye Glu Arg Arg Glu His 100 105 110 GTT TGT GAG CAC TGT CAC AGT CAT GGA ATG GGG CAT GAT ATG AAT ATC 384 Val Cyβ Glu His Cys His Ser Hie Gly Met Gly His Asp Met Asn He 115 120 125 GAG GAG AAA AGA ATT CCT GCA TTC CTG GTA TTA GCT ATT CTG ATA GTA 432 Glu Glu Lys Arg He Pro Ala Phe Leu Val Leu Ala He Leu He Val 130 135 140 TAT ACA GCG TTT GGC GGT GTC CTA ATG TCA AAA TTA GAG CCG TGG TCT 480 Tyr Thr Ala Phe Gly Gly Val Leu Met Ser Lye Leu Glu Pro Trp Ser 145 150 155 160 TTC TTC ACT TCA TTC TAC TGG TCC TTC ATT ACA ATG ACT ACT GTC GGG 528 Phe Phe Thr Ser Phe Tyr Trp Ser Phe He Thr Met Thr Thr Val Gly 165 170 175 TTT GGC GAC TTG ATG CCC AGA AGG GAC GGA TAC ATG TAT ATC ATA TTG 576 Phe Gly Aβp Leu Met Pro Arg Arg Asp Gly Tyr Met Tyr He He Leu 180 185 190 CTC TAT ATC ATT TTA GGT AAA TTT TCA ATG AAA AAA AAA CAA AAA TTC 624 Leu Tyr He He Leu Gly Lys Phe Ser Met Lys Lys Lys Gin Lye Phe 195 200 205 AAA ATA TTT TTA GGT CTT GCA ATA ACT ACA ATG TGC ATT GAT TTG GTA 672 Lys He Phe Leu Gly Leu Ala He Thr Thr Met Cys He Aβp Leu Val 210 215 220 GGA GTA CAG TAT ATT CGA AAG ATT CAT TAT TTC GGA AGA AAA ATT CAA 720 Gly Val Gin Tyr He Arg Lys He His Tyr Phe Gly Arg Lys He Gin 225 230 235 240 GAC GCT AGA TCT GCA TTG GCG GTT GTA GGA GGA AAG GTA GTC CTT GTA 768 Aβp Ala Arg Ser Ala Leu Ala Val Val Gly Gly Lys Val Val Leu Val 245 250 255 TCA GAA CTC TAC GCA AAT TTA ATG CAA AAG CGA GCT CGT AAC ATG TCC 816 Ser Glu Leu Tyr Ala Aβn Leu Met Gin Lys Arg Ala Arg Aβn Met Ser 260 265 270 CGA GAA GCT TTT ATA GTG GAG AAT CTC TAT GTT TCC AAA CAC ATC ATA 864 Arg Glu Ala Phe He Val Glu Asn Leu Tyr Val Ser Lye His He He 275 280 285 CCA TTC ATA CCA ACT GAT ATC CGA TGT ATT CGA TAT ATT GAT CAA ACT 912 Pro Phe He Pro Thr Aβp He Arg Cyβ He Arg Tyr He Aβp Gin Thr 290 295 300 GCC GAT GCT GCT ACC ATT TCC ACG TCA TCG TCT GCA ATT GAT ATG CAA 960 Ala Aβp Ala Ala Thr He Ser Thr Ser Ser Ser Ala He Aβp Met Gin 305 310 315 320 AGT TGT AGA TTT TGT CAT TCA AGA TAT TCT CTC AAT CGT GCA TTC AAA 1008 Ser Cyβ Arg Phe Cyβ Hie Ser Arg Tyr Ser Leu Aβn Arg Ala Phe Lye 325 330 335 TAG 1011 (2) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 336 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: Met Ser Aβp Gin Leu Phe Val Ala Phe Glu Lys Tyr Phe Leu Thr Ser 1 5 10 15 Aβn Glu Val Lye Lye Aβn Ala Ala Thr Glu Thr Trp Thr Phe Ser Ser 20 25 30 Ser He Phe Phe Ala Val Thr Val Val Thr Thr He Gly Tyr Gly A β n 35 40 45 Pro Val Pro Val Thr Asn He Gly Arg He Trp Cys He Leu Phe Ser 50 55 60 Leu Leu Gly He Pro Leu Thr Leu Val Thr He Ala Asp Leu Ala Gly 65 70 75 80 Lys Phe Leu Ser Glu His Leu Val Trp Leu Tyr Gly Asn Tyr Leu Lys 85 90 95 Leu Lys Tyr Leu He Leu Ser Arg His Arg Lys Glu Arg Arg Glu His 100 105 HO Val Cys Glu His Cys His Ser His Gly Met Gly His Aβp Met A β n He 115 120 125 Glu Glu Lys Arg He Pro Ala Phe Leu Val Leu Ala He Leu He Val 130 135 140 Tyr Thr Ala Phe Gly Gly Val Leu Met Ser Lys Leu Glu Pro Trp Ser 145 150 155 160 Phe Phe Thr Ser Phe Tyr Trp Ser Phe He Thr Met Thr Thr Val Gly 165 170 175 Phe Gly Aβp Leu Met Pro Arg Arg Aβp Gly Tyr Met Tyr He He Leu 180 185 190 Leu Tyr He He Leu Gly Lye Phe Ser Met Lye Lys Lys Gin Lys Phe 195 200 205 Lys He Phe Leu Gly Leu Ala He Thr Thr Met Cyβ He Aβp Leu Val 210 215 220 Gly Val Gin Tyr He Arg Lye He His Tyr Phe Gly Arg Lye He Gin 225 230 235 240 Aβp Ala Arg Ser Ala Leu Ala Val Val Gly Gly Lye Val Val Leu Val 245 250 255 Ser Glu Leu Tyr Ala Aβn Leu Met Gin Lye Arg Ala Arg A β n Met Ser 260 265 270 Arg Glu Ala Phe He Val Glu Aβn Leu Tyr Val Ser Lye Hie He He 275 280 285 Pro Phe He Pro Thr Aβp He Arg Cyβ He Arg Tyr He Aβp Gin Thr 290 295 300 Ala Aβp Ala Ala Thr He Ser Thr Ser Ser Ser Ala He Aβp Met Gin 305 310 315 320 Ser Cyβ Arg Phe Cys His Ser Arg Tyr Ser Leu Asn Arg Ala Phe Lye 325 330 335 (2) INFORMATION FOR SEQ ID NO:5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 51 baβe pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: TCCATTTTCT TTGCCGTAAC CGTCGTCACT ACCATCGGAT ACGGTAATCC A 51 (2) INFORMATION FOR SEQ ID NO:6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 51 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: TCATTCTACT GGTCCTTCAT TACAATGACT ACTGTCGGGT TTGGCGACTT G 51 (2) INFORMATION FOR SEQ ID NO:7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: Ala Phe Leu Phe Ser He Glu Thr Gin Thr Thr He Gly Tyr Gly Phe 1 5 10 15 Arg Cys Val Thr Aβp Glu Cyβ Pro 20 (2) INFORMATION FOR SEQ ID NO:8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: Ala Phe Leu Phe Ser Leu Glu Thr Gin Val Thr He Gly Tyr Gly Phe 1 5 10 15 Arg Cyβ Val Thr Glu Gin Cyβ Ala 20 (2} INFORMATION FOR SEQ ID NO:9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: Ala Phe Leu Phe Phe He Glu Thr Glu Ala Thr He Gly Tyr Gly Tyr 1 5 10 15 Arg Tyr He Thr Aβp Hie Cyβ Pro 20 (2) INFORMATION FOR SEQ ID NO:10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: Ala Phe Phe Phe Ala Phe Thr Val Cys Ser Thr Val Gly Tyr Gly Asn 1 5 10 15 He Ser Pro Thr Thr Phe Ala Gly 20 (2) INFORMATION FOR SEQ ID NO:11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: Ala Phe Trp Trp Ala Val Val Thr Met Thr Thr Val Gly Tyr Gly Aβp 1 5 10 15 Met Thr Pro Val Gly Phe Trp Gly 20 (2) INFORMATION FOR SEQ ID NO:12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: Ala Phe Trp Tyr Thr He Val Thr Met Thr Thr Leu Gly Tyr Gly A β p 1 5 10 15 Met Val Pro Glu Thr He Ala Gly 20 (2) INFORMATION FOR SEQ ID NO:13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: Ala Phe Trp Trp Ala Gly He Thr Met Thr Thr Val Gly Tyr Gly A β p 1 5 10 15 He Cyβ Pro Thr Thr Ala Leu Gly 20 ( 2) INFORMATION FOR SEQ ID NO:14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: Gly Leu Trp Trp Ala Leu Val Thr Met Thr Thr Val Gly Tyr Gly Aβp 1 5 10 15 Met Ala Pro Lys Thr Tyr He Gly 20 (2) INFORMATION FOR SEQ ID NO:15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 amino acidβ (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: Ala Leu Tyr Phe Thr Met Thr Cys Met Thr Ser Val Gly Phe Gly Aβn 1 5 10 15 Val Ala Ala Glu Thr Aβp λβn Glu 20 (2) INFORMATION FOR SEQ ID NO:16: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 amino acidβ (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: Cyβ Val Tyr Phe Leu He Val Thr Met Ser Thr Val Gly Tyr Gly λβp 1 5 10 15 Val Tyr Cyβ Glu Thr Val Leu Gly 20 (2) INFORMATION FOR SEQ ID NO:17: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 amino acidβ (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: Ser Leu Tyr Thr Ser Tyr Val Thr Thr Thr Thr He Gly Phe Gly Aβp 1 5 10 15 Tyr Val Pro Thr Phe Gly Ala Aβn 20 (2) INFORMATION FOR SEQ ID NO:18: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear ( i) SEQUENCE DESCRIPTION: SEQ ID NO:18: Ala Phe Phe Phe Ala Phe Thr Val Cys Ser Thr Val Gly Tyr Gly A β n 1 5 10 15 He Ser Pro Thr Thr Phe Ala Gly 20 (2) INFORMATION FOR SEQ ID NO:19: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: Ser He Phe Phe Ala Val Thr Val Val Thr Thr He Gly Tyr Gly λ β n 1 5 10 15 Pro Val Pro Val Thr Asn Thr Gly 20 (2) INFORMATION FOR SEQ ID NO:20: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 amino acidβ (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: Ser Leu Tyr Thr Ser Tyr Val Thr Thr Thr Thr He Gly Phe Gly A β p 1 5 10 15 Tyr Val Pro Thr Phe Gly Ala Aβn 20 (2) INFORMATION FOR SEQ ID NO:21: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 amino acidβ (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: Ser Phe Tyr Trp Ser Phe He Thi. Met Thr Thr Val Gly Phe Gly Asp 1 5 10 5 Leu Met Pro Arg Arg Aβp Gly Tyr 20 (2) INFORMATION FOR SEQ ID NO:22: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 33 baβe pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: ATAAAGCTTA AAAATGTCGC CGAλTCGλTG GλT 33 (2) INFORMλTION FOR SEQ ID NO:23: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 baβe pair β (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: AGCTCTAGAC CTCCATCTGG AAGCCCATGT 30 (2) INFORMATION FOR SEQ ID NO:24: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 baβe pair β (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: AAAλAGCTTA AAATGGCACλ CλTC CG 27 (2) INFORMATION FOR SEQ ID NO:25: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 baβe pair β (B) TYPE: nucleic acid (C) STRANDEDNESS: βingle (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25: AAλCTCGλGT CATACCTGTG GλCT 4 (2) INFORMλTION FOR SEQ ID NO:26: (i) SEQUENCE CHλRλCTERISTICS: (λ) LENGTH: 27 baβe pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: AAAAAGCTTA AAATGGTCGG GCAATTG 27 (2) INFORMATION FOR SEQ ID NO:27: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: AλλAGCATGC TCATCTGGAT GGGCA 25 (2) INFORMATION FOR SEQ ID NO:28: (i) SEQUENCE CHARλCTERISTICS: (λ) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: AAAAAGCTTA AAATGGCCTC GGTCGCC 27 (2) INFORMATION FOR SEQ ID NO:29: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairβ (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: TTTTCTΛGλC TλCATCGTTG TCTT 24 (2) INFORMλTION FOR SEQ ID NO:30: (i) SEQUENCE CHλRλCTERISTICS: (λ) LENGTH: 27 baβe pairβ (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear ( i) SEQUENCE DESCRIPTION: SEQ ID NO:30: AAAAAGCTTA AλλTGλλTCT GλTCAAC 27 (2) INFORMλTION FOR SEQ ID NO:31: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 baβe pairβ (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: AλλTCTλGλT TAGTCGAAλC TGAλ 24 (2) INFORMλTION FOR SEQ ID NO:32: (i) SEQUENCE CHλRλCTERISTICS: (λ) LENGTH: 24 baβe pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: AAAAAGCTTA AλλTGCCTGG CGGλ 24 (2) INFORMATION FOR SEQ ID NO:33: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairβ (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: AλλTCTλGλG GCTACAGGλA GTCC 24 (2) INFORMλTION FOR SEQ ID NO: 4: (i) SEQUENCE CHARλCTERISTICS: (A) LENGTH: 27 baβe pairβ (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: GGGGGTACCλ AAATGTCGGG GTGTGλT 27 (2) INFORMλTION FOR SEQ ID NO:35: (i) SEQUENCE CHλRλCTERISTICS: (A) LENGTH: 25 baβe pairβ (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35: TTTTTCTAGλ TCλλGλGTTλ TCATC 25 (2) INFORMλTION FOR SEQ ID NO:36: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1529 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SE Q UENCE DESCRIPTION: SEQ ID NO:36: λ β x λβ p λβ p λ la His λ βx A sp Asp Ala Asx His Ala Asx His Ala A β p 1 5 0 15 A β x Ala A β p Aβ x His Aβp His Ala Ala His Ala A β x Asp Ala Asx His 20 25 30 A β x λla Aβ x His Ala His Ala Asp Aβp Aβp Ala Ala His Ala His His 35 40 4S λla λla λsp λβ x Hie λβp λ βp His λla λβx λ β x A β p Ala λ β p λβ x His 50 55 60 λβ x Aβ p A β p λ la Hie λ sx λ βx λla λβx His λla λ β p His λ la λla λβ x 65 70 75 λ β p λβ p λ β x λ β x λ βp λβx λβx λla λβp His λ β p His λβ p λβ p His λ β p 85 9° 95 λ β p λla λla Hie Hie λβx λβp λβp λla λla Ala Hie A β p A β p Hie Ala 100 105 H° Ala His λ la λ la His λ sx λ la λla λβx λβx λ β p λla His λ β x λβ p λla 115 120 I 25 Ala A β x Ala λ β x λ β x Hie λla Hie λβp λβx λ β x β p Hie His λβ p λla 130 135 "0 λ β p λ β x λla λ β p λβp λla λla λβp λβx Ala Hie Hie Hie A β x λ β x λla 145 150 X55 λla λep λla λβ p λ la λ βx λla λla λβp Hie λla λ β x Ala Ala Hie λla 165 I' 0 Hie His λ β p Hie λ βx Hie λla λβx Hie λβp A β p A β x Hie A β x A β p Hie 180 185 λ β x λ la Hie His λ βx Hie Hie λβp Hie Hie λ β p A β p Ala Hie Hie λβ x 195 200 205 λ β p λ β p λla λ β p Hie Hie λβx His Hie λβx Hie λ β p λ β x λ β x λla Hie 210 215 220 λβ p λβ p λla λla Hie λla Hie λβp Hie Hie λ β x λla Hie Hie λla λjx 225 230 235 Hie λla Hie λβ x λ βp λ βx λ βx λβx λβx Hie λ β p Ala A β p Hie Ala Hie 245 250 His λla Hie Hie λβ p Hie λ.p Ala Al. Hie Hie Ala Hie Hie A β p λ.p 260 265 27 0 Hie Hie λβ x λ la λla λla λβx λβx Aβp Aβp Ala Hie λ β p A β p A β x λ β p 275 280 285 Hie Aβp λsp λla λβp λla λ β x Ala Hie Aβp λβp Hie λla Hie Hie A β x 290 295 300 λ β p λla λla λla λβ p λla λla Hie Hie λβx Ala Hie A β p λ β p λβ p λl. 305 310 31S Hie λβ x Hie Hie Hie λβp λla λβx λβp λla Hie Hie λ β p λβ p λ β x Hie 325 330 •*■ " λ β x λ la λβ x λla λla λβx Hie Hie λβx λβx λla λla λla λ -x λ ep λ .x 340 345 λla Ala Asp Asx Ala His His Ala Ala His Asp His His His His Asx 355 360 365 Ala Ala Asx Asx His Asp His His Asx Asx His Asx Ala His His λla 370 375 380 His His Asp Aβx His Asp Aβp His Asx Asx Asx Asx His Asp Ala His 385 390 3 95 400 Asx His Ala Asx Aβx Asp Asx Aβp His Aβx His His His Aβp Asx His 405 410 415 Aβx Asx Aβp Asp His His His Asp λβx His His Asx Ala la Asx Ala 420 425 430 His Aβx Aβp His Asx Ala Ala Aβx Asx His Aβp Ala Aβx λla Ala His 435 440 445 λsx λla Hie Hie λβx His λsx λla His λβx λβx His λsx λβp His λβx 450 455 460 λla Hie Hie λβp Hie Hie λβx Hie λβp λβp λla λβp λβx λex λβx λla 465 470 475 480 λβx Hie λβp λla λla λβp λla His His λβx λβx λla Hie λβp Hie λβx 485 490 495 λβx Hie λep λβp Hie Hie Hie Hie Hie λβx Hie λβp λβp λβp Hie λla 500 505 510 λla λβx Hie λβx Hie Hie λβp λβp λla λla Hie λβp λβp λβx λβp λla 515 520 525 λβx Hie Hie λβx λla λla Hie λex λla λla Hie Hie Hie Hie λla λβp 530 535 540 λβx λla λla Hie λβp λβp λβp λβx Hie λla Hie λla Hie λβp λla la 545 550 555 560 λla λla λβx Hie λβx λβp λβp Hie Hie Hie λla Hie λβp λβp λla λβp 565 570 575 Hie Hie λβp λβp Hie λβp λβp λβx λβp λla Hie Hie Hie Hie λex λβx 580 585 590 λβp λβx λla λβx λla λβx λβx Hie λla λβx λla Hie Hie λβp λβp λβx 595 600 605 λβp λβx Hie λβx Hie λβx Hie Hie λβp λla Hie Hie λβp λβp λla λla 610 615 620 λla λβx λla λla λβx λla Hie Hie Hie λβp λβx λβp λβp λla Hie Hie 625 630 635 640 λβp Aβx Hie Aβp Ala Hie λla λla λβx λβp Aβp Hie Hie Aβp Hie Aβp 645 650 655 λla λla λla λla His λβx λβp λsp λla λsp His λβp λla λβx λβx Hie 660 665 670 λla Hie Hie Hie Hie λβx Hie Aβp Ala λla Hie Aβp Ala Hie λβp λla 675 680 685 λβp λβx λβx Hie λla λla λla λβx Hie His λβp Hie Hie λ β p λ β x λla 690 695 700 λla Hie λβx λβp Hie λβx His λla His His His λβx λβp λβp λβx λla 70S 710 715 720 His Ala His Asx Asp His His Asp Asp His Asp Ala His Aβx Asx His 725 730 735 His Asx His Aβp Aβp His Aβp His Aβp His Asp Asx Ala Ala His His 740 745 750 Asp Asx Ala Asx His His Hie His Asx His His His Ala His Aβx Ala 755 760 765 Aβx Ala Ala Ala Asp Aβx Ala Ala Aβp Ala His His His Aβx His Ala 770 775 780 Aβx Aβx Ala Aβx Hie Ala His Aβx λla Aβx Aβx Ala Hie Aβx Ala Ala 785 790 795 800 λla λla Ala Asp Aβp Ala Ala Hie Aβp Aβp Ala Ala Ala His His Aβx 805 810 815 λβp Asx Asp Ala Ala λla λla Aβp Aβp Aβx Aβp Aβp Ala λla λla λβx 820 825 830 λβx λβp λla λβx λla λβp λla λβx λβx Aβp His λβx Hie Aβx λsx λla 835 840 845 His λβx λβx λla Hie Hie λβx Hie Hie Hie λβp λβp λla λβx λβx λla 850 855 860 λβx λla Hie Hie λβx λla λβx λla λla λla λβx Hie λβp Hie Hie λla 865 870 875 880 Hie λβp λβp λβp λβx λla λla λβx Hie λβx Hie Hie λβx λla λla Hie 885 890 895 λβp His His Hie Aβp Aβp Hie Hie Aβx Hie Aβp λβx Hie Hie Hie λ β p 900 905 910 λβx Aβx λβp Hie Hie λla λβx Hie λβx Hie Aβx λla λβx λla λla Hie 915 920 925 λβx Hie Hie Aβx Aβx Aβx Hie λβp Hie Hie λla Hie λβp λla Hie λ β p 930 935 940 λla Aβx Aβx Asp Hie Hie λla λβx λβx λla λβx Hie λβx λβp Hie λ β p 945 950 955 960 λβp λla λβp λla Hie Hie λβx λβx λβp λβp λβx Hie Hie λβx A β x Hie 965 970 975 Ala Ala λla λβx λla λla λla Ala Hie Hie Ala Hie λβp Hie λβx Hie 980 985 990 λβp λla λla λla λla Aβx Aβp Ala Hie Aβp Ala Ala Ala Hie Aβx Hie 995 1000 1005 Ala Aβx λla λβx Hie Hie Hie λβp λβx Hie λβx λla λβx λla λla λ β p 1010 1015 1020 Hie Hie Hie λβx λex Hie λβx Hie Hie λβx Hie Hie λβx λla λla Hie 1025 1030 1035 1040 Hie Hie λβp Hie Hie λβx λβp λβx Hie λβp λβx Aβp Ala Hie Hie Asp 1045 1050 1055 λβp Ala Hie Aβx Aβx Ala Hie Ala Aβp Hie Hie λβp λβx Hie Hie λ β x 1060 1065 1070 λβx Ala Ala Aβp Ala Hie Hie λβx λla λβx Hie λβp λβp λβx λβx λ β p 1075 1080 1085 λla Aβx Ala Asx Asx A sx Aβx Asp His His Ala Asx Asx His Asx Ala 1090 1095 1100 Ala Ala His His Hie Aβp Aβx Aβx Ala His Ala Ala His A β x His His 1105 1110 1115 1120 Asp Aβx Aβp Hie A sp Aβx His Asx His His His His Asx Ala His His 1125 1130 1135 Asx λβx Ala His His Asx His His Asx His His Asx His His Asx Hie 1140 1145 1150 Aβp Aβx Ala Ala Aβx Hie Ala His Asx Asp His A β p His A β x A β x Ala 1155 1160 1165 λβp Ala Aβx Aβp Aβx Aβp Hie Aβp Aβx His His Ala His Asx Asx His 1170 1175 1180 λβp His His Hie Hie λ βx Hie Hie His Asp Ala λ β p His A β x His Ala 1185 1190 1195 1200 λ βx Hie Aβp Ala λβx λla Hie Hie Hie Hie Hie λ β p Hie A β p Ala Hie 1205 1210 1215 Hie λ βp λ βp Hie λβp λβp λla λla Hie Hie λβp λ β x λ β x λla Hie λ β p 1220 1225 1230 His λ sx Hie Hie Hie Hie Hie λβx λla λex λla Hie λ β p λ β p λla Hie 1235 1240 1245 λla λ βx λ βx Hie λ βx λla λβp λβx Aβp Aβx Hie A β x Hie λ β p A β p λ β x 1250 1255 1260 λla λla Hie λβp Aβp Aβp λla Hie λla λβx λla λ β x Hie λ β x λ β x λla 1265 1270 1275 1280 λla Ala Aβx Aβp Hie λβx λβp Hie λβp λβx λβx λla Hie λ β x Hie λ β x 1285 1290 1295 λla λ βx Hie λla Aβx Hie Aβx Ala Ala Aβp Ala Hie Hie Hie λ β p λ β x 1300 1305 1310 λβx λ βp Aβx Hie Aβx Ala λβp λβx Hie Hie Hie λ β x λ β x λ β p Hie Hie 1315 1320 1325 Hie λ βp λ βx λ la λ βx Hie Asx Hie Hie Ala Hie A β p A β p Hie Hie λβ p 1330 1335 1340 λβp λ βx λβx Hie Hie λβx Hie λβx λla λβx λβx λ β p λ β p Hie λ β p λ β p 1345 1350 1355 1360 λβx Aβx His λ βp Hie Aβp Aβp λβx λβx λβx λβp Hie Hie λ β p Hie Hie 1365 1370 1375 λla Hie Hie λ βp λla λβp Asx Ala λβx Hie Hie Asx Asp Hie λβ p λ β p 1380 1385 1390 λla Aβx Ala λla λβp Aβx λsx λla λla Aβp His Ala Hie A β x Hie Hie 1395 1400 1405 Ala Hie Ala λla λla Hie λla Hie Hie Hie λla Hie λla λ β p λβ x λ la 1410 1415 1420 Hie Hie λla λβp λla λβp Hie λla Hie λla λβx Hie Hie λ β p Hie Hie 1425 1430 1435 1440 λla Hie λ la Hie λβp Hie Hie λβp Hie Hie Hie Hie Hie λla Hie Hie 1445 1450 1455 Ala Ala Asp Aβx His Asp His Aβp Aβp Ala Ala His Ala Ala Ala Ala 1460 1465 1470 His Ala Ala His His Ala His His Ala Ala Ala Ala Ala Ala Ala Ala 1475 1480 1485 Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Asp Aβx Aβp 1490 1495 1500 Aβx Aβx Aβp Aβx His Aβx Aβp Ala Asp Aβx Ala His Hie Aβx Ala His 1505 1510 1515 1520 λβp Aβp Ala Aβp Ala Aβp Ala Ala Ala 1525 (2) INFORMATION FOR SEQ ID NO:37: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 479 amino acidβ (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37: Met Ser Pro Aβn Arg Trp He Leu Leu Leu He Phe Tyr He Ser Tyr 1 5 10 15 Leu Met Phe Gly Ala λla He Tyr Tyr Hie He Glu Hie Gly Glu Glu 20 25 30 Lye He Ser λrg λla Glu Gin Arg Lye Ala Gin He λla He λβn Glu 35 40 45 Tyr Leu Leu Glu Glu Leu Gly Aβp Lye Aβn Thr Thr Thr Gin Aβp Glu 50 55 60 He Leu Gin λrg He Ser λβp Tyr Cyβ λβp Lye Pro Val Thr Leu Pro 65 70 75 80 Pro Thr Tyr λβp λβp Thr Pro Tyr Thr Trp Thr Phe Tyr Hie λla Phe 85 90 95 Phe Phe Ala Phe Thr Val Cye Ser Thr Val Gly Tyr Gly Aβn He Ser 100 105 HO Pro Thr Thr Phe λla Gly λrg Met He Met He λla Tyr Ser Val He 115 120 125 Gly He Pro Val λβn Gly He Leu Phe λla Gly Leu Gly Glu Tyr Phe 130 135 140 Gly Arg Thr Phe Glu Ala He Tyr Arg Arg Tyr Lye Lye Tyr Lye Met 145 150 155 160 Ser Thr Aβp Met Hie Tyr Val Pro Pro Gin Leu Gly Leu He Thr Thr 165 170 175 Val Val He Ala Leu He Pro Gly He Ala Leu Phe Leu Val Leu Pro 180 185 190 Cyβ Val Gly Val Hie Leu Leu λrg Glu Leu Gly Leu Ser Ser He Ser 195 200 205 Leu Tyr Tyr Ser Tyr Val Thr He Thr Thr He Gly Phe Gly A β p Tyr 210 215 220 Val Pro Thr Phe Gly Ala Aβn Gin Pro Lys Glu Phe Gly Gly Trp Phe 225 230 235 240 Val Val Tyr Gin He Phe Val He Val Trp Phe He Phe Ser Leu Gly 245 250 255 Tyr Leu Val Met He Met Thr Phe He Thr Arg Gly Leu Gin Ser Lye 260 265 270 Lys Leu Ala Tyr Leu Glu Gin Gin Leu Ser Ser Aβn Leu Lys Ala Thr 275 280 285 Gin Aβn Arg He Trp Ser Gly Val Thr Lye Aβp Val Gly Tyr Leu Arg 290 295 300 λrg Met Leu Aβn Glu Leu Tyr He Leu Lye Val Lye Pro Val Tyr Thr 305 310 315 320 Aβp Val Aβp He Ala Tyr Thr Leu Pro Arg Ser λβn Ser Pro Leu Ser 325 330 335 Met Tyr Arg Val Glu Pro Ala Pro He Pro Ser λrg Lye λrg Ala Phe 340 345 350 Ser Val Cyβ Ala Aβp Met Val Gly λla Gin λrg Glu λla Gly Met Val 355 360 365 Hie λla λβn Ser λβp Thr λβp Leu Thr Lye Leu λβp λrg Glu Lye Thr 370 375 380 Phe Glu Thr λla Glu λla Tyr Hie Gin Thr Thr λβp Leu Leu λla Lye 385 390 395 400 Val Val λβn λla Leu λla Thr Val Lye Pro Pro Pro λla Leu Gin Glu 405 410 415 λβp λla λla Leu Tyr Gly Gly Tyr Hie Gly Phe Ser λβp Ser Gin He 420 425 430 Leu λla Ser Glu Trp Ser Phe Ser Thr Val λβn Glu Phe Thr Ser Pro 435 440 445 λrg λrg Pro λrg λla λrg λla Cye Ser λβp Phe λen Leu Glu λla Pro 450 455 460 λrg Trp Gin Ser Glu λrg Pro Leu λrg Ser Ser Hie λ β n Glu Trp 465 470 475 (2) INFORMλTION FOR SSQ ID NO:38: (i) SEQUENCE CHλRλCTERISTICS: (λ) LENGTH: 335 amino acidβ (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38: Met Ser Aβp Gin Leu Phe Val λla Phe Glu Lye Tyr Phe Leu Thr Ser 1 5 10 15 λβn Glu Val Lye Lye λβn λla λla Thr Glu Thr Trp Thr Phe Ser Ser 20 25 30 Ser He Phe Phe λla Val Thr Val Val Thr Thr He Gly Tyr Gly λ β n 35 40 45 Pro Val Pro Val Thr Aβn He Gly Arg He Trp l e ι_eu t-ne ser ι_eu 50 55 60 He Gly He Pro Leu Thr Leu Val Thr He Ala Leu Ala Gly Lys Phe 65 70 75 80 Leu Ser Glu His Leu Val Trp Leu Tyr Gly Aβn Tyr Leu Lys Leu Lye 85 90 95 Tyr Leu He Leu Ser Arg His Arg Lys Glu Arg Arg Glu His Val Cyβ 100 105 110 Glu Hie Cyβ His Ser His Gly Met Gly Hie Aβp Met λβn He Glu Glu 115 120 125 Lye Arg He Pro Ala Phe Leu Val Leu Ala He Leu He Val Tyr Thr 130 135 140 Ala Phe Gly Gly Val Leu Met Ser Lye Leu Glu Pro Trp Ser Phe Phe 145 150 155 160 Thr Ser Phe Tyr Trp Ser Phe He Thr Met Thr Thr Val Gly Phe Gly 165 170 175 λβp Leu Met Pro λrg λrg λβp Gly Tyr Met Tyr He He Leu Leu Tyr 180 185 190 He He Leu Gly Lye Phe Ser Met Lye Lye Lye Gin Lye Phe Lye He 195 200 205 Phe Leu Gly Leu Ala He Thr Thr Met Cyβ He Aβp Leu Val Gly Val 210 215 220 Gin Tyr He Arg Lye He Hie Tyr Phe Gly λrg Lye He Gin λβp λla 225 230 235 240 λrg Ser λla Leu λla Val Val Gly Gly Lye Val Val Leu Val Ser Glu 245 250 255 Leu Tyr λla Aβn Leu Met Gin Lye Arg λla λrg λβn Met Ser λrg Glu 260 265 270 λla Phe He Val Glu λβn Leu Tyr Val Ser Lye Hie He He Pro Phe 275 280 285 He Pro Thr Aβp He Arg Cye He Arg Tyr He Aβp Gin Thr λla λβp 290 295 300 λla λla Thr He Ser Thr Ser Ser Ser λla He λβp Met Gin Ser Cy β 305 310 315 320 λrg Phe Cye Hie Ser Arg Tyr Ser Leu λβn λrg λla Phe Lye Xaa 325 330 335

Claims: What is claimed is: 1. A potassium channel comprising four hydrophobic domains capable of forming transmembrane helices, wherein (i) a first pore-forming domain is interposed between a first and a second transmembrane helix; and (ii) a second pore-forming domain is interposed between a third and a fourth transmembrane helix. 2. The potassium channel of claim 1 wherein each pore- forming domain comprises a potassium selective peptide motif. 3. The potassium channel of claim 2 wherein the peptide motif is selected from the group consisting of a Y/F-G dipeptide motif and a G-Y/F-G tripeptide motif. 4. The potassium channel of claim 3 wherein at least one pore-forming domain is positioned proximal to an exterior portion of a cell membrane. 5. The potassium channel of claim 4 further comprising an amino-terminal glycosylation site. 6. The potassium channel of claim 5 wherein said glycosylation site is asparagine-linked. 7. The potassium channel of claim 6 characterized in that it belongs to a class of invertebrates. 8. The potassium channel of claim 7 characterized in that it is insect-derived. 9. The potassium channel of claim 7 characterized in that it is nematode-derived. 10. An isolated nucleotide sequence capable of encoding DmORF-1. 11. The isolated nucleotide sequence of Claim 10 comprising the nucleotide sequence depicted in Seq. I.D. No. 1. 12. An isolated nucleotide sequence capable of encoding CORK. 13. The isolated nucleotide sequence of Claim 12 encoding for the protein depicted in Sequence I.D. No. 36. 14. An expression vector capable of expressing a heterologous potassium channel in a cell membrane of a yeast cell comprising the nucleotide sequence of Claim 10. 15. An expression vector capable of expressing a heterologous, potassium channel in a cell membrane of a yeast cell comprising the nucleotide sequence of Claim 11. 16. An expression vector capable of expressing a heterologous potassium channel in a cell membrane of a yeast cell comprising the nucleotide sequence of Claim 12. 17. An expression vector capable of expressing a heterologous potassium channel in a cell membrane of a yeast cell wherein the potassium channel comprises the amino acid sequence of Claim 13. 18. A transformed yeast cell comprising the nucleotide sequences of Claims 10, 11, 12 or 13. 19. A transformed yeast cell comprising the expression vector of claims 14, 15, 16 or 17. 20. A method of assaying substances to determine effects on cell growth, the method comprising the steps of: a. preparing cultures of yeast cells in liquid medium lacking uracil, the liquid medium consisting of a concentration of KCl adequate to support growth of potassium-dependent mutant strains; b. plating the yeast cells in uracil-free agar medium, the agar medium consisting of sufficient KCl to selectively support growth of potassium- dependent mutant strains containing a heterologous potassium channel of claim 1; c. applying substances to the agar plate; d. incubating the agar plate to permit growth; and e. identifying zones of growth around the substances, wherein the level of growth indicates whether or not activity of the heterologous potassium channel has been modulated as compared to control. 21. The yeast cell of Claim 20 further comprising a nucleotide sequence encoding RAK, or a nucleotide sequence of Claim 10, 11, 12 or 13. 22. The method of claim 20, wherein said effect on cell growth is modulated by activation of the potassium channel. 23. The method of claim 20, wherein said effect on cell growth is modulated by inhibition of said potassium channel. 2 . A method of selectively inhibiting insect pests by applying to such insect pests a substance capable of inhibiting a potassium channel substantially homologous to that encoded by the nucleotide sequence of claim 10. 25. A method of selectively inhibiting nematode pests by applying to such pests a substance capable of inhibiting a potassium channel substantially homologous to that encoded by the nucleotide sequence of claim 12. 26. A method of modulating the activity of a potassium channel positioned in a cellular membrane and comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 36, by contacting said cellular membrane with a substance, in an amount and for a period of time sufficient to inhibit the ability of potassium ions to pass through said channel.

Inventors: PAUSCH MARK HENRY, PRICE LAURA ALICIA, PAUSCH, MARK, HENRY, PRICE, LAURA, ALICIA
Applicants: AMERICAN CYANAMID CO, PAUSCH MARK HENRY, PRICE LAURA ALICIA, AMERICAN CYANAMID COMPANY, PAUSCH, MARK, HENRY, PRICE, LAURA, ALICIA
Application year: 1995
Main classification: C07K14
IPC codes: A01N63, C07K14, C12N1, C12N15, C12R1