AU2775502A - Insect sodium channels from insecticide-susceptible and insecticide-resistant house flies - Google Patents

Description

P/00/011 Regulation 3.2

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT Invention Title: INSECT SODIUM CHANNELS FROM INSECTICIDE-SUSCEPTIBLE AND INSECTICIDE- RESISTANT HOUSE FLIES Applicant: CORNELL RESEARCH FOUNDATION, INC.

The following statement is a full description of this invention, including the best method of performing it known to me: la INSECT SODIUM CHANNELS FROM INSECTICIDE-SUSCEPTIBLE AND INSECTICIDE-RESISTANT HOUSE FLIES FIELD OF THE INVENTION The present invention relates generally to insect sodium channel proteins, and more particularly to insecticide-susceptible and insecticide-resistant voltagesensitive sodium channels of the house fly Musca domestica.

BACKGROUND OF THE INVENTION Throughout this application various :20 publications are referenced, many in parenthesis. Full citations for these publications are provided at the end of the Detailed Description. The disclosures of these publications in their entireties are hereby incorporated by reference in this application.

25 Cell membranes must allow passage of various polar molecules, including ions, sugars, amino acids, and nucleotides. Special membrane proteins are responsible for transferring such molecules across cell membranes.

These proteins, referred to as membrane transport proteins, occur in many forms and in all types of biological membranes. Each protein is specific in that it transports a particular class of molecules (such as ions, sugars, or amino acids) and often only certain molecular species of the class. All membrane transport proteins that have been studied in detail have been found to be multipass transmembrane proteins. By forming a continuous protein pathway across the membrane, these proteins enable the specific molecules to cross the membrane without WO 98/28446 PCT/US97/24256 -2 coming into direct contact with the hydrophobic interior of the lipid bilayer of the plasma membrane.

There are two major classes of membrane transport proteins: carrier proteins and channel proteins. Carrier proteins bind the specific molecule to be transported and undergo a series of conformational changes in order to transfer the bound molecule across the membrane. Channel proteins, on the other hand, need. not bind the molecule. Instead, they form hydrophilic pores that extend across the lipid bilayer; when these pores are open, they allow specific molecules (usually inorganic ions of appropriate size and charge) to pass through them and thereby cross the membrane. Transport through channel proteins occurs at a much faster rate than transport 15 mediated by carrierproteins.

Channel proteins which are concerned specifically with inorganic ion transport are referred to as ion channels, and include ion channels for sodium, potassium, calcium, and chloride ions. Ion channels which open in response to a change in the voltage across the e membrane are referred to as voltage-sensitive ion channels.

*"The sodium channel is one of the most thoroughly characterized of the voltage-sensitive channels (see Fig. 1 for a model of a voltage-sensitive sodium channel). In vertebrates, sodium channels in the brain, muscle, and other tissues are large membrane glycoprotein complexes composed of an alpha subunit (230-270 kDa) and 1-2 tightly associated smaller (33-38 kDa) beta subunits (reviewed by Catterall 1992). The large alpha subunit forms the ion permeable pore while the smaller subunits play key roles in the regulation of channel function (Isom et al. 1992; reviewed by Isom et al. 1994). The alpha subunit is common to purified channel preparations from Electrophorus electricus (electric eel) electric organ WO 98/28446 PCTIUS97/24256 -3 (Noda et al. 1984), rat brain (Noda et al. 1986), rat skeletal muscle (Barchi 1988) and chick heart muscle (Catterall 1986). Other studies have revealed the existence of multiple closely related isoforms of the sodium channel found in different animal species, in different tissues within the same species, and even in the same tissue (Catterall et al. 1981; Frelin et al. 1984; Rogart 1986; Moczydlowski et al. 1986).

The structure of invertebrate sodium channels is not as well defined. Gene cloning studies have established the existence of alpha subunits of structure similar to those described for vertebrates (Loughney et al. 1989; Ramaswami and Tanouye 1989; Okamoto et al.

1987). Analysis of the para behavioral mutant (paralytic; 15 Suzuki et al. 1971) of Drosophila melanogaster revealed that the para gene encodes a Drosophila sodium channel alpha subunit (Loughney et al. 1989). The entire para cDNA sequence was determined (Loughney et al. 1989; Thackeray and Ganetzky 1994).

The kdr mutant of the house fly Musca domestica has also been studied. The kdr insecticide resistance trait of the house fly confers reduced neuronal sensitivity to the rapid paralytic and lethal actions of S'. DDT and pyrethroid insecticides (Soderlund and Bloomquist 1990). Because these insecticides are known to modify neuronal excitability by altering the inactivation kinetics of voltage-sensitive sodium channels (Soderlund and Bloomquist 1989; Bloomquist 1993), efforts to identify the molecular basis of kdr resistance have focused on the pharmacology and structure of this target.

Recently, tight genetic linkage between the kdr trait and a restriction fragment length polymorphism located within a segment of the house fly homolog of the para gene of Drosophila melanogaster was demonstrated (Knipple et al. 1994). Similar linkage studies have also WO 98/28446 PCT/US97/24256 4 documented tight linkage of the super-kdr resistance trait of the house fly (Williamson et al. 1993) to molecular markers lying within the para-homologous voltage-sensitive sodium channel gene.

Elucidation of the structure of the house fly sodium channel gene will enable the screening of potential insecticidal agents which act upon the sodium channel.

A need continues to exist, therefore, for the determination of the primary structure of the house fly sodium channel, i.e. the nucleotide and amino acid sequences of the channel.

o* SUMMARY OF INVENTION To this end, the subject invention provides the 15 6318 nucleotide coding sequence (SEQ ID NO:1) of the voltage-sensitive sodium channel gene from insecticidesusceptible (NAIDM strain) house flies (Musca domestica), determined by automated direct DNA sequencing of PCR fragments obtained by amplification on first strand cDNA from adult heads. The deduced 2105-residue amino acid sequence (SEQ ID NO:3) exhibits overall structure and organization typical of sodium channel alpha subunit genes and is 90.0% identical to that of the D. melanogaster para gene product. There is no evidence for the existence of multiple splice variants among voltage-sensitive sodium channel cDNAs obtained from adult house fly head preparations. Comparison of the coding sequence of the voltage-sensitive sodium channel gene of the kdr insecticide-resistant house fly strain (538ge strain) to that of the NAIDM strain reveals 12 amino acid differences in the 538ge strain. The amino acid sequence (SEQ ID NO:4) of the Kdr strain is only 2104 residues in length, as a result of five amino acid substitutions, four (4) amino acid deletions, and three amino acid insertions as compared to the 2105-residue amino acid sequence (SEQ WO 98/28446 PCT/US97/24256 5 ID NO:3) of the NAIDM strain. The nucleotide sequence (SEQ ID NO:2) of the Kdr strain is therefore 6315 nucleotides in length, which is three nucleotides shorter than the nucleotide sequence (SEQ ID NO:1) of the NAIDM strain.

More particularly, the subject invention provides an isolated nucleic acid molecule encoding a voltage-sensitive sodium channel of Musca domestica, wherein the voltage-sensitive sodium channel is capable of conferring sensitivity or resistance to an insecticide in Musca domestica. In one embodiment, the nucleic acid molecule confers insecticide susceptibility to the house fly, and in another embodiment the nucleic acid molecule confers insecticide resistance to the house fly. The 15 nucleic acid molecule conferring insecticide resistance is preferably a mutated form of the nucleic acid molecule encoding the insecticide susceptible channel. The .i invention also provides an antisense nucleic acid molecule complementary to mRNA encoding the voltage-sensitive sodium channel of Musca domestica.

The isolated nucleic acid molecules of the invention can be inserted into suitable expression vectors and/or host cells. Expression of the nucleic acid molecules encoding the sodium channels results in production of functional sodium channels in a host cell.

Expression of the antisense nucleic acid molecules or fragments thereof in a host cell results in decreased expression of the functional sodium channels.

The invention further provides a ribozyme having a recognition sequence complementary to a portion of mRNA encoding a voltage-sensitive sodium channel of Musca domestica. The ribozyme can be introduced into a cell to also achieve decreased expression of sodium channels in the cell.

WO 98/28446 PCT/US97/24256 6- The invention further provides a method of screening a chemical agent for the ability of the chemical agent to modify sodium channel function, and a method of obtaining DNA encoding a voltage-sensitive sodium channel of Musca domestica.

Further provided is an isolated nucleic acid molecule encoding a voltage-sensitive sodium channel of an insect, wherein the nucleic acid molecule encodes a first amino acid sequence having at least 95% amino acid identity to a second amino acid sequence. The second amino acid sequence is, in two preferred embodiments, SEQ .ID NO:3 or SEQ ID NO:4.

The invention also provides an isolated voltage-sensitive sodium channel of Musca domestica, and 15 antibodies or antibody fragments specific for the sodium channel. The antibodies or antibody fragments can be used to detect the presence of the sodium channel in samples.

Further provided is an isolated voltagesensitive sodium channel of Musca domestica, wherein the voltage-sensitive sodium channel is comprised of a protein having a first amino acid sequence with at least 95% amino acid identity to a second amino acid sequence. In two preferred embodiments, the second amino acid sequence is SEQ ID NO:3 or SEQ ID NO:4.

Also provided by the subject invention is a plasmid designated pPJII and deposited with the ATCC under Accession No.97831, as well as a KpnI/AatII restriction fragment of about 3620 bp of the plasmid designated pPJI1.

Further provided is a plasmid designated pPJI2 and deposited with the ATCC under Accession No. 97832, as well as an AatII/SphII restriction fragment of about 2700 bp of the plasmid designated pPJI2. When the above two restriction fragments are ligated together at their AatII sites, the resulting nucleic acid molecule encodes a voltage-sensitive sodium channel which confers WO 98/28446 PCTIUS97/24256 7 susceptibility to an insecticide in Musca domestica. This resulting nucleic acid molecule is also provided by the subject invention.

BRIEF DESCRIPTION OF THE DRAWINGS These and other features and advantages of this invention will be evident from the following detailed description of preferred embodiments when read in conjunction with the accompanying drawings in which: Fig. 1 is a model of a voltage sensitive sodium channel from mammalian brain in the plasma membrane. The alpha and beta, subunits interact noncovalently; the alpha and beta 2 subunits are linked by disulfide bonds. The branched structures at the outer surface of the channel 15 represent oligosaccharides; Fig. 2 is a diagram of the structural organization of the voltage-sensitive sodium channel coding sequence of Musca domestica (Vsscl) showing repeated homology domains I-IV and putative transmembrane helices (rectangles). Shown below the structural organization are the relative length and location of the previously-described 309-nucleotide exon of Vsscl (Knipple et al. 1994) (exon) and seven overlapping PCR-amplified .*cDNA fragments employed as templates for DNA sequencing; Fig. 3 shows the alignment of the predicted amino acid sequences of Vssc 1 N'D (NAIDM) and Vssc 1 538lg (538ge) with that of the a'b-c-d'e-f-h-i* splice variant of the D. melanogaster para sequence (para) obtained using the DNASTAR computer program (Clustal method). Residues that are identical to the NAIDM sequence in both 538ge and para are indicated as dashes in the latter two sequences; gaps introduced to obtain optimal alignment are indicated as periods The locations of 24 putative helical transmembrane domains IS1, IS2, etc.) and WO 98/28446 PCT/US97/24256 8 four putative pore-forming domains IP, IIP) are marked by solid bars above the NAIDM sequence. Also marked above the NAIDM sequence are possible sites for Nlinked glycosylation cAMP-dependent protein-kinase phosphorylation and protein kinase C phosphorylation and Fig. 4 is a diagram of the Vsscl gene product showing the locations of 12 amino acid differences identified in the Vsscl 5 3 Sge sequence, including 5 amino acid substitutions, 4 amino acid deletions, and 3 amino acid insertions in the Vssc15 3 8 ge sequence as compared to the Vssc 1 N A IDM sequence DETAILED DESCRIPTION The plasmids designated pPJI1 and pPJI2 have each been deposited pursuant to, and in satisfaction of, the requirements of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure, with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland, 20852 under ATCC Accession No. 97831 (pPJI1) and ATCC Accession No. 97832 (pPJI2). Both deposits were made on December 1996.

As used herein, the term "isolated" when used in conjunction with a nucleic acid molecule refers to: 1) a nucleic acid molecule which has been separated from an organism in a substantially purified form (i.e.

substantially free of other substances originating from that organism), or 2) a nucleic acid molecule having the same nucleotide sequence but not necessarily separated from the organism synthesized nucleic acid molecules). The term "isolated" when used in conjunction with a channel refers to a channel encoded by such an "isolated" nucleic acid molecule, generally expressed in a WO 98/28446 PCTIUS97/24256 9 membrane, such as a plasma membrane within a cell or a synthetic lipid bilayer membrane. The expressed "isolated" channel has the pharmacological properties of a functional sodium channel.

As further used herein, the terms "corresponding to" or "having" or "as shown in" when used in conjunction with a SEQ ID NO for a nucleotide sequence refer to a nucleotide sequence which is substantially the same nucleotide sequence, or derivatives or equivalents thereof (such as deletion and hybrid variants thereof, splice variants thereof, etc.). Nucleotide additions, deletions, and/or substitutions, such as those which do not affect the translation of the DNA molecule, are within the scope of a nucleotide sequence corresponding to or having or as shown in a particular nucleotide sequence the amino acid sequence encoded thereby remains the same). Such additions, deletions, and/or substitutions can be, for example, point mutations made according to 9 e methods known to those skilled in the art. It is also possible to substitute a nucleotide which alters the amino acid sequence encoded thereby, where the amino acid substituted is a conservative substitution or where amino acid homology is conserved. It is also possible to have minor nucleotide additions, deletions, and/or substitutions which do not alter the function of the resulting VSSC. Similarly, the term "corresponding to" or "having" or "as shown in" when used in conjunction with a SEQ ID NO for an amino acid sequence refers to an amino acid sequence which is substantially the same amino acid sequence or derivatives or equivalents thereof. Amino acid additions, deletions, and/or substitutions which do not negate the ability of the resulting protein to form a functional sodium channel are within the scope of an amino acid sequence corresponding to or having or as shown in a particular amino acid sequence. Such additions, WO 98/28446 PCT/US97/24256 10 deletions, and/or substitutions can be, for example, the result of point mutations in the DNA encoding the amino acid sequence, such point mutations made according to methods known to those skilled in the art. Substitutions may be conservative substitutions of amino acids. As used herein, two amino acid residues are conservative substitutions of one another where the two residues are of the same type. In this regard, for purposes of the present invention, proline, alanine, glycine, serine, and threonine, all of which are neutral, weakly hydrophobic residues, are of the same type. Glutamine, glutamic acid, asparagine, and aspartic acid, all of which are acidic, hydrophilic residues, are of the same type. Another type of residue is the basic, hydrophilic amino acid residues, 15 which include histidine, lysine, and arginine. Leucine, isoleucine, valine, and methionine all of which are hydrophobic, aliphatic amino acid residues, form yet another type of residue. Yet another type of residue consists of phenylalanine, tyrosine, and tryptophan, all of which are hydrophobic, aromatic residues. Further descriptions of the concept of conservative substitutions are given by French and Robson 1983, Taylor 1986, and Bordo and Argos 1991.

As further used herein, the term "corresponding to" or "having" or "as shown in" or "consisting of" when used in conjunction with a SEQ ID NO for a nucleotide or amino acid sequence is intended to cover linear or cyclic versions of the recited sequence (cyclic referring to entirely cyclic versions or versions in which only a portion of the molecule is cyclic, including, for example, a single amino acid cyclic upon itself), and is intended to cover derivative or modified nucleotides or amino acids within the recited sequence. For example, those skilled in the art will readily understand that an adenine nucleotide could be replaced with a methyladenine, or a WO 98/28446 PCTUS97124256 11 cytosine nucleotide could be replaced with a methylcytosine, if a methyl side chain is desirable.

Nucleotide sequences having a given SEQ ID NO are intended to encompass nucleotide sequences containing these and like derivative or modified nucleotides, as well as cyclic variations. As a further example, those skilled in the art will readily understand that an asparagine residue could be replaced with an ethylasparagine if an ethyl side chain is desired, a lysine residue could be replaced with a hydroxylysine if an OH side chain is desired, or a valine residue could be replaced with a methylvaline if a methyl side chain is desired. Amino acid sequences having a given SEQ ID NO are intended to encompass amino acid sequences containing these and like derivative or modified 15 amino acids, as well as cyclic variations. Cyclic, as used herein, also refers to cyclic versions of the derivative or modified nucleotides and amino acids.

The function of the encoded sodium channel can be assayed according to methods known in the art, such as by voltage clamp analysis of the channel following the functional expression of the channel in oocytes of the frog Xenopus laevis (see Taglialatela et al. 1992 and Stuhmer 1992 for a general discussion of the voltage clamp analysis of receptors and ion channels expressed in Xenopus oocytes). As used herein, "functional expression" refers to the synthesis and any necessary posttranslational processing of a sodium channel molecule in a host cell so that the channel is inserted properly in the cell membrane and is capable of conducting sodium ions in response to an experimentally-imposed change in the cell membrane potential or upon exposure to appropriate pharmacological agents.

As further used herein, "sensitivity" and "resistance" refer to the relative responses of genetically-defined insect populations to the paralytic or WO 98/28446 PCT/US97/24256 12 lethal actions of a test insecticide. For example, a dose of DDT [1,1-bis-(4-chlorophenyl)- 2 2 2 -trichloroethane] of approximately 0.02 pg per adult fly will kill approximately 50% of the treated individuals of a susceptible (Cooper-S) house fly strain, whereas doses of approximately 0.5 pg per adult fly are required to kill approximately 50% of the treated individuals of a resistant (538ge) house fly strain (Sawicki 1978). The absolute doses that define susceptibility and resistance vary with the insect species and genetically defined populations examined, the test insecticide employed, and the method of exposure. In general, an insect strain or population is considered "resistant" if it exhibits tolerance to a test insecticide (assessed as the dose required to poison 50% of a treated population or group) that is at least 10 times greater than the tolerance of an appropriate reference, or "susceptible" population. Test insecticides include not only DDT but also analogs of DDT methoxychlor, perthane) and pyrethroid insecticides deltamethrin, fenvalerate, resmethrin, permethrin).

As also used herein, insects include Musca domestica (the house fly), the fruit or vinegar fly (Drosophila melanogaster), and various other insect species of agricultural, medical or veterinary importance, such as Heliothis virescens (the tobacco budworm), Leptinotarsa decemlineata (the Colorado potato beetle), Blattella germanica (the German cockroach), and Aedes aegypti (the yellow fever mosquito).

The subject invention provides an isolated nucleic acid molecule encoding a voltage-sensitive sodium channel (VSSC) of Musca domestica, wherein the VSSC is capable of conferring sensitivity or resistance to an insecticide in Musca domestica. The nucleic acid molecule can be deoxyribonucleic acid (DNA) or ribonucleic acid WO 98/28446 PCTIUS9/24256 13 (RNA, including messenger RNA or mRNA), genomic or recombinant, biologically isolated or synthetic.

The DNA molecule can be a cDNA molecule, which.

is a DNA copy of a messenger RNA (mRNA) encoding-the

VSSC.

In one embodiment, the VSSC confers insecticide susceptibility to Musca domestica. An example of such an insecticide susceptible VSSC is the channel encoded by the nucleotide sequence as shown in SEQ ID NO:1. SEQ ID NO:1 is the DNA sequence of one allele of the VSSC of Musca domestica. The amino acid sequence encoded by this allele is shown in SEQ ID NO:3.

In another embodiment, the VSSC confers insecticide resistance to Musca domestica. An example of Ssuch an insecticide resistant VSSC is the channel encoded 15 by the nucleotide sequence as shown in SEQ ID NO:2. SEQ ID NO:2 is the DNA sequence of another allele of the VSSC of Musca domestica characteristic of the kdr insecticide resistant strain. The amino acid sequence encoded by this emutant allele is shown in SEQ ID NO:4.

The insecticide resistant allele preferably has the nucleotide sequence of a second nucleic acid molecule with one or more mutations therein, wherein the second nucleic acid molecule encodes an insecticide sensitive VSSC and wherein one or more mutations in the second nucleic acid molecule render the resulting VSSC resistant to an insecticide (hence the term "mutant" allele). In one embodiment, the mutant allele (having amino acid SEQ ID NO:4) has the amino acid sequence encoded by the susceptibility allele (amino acid SEQ ID NO:3) with amino acid differences as follows: a substitution of phenylalanine for leucine at amino acid residue 1014 of SEQ ID NO:3; a substitution of isoleucine for methionine at amino acid residue 1140 of SEQ ID NO:3; a substitution of aspartic acid for glycine at amino acid residue 2023 of SEQ ID NO:3; a deletion of amino acid residues 2031-2034 WO 98/28446 PCT/US97/24256 14 of SEQ ID NO:3 (glycine-alanine-threonine-alanine); a substitution of threonine for serine at amino acid residue 2042 of SEQ ID NO:3; a substitution of alanine for valineat amino acid residue 2054 of SEQ ID NO:3; and an insertion of three amino acid residues (asparagineglycine-glycine) after amino acid residue 2055 of SEQ ID NO:3 (between amino acid residues 2055 and 2056 of SEQ ID NO:3). One or more of these amino acid differences can be included in an insecticide resistant VSSC. Other suitable sites for mutations can be identified by conventional, molecular genetic approaches, such as the identification of amino acid sequence substitutions/insertions/deletions in the VSSC sequences of other insecticide-resistant house fly strains.

15 The invention also provides an antisense nucleic acid molecule that is complementary to the mRNA encoding the VSSC, or a fragment thereof. Antisense nucleic acid molecules can be RNA or single-stranded

DNA.

Antisense molecules can be complementary to the entire DNA molecule encoding the VSSC, i.e. of the same nucleotide length as the entire molecule. It may be desirable, however, to work with a shorter molecule. In this instance, fragments of the entire antisense molecule can be used. Suitable fragments are capable of hybridizing to 25 the mRNA encoding the entire molecule, and preferably consist of at least twenty nucleotides. These antisense molecules and fragments thereof can be used to reduce steady state levels of a VSSC gene product of Musca domestica, by introducing into cells an RNA or singlestranded DNA molecule that is complementary to the mRNA of the VSSC by introducing an antisense molecule). The antisense molecule can base-pair with the mRNA of the VSSC, preventing translation of the mRNA into protein.

Thus, an antisense molecule to the VSSC of Musca domestica WO 98/28446 PCT/US97/24256 15 can prevent translation of mRNA encoding the VSSC into a functional sodium channel protein.

More particularly, an antisense molecule complementary to mRNA encoding a VSSC of Musca domestica, or a fragment thereof, can be used to decrease expression of a functional VSSC of Musca domestica. A cell with a first level of expression of a functional VSSC of Musca domestica is first selected, and then the antisense molecule (or fragment thereof) is introduced into the cell. .The antisense molecule (or fragment thereof) blocks expression of functional VSSCs of Musca domestica, resulting in a second level of expression of a functional VSSC of Musca domestica in the cell. The second level is less than the initial first level.

15 Antisense molecules can be introduced into cells by any suitable means. Suitable cells include e Xenopus oocytes which are useful host cells for studying the expression of the encoded sodium channel, and various insect cells, including but not limited to the insect cell lines Drosophila Schneider (Johansen et al. 1989), Drosophila K, (Sang 1981), Sf9 (Smith et al. 1983), and High Five® (see U.S. Patent No. 5,300,435). In one embodiment, the antisense RNA molecule is injected directly into the cellular cytoplasm, where the RNA interferes with translation. A vector may also be used for introduction of the antisense molecule into a cell.

Such vectors include various plasmid and viral vectors.

For a general discussion of antisense molecules and their use, see Han et al. 1991 and Rossi 1995.

The invention further provides a special category of antisense RNA molecules, known as ribozymes, having recognition sequences complementary to specific regions of the mRNA encoding the VSSC of Musca domestica.

Ribozymes not only complex with target sequences via complementary antisense sequences but also catalyze the WO 98/28446 PCT/US97/24256 16 hydrolysis, or cleavage, of the template mRNA molecule.

Examples, which are not intended to be limiting, of suitable regions of the mRNA template to be targeted by ribozymes are any of the regions encoding the 24 putative transmembrane domains of the VSSC of Musca domestica.

Expression of a ribozyme in a cell can inhibit gene expression (such as the expression of a VSSC of Musca domestica). More particularly, a ribozyme having a recognition sequence complementary to a region of a mRNA encoding a VSSC of Musca domestica can be used to decrease expression of a functional VSSC of Musca domestica.

A

cell with a first level of expression of a functional

VSSC

of Musca domestica is first selected, and then the ribozyme is introduced into the cell. The ribozyme in the 15 cell decreases expression of a functional VSSC of Musca Sdomestica in the cell, because mRNA encoding the VSSC is cleaved and cannot be translated.

Ribozymes can be introduced into cells by any suitable means. Suitable cells include Xenopus oocytes which are useful host cells for studying the expression of the encoded sodium channel, and various insect cells, including but not limited to the insect cell lines Drosophila Schneider, Drosophila K, Sf9, and High Five®.

In one embodiment, the ribozyme is injected directly into the cellular cytoplasm, where the ribozyme cleaves the mRNA and thereby interferes with translation. A vector may be used for introduction of the ribozyme into a cell.

Such vectors include various plasmid and viral vectors (note that the DNA encoding the ribozyme does not need to be "incorporated" into the genome of the host cell; it could be expressed in a host cell infected by a viral vector, with the vector expressing the ribozyme, for instance). For a general discussion of ribozymes and their use, see Sarver et al. 1990, Chrisey et al. 1991, Rossi et al. 1992, and Christoffersen et al. 1995.

WO 98/28446 PCTIUS97/24256 17 The nucleic acid molecules of the subject invention can be expressed in suitable host cells using conventional techniques. Any suitable host and/or vector system can be used to express the VSSCs. These include, but are not limited to, eukaryotic hosts such as mammalian cells Hela cells, Cv-1 cells, COS cells), Xenopus oocytes, and insect cells insect cell lines such as Drosophila Schneider, Drosophila Sf9, and High Five®).

Techniques for introducing the nucleic acid molecules into the host cells may involve the use of expression vectors which comprise the nucleic acid molecules. These expression vectors (such as plasmids and viruses; viruses including bacteriophage) can then be used to introduce the nucleic acid molecules into suitable host 15 cells. For example, sodium channel expression is often studied in Xenopus oocytes. DNA encoding the VSSC can be injected into the oocyte nucleus or transformed into the oocyte using a suitable vector, or mRNA encoding the VSSC can be injected directly into the oocyte, in order to obtain expression of a functional VSSC in the oocyte. It may be beneficial when expressing the sodium channels of the subject invention in Xenopus oocytes to coexpress a nucleic acid molecule encoding a tipE protein (Feng et al.

1995). Tip E has been found to be necessary to obtain 25 expression of some sodium channels in Xenopus oocytes (Feng et al. 1995).

Various methods are known in the art for introducing nucleic acid molecules into host cells. One method is microinjection, in which DNA is injected directly into the nucleus of cells through fine glass needles (or RNA is injected directly into the cytoplasm of cells). Alternatively, DNA can be incubated with an inert carbohydrate polymer (dextran) to which a positively charged chemical group (DEAE, for diethylaminoethyl) has been coupled. The DNA sticks to the DEAE-dextran via its WO 98/28446 PCT/US97/24256 18 negatively charged phosphate groups. These large DNAcontaining particles stick in turn to the surfaces of cells, which are thought to take them in by a process known as endocytosis. Some of the DNA evades destruction in the cytoplasm of the cell and escapes to the nucleus, where it can be transcribed into RNA like any other gene in the cell. In another method, cells efficiently take in DNA in the form of a precipitate with calcium phosphate.

In electroporation, cells are placed in a solution containing DNA and subjected to a brief electrical pulse that causes holes to open transiently in their membranes.

DNA enters through the holes directly into the cytoplasm, bypassing the endocytotic vesicles through which they pass in the DEAE-dextran and calcium phosphate procedures (passage through these vesicles may sometimes destroy or damage DNA). DNA can also be incorporated into artificial lipid vesicles, liposomes, which fuse with the cell membrane, delivering their contents directly into the cytoplasm. In an even more direct approach, used primarily with plant cells and tissues, DNA is absorbed to the surface of tungsten microprojectiles and fired into cells with a device resembling a shotgun.

**Several of these methods, microinjection, electroporation, and liposome fusion, have been adapted to introduce proteins into cells. For review, see Mannino and Gould-Fogerite 1988, Shigekawa and Dower 1988, Capecchi 1980, and Klein et al. 1987.

Further methods for introducing nucleic acid molecules into cells involve the use of viral vectors.

Since viral growth depends on the ability to get the viral genome into cells, viruses have devised clever and efficient methods for doing it. One such virus widely used for protein production is an insect virus, baculovirus. Baculovirus attracted the attention of researchers because during infection, it produces one of WO 98/28446 PCT/US97/24256 19 its structural proteins (the coat protein) to spectacular levels. If a foreign gene were to be substituted for this viral gene, it too ought to be produced at high level.

Baculovirus, like vaccinia, is very large, and therefore foreign genes must be placed in the viral genome by recombination. To express a foreign gene in baculovirus, the gene of interest is cloned in place of the viral coat protein gene in a plasmid carrying a small portion of the viral genome. The recombinant plasmid is cotransfected into insect cells with wild-type baculovirus DNA. At a low frequency, the plasmid and viral DNAs recombine through homologous sequences, resulting in the insertion of the foreign gene into the viral genome. Virus plaques develop, and the plaques containing recombinant virus look different because they lack the coat protein. The plaques with recombinant virus are picked and expanded. This virus stock is then used to infect a fresh culture of insect cells, resulting in high expression of the foreign protein. For a review of baculovirus vectors, see Miller 20 (1989). Various viral vectors have also been used to transform mammalian cells, such as bacteriophage, vaccinia virus, adenovirus, and retrovirus.

As indicated, some of these methods of transforming a cell require the use of an intermediate 25 plasmid vector. U.S. Patent No. 4,237,224 to Cohen and Boyer describes the production of expression systems in the form of recombinant plasmids using restriction enzyme cleavage and ligation with DNA ligase. These recombinant plasmids are then introduced by means of transformation and replicated in unicellular cultures including procaryotic organisms and eucaryotic cells grown in tissue culture. The DNA sequences are cloned into the plasmid vector using standard cloning procedures known in the art, as described by Sambrook et al. (1989).

WO 98/28446 PCT/US97/24256 20 Host cells into which the nucleic acid encoding the VSSC has been introduced can be used to produce (i.e.

to functionally express) the voltage-sensitive sodium channel.

Having identified the nucleic acid molecules encoding VSSCs and methods for expressing functional channels encoded thereby, the invention further provides a method of screening a chemical agent for the ability of the chemical agent to modify sodium channel function. The method comprises introducing a nucleic acid molecule encoding the VSSC into a host cell, and expressing the VSSC encoded by the molecule in the host cell. The expression results in the functional expression of a VSSC in the membrane of the host cell. The cell is then 15 exposed to a chemical agent and evaluated to determine if C the chemical agent modifies the function of the VSSC.

From this evaluation, chemical agents effective in altering the function of the sodium channel can be found.

Such agents may be, for example, tetrodotoxin, 20 veratridine, and scorpion venom toxins. -Additional agents can be found in Soderlund and Knipple 1994.

Cells transformed to include the VSSC according to the subject invention can be exposed to various potential insecticides and pesticides and evaluated for their susceptibility to the agents to develop and identify insect control agents that will not cause adverse effects to vertebrate species. Exemplary methods of screening are described in Eldefrawi et al. 1987 and Rauh et al. 1990.

The evaluation of the function of the sodium channel can be by any means known in the art. In one embodiment, the evaluation comprises monitoring sodium transport through the VSSC. Sodium transport can be monitored by preincubating cells in a medium containing one or more chemical agents, adding a medium containing radiosodium 22 Na) incubating the cells further in this medium, and WO 98/28446 PCT/US97/24256 21 isolating cells by filtration. Sodium transport is detected by the measurement of 22 Na* within the cells by liquid scintillation counting or other radiometric techniques (Bloomquist and Soderlund 1988).

Alternatively, ["C]guanidinium ion can be employed as the radiotracer in the place of sodium using the same procedure (Jacques et al. 1978). In another embodiment, the function of the VSSC can be evaluated by preincubating cells to equilibrium with a sodium-selective fluorescent chelating agent SBFI [sodium-binding benzofuran isophthalate]), washing the cells, exposing the cells to a test agent, and monitoring the increase in intracellular sodium by measuring the fluorescence of the SBFI-sodium complex (Deri and Adam-Vizi 1993).

The nucleic acid molecules of the subject invention can be used either as probes or for the design of primers to obtain DNA encoding other VSSCs by either cloning and colony/plaque hybridization or amplification using the polymerase chain reaction (PCR).

Specific probes derived from SEQ ID NOs 1 or 2 can be employed to identify colonies or plaques containing cloned DNA encoding a member of the VSSC family using known methods (see Sambrook et al. 1989). One skilled in the art will recognize that by employing such probes under high stringency conditions (for example, hybridization at 42 0 C with 5X SSPC and 50% formamide, washing at 50-650C with 0.5X SSPC), sequences having regions which are greater than 90% identical to the probe can be obtained.

Sequences with lower percent identity to the probe, which also encode VSSCs, can be obtained by lowering the stringency of hybridization and washing (for example, by reducing the hybridization and wash temperatures or reducing the amount of formamide employed).

More particularly, in one embodiment, the method comprises selection of a DNA molecule encoding a WO 98/28446 PCT/US97/24256 22 VSSC of an insect, or a fragment thereof, the DNA molecule having a nucleotide sequence selected from the group consisting of SEQ ID NO:1 and SEQ ID NO:2, and designing an oligonucleotide probe for a VSSC based on SEQ-ID NO:1 or SEQ ID NO:2. A genomic or cDNA library of an insect is then probed with the oligonucleotide probe, and clones are obtained from the library that are recognized by the oligonucleotide probe so as to obtain DNA encoding another

VSSC.

Specific primers derived from SEQ ID NOs 1 or 2 can be used in PCR to amplify a DNA sequence encoding a member of the VSSC family using known methods (see Innis et al. 1990). One skilled in the art will recognize that by employing such primers under high stringency conditions (for example, annealing at 50-60 0 C, depending on the length and specific nucleotide content of the primers employed), sequences having regions greater than identical to the primers will be amplified.

More particularly, in a further embodiment the 20 method comprises selection of a DNA molecule encoding a VSSC of an insect, or a fragment thereof, the DNA molecule having a nucleotide sequence selected from the group consisting of SEQ ID NO:1 and SEQ ID NO:2, designing degenerate oligonucleotide primers based on regions of SEQ ID NO:1 or SEQ ID NO:2, and employing such primers in the polymerase chain reaction using as a template a DNA sample to be screened for the presence of VSSC-encoding sequences. The resulting PCR products can be isolated and sequenced to identify DNA fragments that encode polypeptide sequences corresponding to the targeted region of a VSSC.

Various modifications of the nucleic acid and amino acid sequences disclosed herein are covered by the subject invention. These varied sequences still encode a functional VSSC. The invention thus further provides an WO 98/28446 PCT/US97/24256 23 isolated nucleic acid molecule encoding a VSSC of an insect, the nucleic acid molecule encoding a first amino acid sequence having at least 95% amino acid identity to a second amino acid sequence, the second amino acid sequence being as shown in SEQ ID NO:3. The resulting encoded VSSC is susceptible to an insecticide. The invention also provides an isolated nucleic acid molecule encoding a VSSC of an insect, the nucleic acid molecule encoding a first amino acid sequence having at least 95% amino acid identity to a second amino acid sequence, the second amino acid sequence being as shown in SEQ ID NO:4. The resulting VSSC is resistant to an insecticide.

The invention further provides isolated voltage-sensitive sodium channels of Musca domestica, wherein the VSSC is capable of conferring sensitivity or resistance to an insecticide in Musca domestica. In one embodiment, the VSSC confers susceptibility to an insecticide in Musca domestica, such as the VSSC encoded by the nucleotide sequence as shown in SEQ ID NO:1 (which 20 encodes an amino acid sequence as shown in SEQ ID NO:3) In a further embodiment, the VSSC confers resistance to an insecticide in Musca domestica, such as the VSSC encoded by the nucleotide sequence as shown in SEQ ID NO:3 (which encodes an amino acid sequence as shown in SEQ ID NO:4) Preferably, the insecticide resistant VSSC is encoded by a nucleic acid molecule having the nucleotide sequence of a second nucleic acid molecule with one or more mutations therein, wherein the second nucleic acid molecule encodes an insecticide sensitive VSSC, and wherein the one or more mutations in the second nucleic acid molecule render the resulting voltage-sensitive sodium channel resistant to an insecticide. For example, the nucleotide sequence of the second nucleic acid molecule may encode amino acid SEQ ID NO:3, and the insecticide resistant VSSC may have that amino acid sequence with one or more differences therein WO 98/28446 PCT/US97/24256 24 as follows: a substitution of phenylalanine for leucine at amino acid residue 1014 of SEQ ID NO:3; a substitution of isoleucine for methionine at amino acid residue 1140 of SEQ ID NO:3; a substitution of aspartic acid for-glycine at amino acid residue 2023 of SEQ ID NO:3; a deletion of amino acid residues 2031-2034 of SEQ ID NO:3 (glycinealanine-threonine-alanine); a substitution of threonine for serine at amino acid residue 2042 of SEQ ID NO:3; a substitution of alanine for valine at amino acid residue 2054 of SEQ ID NO:3; and an insertion of three amino acid residues (asparagine-glycine-glycine) after amino acid residue 2055 of SEQ ID NO:3 (between amino acid residues 2055 and 2056 of SEQ ID NO:3).

A variety of methodologies known in the art can be utilized to obtain an isolated VSSC according to the subject invention. In one method, the channel protein is purified from tissues or cells which naturally produce the channel protein. One skilled in the art can readily follow known methods for isolating proteins in order to obtain a member of the VSSC protein family, free of natural contaminants. These include, but are not limited to, immunochromatography, HPLC, size-exclusion chromatography, ion-exchange chromatography, and immunoaffinity chromatography. In another embodiment, a member of the VSSC family can be purified from cells which have been altered to express the channel protein. As used herein, a cell is said to be "altered to express the channel protein" when the cell, through genetic manipulation, is made to produce the channel protein which it normally does not produce or which the cell normally produces at low levels. One skilled in the art can readily adapt procedures for introducing and expressing either genomic, cDNA or synthetic sequences into. either eukaryotic or prokaryotic cells in order to generate a WO 98/28446 PCT/US97/24256 25 cell which produces a member of the VSSC family utilizing the sequences disclosed herein.

A VSSC as defined herein includes molecules encoding VSSCs encoded by an amino acid sequence-having at least 95% amino acid identity to SEQ ID NO:3 or to SEQ ID NO:4.

Antibodies can be raised to the voltagesensitive sodium channel. Antibodies of the subject invention include polyclonal antibodies and monoclonal antibodies capable of binding to the channel protein, as well as fragments of these antibodies, and humanized forms. Humanized forms of the antibodies of the subject invention may be generated using one of the procedures known in the art such as chimerization. Fragments of the antibodies of the present invention include, but are not limited to, the Fab, the Fab2, and the Fd fragments.

The invention also provides hybridomas which are capable of producing the above-described antibodies.

A hybridoma is an immortalized cell line which is capable 20 of secreting a specific monoclonal antibody.

In general, techniques for preparing polyclonal and monoclonal antibodies as well as hybridomas capable of producing the desired antibody are well known in the art (see Campbell 1984 and St. Groth et al. 1980). Any animal (mouse, rabbit, etc.) which is known to produce antibodies can be immunized with the antigenic channel protein (or an antigenic fragment thereof). Methods for immunization are well known in the art. Such methods include subcutaneous or intraperitoneal injection of the protein. One skilled in the art will recognize that the amount of the channel protein used for immunization will vary based on the animal which is immunized, the antigenicity of the protein, and the site of injection.

The protein which is used as an immunogen may be modified or administered in an adjuvant in order to WO 98/28446 PCT/US97/24256 26 increase the protein's antigenicity. Methods of increasing the antigenicity of a protein are well known in the art and include, but are not limited to, coupling the antigen with a heterologous protein (such as a globulin or beta-galactosidase) or through the inclusion of an adjuvant during immunization.

For monoclonal antibodies, spleen cells from the immunized animals are removed, fused with myeloma cells, such as SP2/O-Ag 15 myeloma cells, and allowed to become monoclonal antibody producing hybridoma cells.

Any one of a number of methods well known in the art can be used to identify the hybridoma cell which produces an antibody with the desired characteristics.

These include screening the hybridomas with an ELISA assay, western blot analysis, or radioimmunoassay (Lutz et al. 1988).

H ybridomas secreting the desired antibodies are cloned and the class and subclass are determined using procedures known in the art (Campbell 1984).

20 For polyclonal antibodies, antibody containing antisera is isolated from the immunized animal and is screened for the presence of antibodies with the desired specificity using one of the above-described procedures.

The present invention further provides the above-described antibodies in detectably labeled form.

Antibodies can be detectably labeled through the use of radioisotopes, affinity labels (such as biotin, avidin, etc.), enzymatic labels (such as horseradish peroxidase, alkaline phosphatase, etc.), fluorescent labels (such as FITC or rhodamine, etc.), paramagnetic atoms, etc.

Procedures for accomplishing such labeling are well known in the art, for example see Sternberger et al. 1970, Bayer et al. 1979, Engval et al. 1972, and Goding 1976.

The labeled antibodies or fragments thereof of the present invention can be used for in vitro, in vivo, WO 98/28446 PCTIUS97/24256 27 and in situ assays to identify cells or tissues which express a VSSC, to identify samples containing the VSSC proteins, or to detect the presence of a VSSC in a sample.

More particularly, the antibodies or fragments thereof can thus be used to detect the presence of a VSSC in a sample, by contacting the sample with the antibody or fragment thereof. The antibody or fragment thereof binds to any VSSC present in the sample, forming a complex therewith.

The complex can then be detected, thereby detecting the presence of the VSSC in the sample.

Fragments of the nucleic acid molecules encoding a VSSC are also provided, and are best defined in the context of amino acid sequence relationships among members of the VSSC sequence family and information on the function of specific VSSC domains. For example the amino acid sequence encoded by nucleotides 4648-4803 of SEQ ID NOs 1 or 2 encodes an amino acid sequence that is highly conserved among VSSC family members and is identified as the structural component forming the "inactivation gate" of sodium channels. Antibodies prepared-to the polypeptide encoded by this fragment would therefore be expected to be of use as reagents capable of detecting many members of the VSSC family. Such antibodies, if introduced into cells that express VSSCs, would also be expected to modify the normal function of the VSSCs expressed in those cells. In contrast, the amino acid sequence encoded by nucleotides 3079-3852 of SEQ ID NOs 1 or 2 encodes an amino acid sequence that is less well conserved between the VSSCs of the insects Musca domestica and Drosophila melanogaster. Antibodies prepared to the polypeptide encoded by this fragment would therefore be expected to recognize selectively the VSSC from which the fragment was derived.

Also provided by the subject invention is a plasmid designated pPJI1 and deposited with the ATCC under WO 98/28446 PCT[US97/24256 28 Accession No.97831, as well as a KpnI/AatII restriction fragment of about 3620 bp of the plasmid designated pPJIl.

Further provided is a plasmid designated pPJI2 and deposited with the ATCC under Accession No. 97832, as well as an AatII/SphII restriction fragment of about 2700 bp of the plasmid designated pPJI2. When the above two restriction fragments are ligated together at their AatII sites, the resulting nucleic acid molecule encodes a voltage-sensitive sodium channel which confers susceptibility to an insecticide in Musca domestica. This resulting nucleic acid molecule is also provided by the subject invention.

MATERIALS AND METHODS Heads of newly-emerged adult house flies (NAIDM or 538ge strain) (Knipple et al. 1994) were ground to a fine powder under liquid N 2 and extracted with acid guanidinium isothiocyanate/phenol/chloroform to obtain total RNA (Chomczynski and Sacchi 1987), which was 20 fractionated on oligo(dT)-paramagnetic beads (PolyATtract mRNA isolation system; Promega, Madison, WI) to obtain poly(A') RNA. Pools of first strand cDNA were synthesized using either random hexamers (Harvey and Darlison 1991) or oligo(dT) adapted for the 3'-RACE procedure (Frohman and Martin 1989). These cDNA pools were employed as templates in the polymerase chain reaction (PCR) (Saiki et al. 1988) to amplify overlapping cDNA segments spanning the entire Vsscl coding sequence. Mixed-sequence oligonucleotide primers employed for these amplifications comprised all possible sequence combinations encoding short 6-8 residues) regions of amino acid conservation between the para gene of D. melanogaster and rat brain sodium channel I (Loughney et al. 1989; Knipple et al. 1991). In a few cases, mixed-sequence primers were based solely on the D.

melanogaster sequence. Defined-sequence primers were WO 98/28446 PCT/US97/24256 29 derived either from the previously described 309nucleotide exon of the house fly Vsscl gene (Knipple et al. 1994) or from internal sequences of house fly cDNA fragments obtained by amplification with mixed-sequence primers. All primers were synthesized using an Applied Biosystems 392 instrument, deprotected using procedures provided by Applied Biosystems, desalted, and used without further purification. The sequences and designations of these primers are given in Table I. The methods and reagents employed in PCR amplifications are described elsewhere (Knipple et al. 1991; Henderson et al. 1994; Knipple et al. 1994); specific amplification conditions for each cDNA fragment were optimized by varying the annealing temperatures and extension times of the reaction. Following amplification, PCR products were separated from excess primers either by filtration of the reaction mixture through a Centricon-100 concentrator (Amicon, Beverly, MA) or by preparative electrophoresis on agarose gels, excision of the desired product, and 20 extraction from the gel matrix (QIAquick spin column; Qiagen, Chatsworth, CA) prior to use as templates for DNA sequencing.

The DNA sequences of amplified cDNA fragments were determined by automated sequencing with an Applied Biosystems 373 instrument using fluorescently-labeled dideoxynucleotides and Taq DNA polymerase (PCR/Sequencing Kit; Applied Biosystems, Foster City, CA) in a modification of the dideoxynucleotide chain-termination method (Sanger et al. 1977). Sequencing of each amplification product was initiated by using the amplification primers to sequence inward from the termini, and additional primers were synthesized as needed to obtain the complete sequence of each strand. Mixedsequence amplification primers were employed for sequencing at concentrations 10-fold higher than that used WO 98/28446 PCT/US97/24256 30 for defined-sequence primers. All sequence ambiguities and apparent polymorphisms were resolved by performing additional multiple sequencing reactions. The full-length Vsscl coding sequences from the NAIDM and 538ge strains were compiled from 239 and 209 individual sequencing reactions, respectively, and were edited using the SeqEd software program (Applied Biosystems). Complete house fly Vsscl sequences were analyzed and compared with published sodium channel sequences using the DNASTAR software package (DNASTAR, Madison, WI).

EXAMPLE I SEQUENCING OF THE INSECTICIDE SENSITIVE VSSC OF HOUSE FLY As an expedient alternative to conventional iterative screenings of cDNA libraries, a sequencing strategy for the house fly Vsscl gene was based on the PCR 20 amplification and direct automated sequencing of overlapping cDNA fragments (Fig. The point of entry for this strategy was the 309-nucleotide exon of the house fly Vsscl gene identified previously from sequencing of cloned genomic DNA (Knipple et al. 1994). The use of 25 defined-sequence primers from this region (Table I, Al or B2) in combination with mixed-sequence primers encoding conserved amino acid sequences in either region IIS3 (A2) or the extracellular N-terminal domain (Bl) gave cDNA fragments A and B. A second point of entry was established 30 in homology domain IV using a pair of mixed-sequence primers (C1 and C2) to obtain fragment C. A primer (D2) designed from the internal sequence of fragment C, together with a mixed-sequence primer (D1) encoding a conserved amino acid motif in the short linker between homology domains III and IV, gave fragment D. A pair of defined-sequence primers (El, E2) based on internal WO 98/28446 PCT/US97/24256 31 sequences of fragments A and D gave the large fragment E, which spanned most of homology domain II and all of homology domain III. Fragment F, corresponding to the end of the coding sequence, was obtained using a-definedsequence primer (F2) derived from the internal sequence of fragment B and a mixed-sequence primer (Fl) derived from a segment of the D. melanogaster sequence upstream from the translation start site (Loughney et al. 1989). Similarly, fragment G, containing the 3' end of the coding sequence, was obtained using a defined-sequence primer (Gl) derived from the internal sequence of fragment C and a mixedsequence primer (G2) derived from a segment of the D.

melanogaster sequence downstream from the stop codon (Thackeray and Ganetzky 1994).

The complete coding sequence of the Vssc1"AI allele of the house fly, comprising a single open reading S. frame of 6318 nucleotides (SEQ ID NO:1), was determined by automated DNA sequencing using cDNA fragments A G as templates (Fig. This cDNA coded for a 2105-amino acid polypeptide (SEQ ID NO:3) with a predicted molecular weight of 236,671 Daltons that exhibited all of the common structural landmarks found in sodium channel a subunit :genes (Catterall 1992; Kallen et al. 1993) (see Fig. 3), including four large internally homologous subdomains (I- IV), each containing six hydrophobic putative transmembrane helices (S1-S6) and a conserved sequence element between domains S5 and S6 identified as an ion pore-forming domain. The deduced VssclNAID amino acid sequence also contained a conserved element in the S4 region of each homology domain, characterized by a repeated motif of positively-charged amino acids that are thought to form the voltage-sensing element of the channel, and a short segment of conserved sequence between homology domains III and IV that has been identified as the channel inactivation gate (see Fig. The deduced WO98/28446 PCT/US97/24256 32 VssclNAIDM protein contained 10 potential sites for N-linked glycosylation (Kornfeld and Kornfeld 1985), 6 of which occur in putative extracellular regions. These regions of other sodium channel a subunit sequences are also known to contain potential glycosylation sites (Catterall 1992; Kallen et al. 1993).

Vertebrate sodium channels are known to undergo functional regulation as the result of phosphorylation by cAMP-dependent protein kinases at sites in the intracellular linker between homology domains I and II and by protein kinase C at a site in the intracellular linker between homology domains III and IV (Catterall 1992; Kallen et al. 1993). The deduced VssclNAI N protein contained three potential cAMP-dependent protein kinase phosphorylation sites (Kemp and Pearson 1990) (Ser540, Ser557, and Ser628) in the cytoplasmic linker between homology domains I and II. The location of two of these (Ser540 and Ser557 of SEQ ID NO:3) corresponded to the cluster of four sites found in this region of vertebrate brain sodium channels that are implicated in sodium channel regulation (Catterall 1992). The deduced VssclAID protein also contained three additional potential phosphorylation sites (Ser1167, Serl207, and Ser2097 of SEQ ID NO:3) in other putative intracellular domains. The role of these phosphorylation sites in the regulation of insect sodium channels by cAMP-dependent protein kinase is not known. The deduced house fly voltage-sensitive sodium channel protein also contained two potential sites for protein kinase C phosphorylation (Serll91 and Ser1582 of SEQ ID NO:3) (Kemp and Pearson 1990), the latter of which is the conserved site located within the inactivation gate sequence of the cytoplasmic linker between domains III and IV. Although the conservation of this site implicates a role for protein kinase C in the regulation of insect WO 98/28446 PCT/US97/24256 33 sodium channels, such an effect has not been demonstrated experimentally.

The deduced VssclNAID protein was 90.0% identical to the most similar variant of the para gene product of D. melanogaster (SEQ ID NO:19) (Loughney et al.

1989; Thackeray and Ganetzky 1994) (Fig. The level of sequence identity was highest in the N-terminal intracellular domain, the linker between homology domains III and IV, and homology domain IV. The level of sequence identity was lowest in the intracellular C-terminal domain. Alignment of the Vsscl sequence with 12 other sodium channel a subunit sequences found in the GenBank database showed that the Vsscl and para gene products exhibited approximately the same degree of sequence similarity as homologous sodium channel a subunit isoforms from different vertebrate species. These findings confirm Sand extend previous observations (Williamson et al. 1993; Knipple et al. 1994), based on fragmentary genomic DNA and cDNA sequences, of the high degree of sequence similarity 20 between this house fly gene and the para gene of D.

melanogaster and reinforce the conclusion that Vsscl is the homolog of para in the house fly.

In D. melanogaster (Thackeray and Ganetzky 1994; O'Dowd et al. 1995) and Drosophila virilis (Thackeray and Ganetzky 1995), multiple sodium channel a subunit variants, each under specific developmental regulation, are generated from the para gene by the alternative usage of 8 exons (designated a-f, h, and i) located in homology domain II and portions of the cytoplasmic linker regions on either side of this domain.

Given the heterogeneity of sodium channel-encoding sequences found in these Dipteran species, it was surprising to detect only a single sequence variant among the pool of amplified house fly head cDNA fragments. The VssclNIDM sequence contained segments identical to exon a WO 98/28446 PCTI/US97/24256 34 and homologous (21 identical amino acids out of 24) to exon i of D. melanogaster. Recent studies suggest that both of these exons are required for the expression of high sodium current densities in embryonic D. melanogaster neurons (O'Dowd et al. 1995). In the region encoded by either exon c or exon d, the house fly sequence differs from both D. melanogaster sequences but is slightly more similar to exon d (50 identical amino acids out of than to exon c (49 identical amino acids out of 55). The house fly sequence lacked segments homologous to D.

melanogaster exons b, e, and f but contained a segment identical to exon h, which is a variable element found in some D. virilis sequences but not detected in D.

melanogaster. The house fly VssclNAIDM sequence described is thus characterized as structurally homologous to the a*b-c-d'e-f-hli* splice variant of D. melanogaster and D.

virilis. The identification of this molecular form as the predominant sodium channel sequence variant in house fly S* heads was unexpected because it has not been detected 20 among the arrays of splice variants detected in whole embryos or whole adults of either D. melanogaster or D.

virilis.

EXAMPLE II SEQUENCING OF THE INSECTICIDE RESISTANT VSSC OF HOUSE FLY The PCR amplification/ sequencing strategy 30 summarized in Fig. 2 was also employed to determine the sequence of Vsscl cDNAs from heads of the 538ge house fly strain that carries the kdr trait. The nucleotide sequence of the VSSC of the 538ge house fly is shown in SEQ ID NO:2, and the amino acid sequence is shown in SEQ ID NO:4. The amino acid sequence of 2104 residues (SEQ ID NO:4) encoded by the Vsscls 38se cDNA contained 12 amino acid WO 98/28446 PCT/US97/24256 35 differences compared to that of the VssclNAIDM sequence (SEQ ID NO:3) as follows: a substitution of phenylalanine for leucine at amino acid residue 1014 of SEQ ID NO:3; a substitution of isoleucine for methionine at amino acid residue 1140 of SEQ ID NO:3; a substitution of aspartic acid for glycine at amino acid residue 2023 of SEQ ID NO:3; a deletion of amino acid residues 2031-2034 of SEQ ID NO:3 (glycine-alanine-threonine-alanine); a substitution of threonine for serine at amino acid residue 2042 of SEQ ID NO:3; a substitution of alanine for valine at amino acid residue 2054 of SEQ ID NO:3; and an insertion of three amino acid residues (asparagineglycine-glycine) after amino acid residue 2055 of SEQ ID NO:3 (between amino acid residues 2055 and 2056 of SEQ ID NO:3). A comparison of the VssC153Bge (SEQ ID NO:4) and VsscNAI D (SEQ ID NO:3) amino acid sequences to the para sequence of the Canton-S strain of D. melanogaster (SEQ ID NO:19) is shown in Fig. 3. The locations and amino acid sequence context of the differences are shown in Fig. 4.

In Fig. 4, S refers to the NAIDM amino acid sequence (SEQ ID NO:3), and R refers to the kdr sequence (SEQ ID NO:4).

Dashes indicate that the Kdr sequence has the identical residue at that position as does the NAIDM sequence. The difference labeled 1 shows amino acids 1009-1019 of SEQ ID NO:3, with the amino acid substitution at residue 1014 shown. The difference labeled 2 shows amino acids 1135- 1145 of SEQ ID NO:3, with the amino acid substitution at residue 1140 shown. The difference labeled 3 shows amino acids 2018-2028 of SEQ ID NO:3, with the amino acid substitution at residue 2023 shown. The difference labeled 4 shows amino acids 2027-2038 of SEQ ID NO:3, with the deletion of residues 2031-2034 shown. The difference labeled 5 shows amino acids 2037-2047 of SEQ ID NO:3, with the amino acid substitution at residue 2042 shown. The difference labeled 6 shows amino acids 2051-2059 of SEQ ID WO 98/28446 PCT/US97/24256 -36 NO:3, with the amino acid substitution at residue 2054 shown and the insertion of three residues between 2055 and 2056 shown.

Although preferred embodiments have been depicted and described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions and the like can be made without departing from the spirit of the invention and these are therefore considered to be within the scope of the invention as defined in the claims which follow.

Table 1. Names and sequences of oligonucleotide primers used in the PCR amplification of partial Vsscl2 cDNAs.

Name Sequence Al SI-CGGTTGGGCTTTCCTGTC-31 SEQ ID A2 5' -GGGAATTCRAADATRTTCCANCCYTC- 3' SEQ ID NO:6 Bi 51-CCCGARGAYATHGAYCYNTAYTA-31 SEQ ID NO:7 B2 51-CGTATCGCCTCCTCCTCG-31 SEQ ID NO:8 C1 5' -GGGTCTAGATHTTYGCNATHTTYGGNATG' 3' SEQ ID NO: 9 C2 5'-GGGGAATTCNGGRTCRAAYTGYTGCCA-3' SEQ ID NO:l0 D1 5'-GGGTCTAGARGANCARAARAARTAYTA-3' SEQ ID NO:1l D2 51-TCATACTTTGGCCCAATGTC-31 SEQ ID NO:12 25 El 51-CCCGAATTAGAGAAGGTGCTG-31 SEQ ID NO:13 E2 5'-ACTATTGCTTGTGGTCGCCAC-31 SEQ ID NO:14 Fl 51-CATCNTTRGCNGCNTAGACNATGAC-31 SEQ ID F2 5'-GATTGAATGGATCGAGCAGCC-31 SEQ ID NO:l6 G1 5" -CGTTTCTCCTTTCATATCTAG-31 SEQ ID NO:17 G2 51-GGAGBGGBGGNCKBGGNCKNGCTCA-31 SEQ ID NO:18 Designation of oligonucleotide mixtures: B=G+T+C; D=G+A+T; H=A+T+C; K=GiT; N=A+C+G+T; R=A+G; Y=C+T.

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Sawicki, R.M. Nature 275 (5679) :443-444 (1978) *.:Shigekawa, K. andoe, W.J. BioTechniques 6:742-751 (1988).

Smith, G.E. et al. Mol Cell Biol 3:2156-216S (1983).

Soderlund, D.M. and Bloomquist, J.R. Annu Rev Entomol 34 :77-96 (198 9).

Soderlund, D.M. and Bloomquist, in Pesticide Resistance in Arthropods (Edited by R.T.Roush and B.E.

WO 98/28446 PCT1US97/24256 Tabashnik), pp. 58-96. Chapman and Hall, New York, NY (1990).

Soderlund, and Knipple, in Molecular Action of Insecticides on Ion Channels, eds. Clark, American Chemical Society, Washington, pp. 97-108 (1994).

St. Groth, et al., J mmunol Methods 35:1-21 (1980).

Sternberger, et al., J Histochem Cytochem 18:315 (1970).

Stuhmer, Methods in Enzyrnology 207:319-339 (1992).

Suzuki et al., Proc Natl Acad Sci USA 68:890-893 (1971).

Taglialatela, et al., Biophys J 61:78-82 (1992).

Taylor, J Theor Biol 119:205-218 (1986).

Thackeray, J.R. and Ganetzky, J Neuroscj 14:2569-2578 (1994).

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Williamson, et al., 14o1 Gen Genet 240:17-22 (1993).

:0 0 :0.:0 0 0* 0 0. are claims pages they appear after the sequence listing WO 98/28446 PCT/US97/24256 41 SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: Cornell Research Foundation, Inc-.

(ii) TITLE OF INVENTION: INSECT SODIUM CHANNELS FROM INSECTICIDE-SUSCEPTIBLE

AND

INSECTICIDE-RESISTANT

HOUSE

FLIES

(iii) NUMBER OF SEQUENCES: 19 (iv) CORRESPONDENCE

ADDRESS:

ADDRESSEE: NIXON, HARGRAVE, DEVANS DOYLE LLP STREET: Clinton Square, P.O. Box 1051 CITY: Rochester STATE: New York COUNTRY: USA ZIP: 14603 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.30 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: FILING DATE:

CLASSIFICATION:

i: (vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: US 08/772,512 FILING DATE: 24-DEC-1996 (viii) ATTORNEY/AGENT INFORMATION: NAME: Goldman, Michael L.

REGISTRATION NUMBER: 30,727 REFERENCE/DOCKET NUMBER: 19603/602 (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: 716-263-1304 TELEFAX: 716-263-1600 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 6318 base pairs TYPE: nucleic acid WO 98/28446 PCTIUS97/24256 -42- STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:

ATGACAGAAG

CGCGAATCAT

GAAAGAAAGA

GGTCCACAGC

AGCTTCCCGC

GTACTGACAT

GCAATGTGGC

CATCCCTTGT

ATGCCGACAA

GAATCAGCTG

GATGCATGGA

GATTTAGGTA

GCCATTGTGC

CGCGATGTGA

ATCTATATGG

GGCAATCTGA

GAGAACGATG

GAGGATTACG

GATTCATTCG

GATCTGTATC

ATCATCTTCC

TATGACGAAT

GAAGCTGAAG

GCTCAAGCGG

AAGAGTCCCA

GATGACAACA

ATTCCGACTC

TGTTACAAAT

GAGCCGCCGA

CGGATCCCAC

CGGAATTGGC

TTGTAGTAAT

TGCTCGATCC

TTTCGTTATT

CGCCCACGGT

TTAAAGTGAT

ATTGGCTGGA

ATCTCGCAGC

CAGGTCTAAA

TAATTTTGAC

GTGTTCTAAC

CCGATGAAAA

GCGAGTCATA

TCTGCCTGCA

GTTGGGCTTT

AGCACGTGCT

TAGGTTCATT

TGCAAAAGAA

AAGCGGCAGC

CTCAGGATGC

CGTACTCTTG

ACAAAGAGAA

GATATCTGAG

CGAACAACGT

AGGAGAGCAG

ACTTGAACAG

CTCCACTCCT

AAGTAAAGGA

ATTCAATCCG

CATTATCACC

CGAATCCACA

GGCACGAGGT

CTTCGTAGTA

TTTGAGAACA

AACCATTGTC

AATGTTTTCC

ACAAAAGTGC

CTGGTTTCTA

TCCGGTGTGC

GGGCTTCGGC

CCTGTCGGCG

GCAAGCAGCT

CTATCTTGTG

GGCCGAAGAA

AGCCAAGGCG

AGCGGATGCC

CATTAGCTAT

GATGTCCATA

GAAGAACGCA

ATCGCTGAAC

ATACGATATG

GGTGTGCCTA

CTCGAGGATA

.AAGGATATTT

ATACGTCGTG

ACTATTCTAA

GAGGTGATAT

TTCATTTTAT

ATAGCTTTAG

TTTAGGGTAC

GGTGCTGTCA

CTGTCGGTGT

ATTAAACGAT

CACAATAGCA

GGGAATGTAT

CCCAATCCCA

TTTCGTCTCA

GGACCCTGGC

AATTTG1ATTT

GAAGAGGCTG

GCCAAACTGG

GCTGCGGCAG

GAACTGTTTG

CGCAGCGTCG

TACCTGTTCG

TCGATCCCTT

TTCGTTTTTC

TAGCCATTTA

CTAATTGTAT

TCACCGGAAT

GCCCGTTTAC

CTTATGTGAC

TGCGAGCTCT

TTGAATCTGT-

TCGCGCTGAT

TCCCCCTGGA

ACAGTTCCAA

CCGGTGCGGG

ACTACGACTA

TGACCCAAGA

ACATGTTGTT

TGGCCATTGT

CCGAGGAGGA

AGGAGCGGGC

CTCTGCATCC

TTGGCGGCGA

AAGTGGAATC

AATGCAGGGC

CTACAGTAAT

TGCCTCAAAA

TATTTTAGTG

TTTAATGATA

CTACACATTT

GTATCTTAGA

CATGGGCATA

GAAAACCGTA

AAAAAATCTA

GGGCCTACAA

CGGCAGTTGG

TTGGTTTACG

ACAATGCGGC

CACCAGTTTC

TTTCTGGGAG

CTTTATAGTC

TGCCATGTCT

GGCGATACGA

CAATGTAGCA

CGAGATGGCA

GAAGGGCAAC

GGAGTCGGTG

GTTTGTTCCG TCCCTTCACC ATGAAAAACA AAAGGAGCTG ATGACGAGGA CGAAGATGAA 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 16?~0 AGCGTTATAC AAAGACAACC AGCACCTACC ACAGCACCCG CTACTAAAGT CCGTAAAGTT WO 98/28446 PCT/US97/24256 43 AGCACGACTT CCTTATCCTT ACCTGGTTCA CCATTTAACC TACGCCGGGG ATCACGTAGT a a a a a

TCACACAAGT

AAGCCATTGG

TCGAATGCCG

TGTAATTTAG

TCACATGGTG

AGCAAATTGC

AGTAGTACGG

ATGGGTCAGG

GAGCCCGTCC

ATTGAACAAG

GATGGTCCCA

TGTGTATGGG

TTCGATCCAT

GCCATGGATC

TTCTTCACGG

TACTACTTCC

GAATTGGGCC

GTATTCAAAT

ACAATGGGTG

GTGATGGGAA

CATGAATTAC

GTGCTGTGCG

TGTATACCCT

TTAGCTTTGC

GATACCAATA

CGTAATATTG

CAACCATCAG

GGCTTGATCA

ATGGAGTTCA

AACAACACAA

CTAAACCATA

ACACAATACG

AAATGGGCGT

TACTGCAAAC

ATATCAGGAT

TAACACCAAT

GTCCGAAGAG

GTTCTAGACA

TTCTTCATAT

ATTTATTGGG

TGGCATGGCG

GCAGTCGCAA

CACACGCAAT

CTGGTGGTGG

CTATCCCGAT

ATTATACAGA

CGAAGCTGGC

AAACTCAAAC

AGTGGTAGAC

CCGCTGGTCG

GCATAGTCGT

CATTCAAGGA

CATCGCCCTC

ACTGTTGTTG

GGTGTGGTTA

TCGTGGAGCT

CTTCATTACC

ATCACGACAT GAATCCGGAA CCACTTTTGC AATTGAAGCC AGGAAGGCTG GAACATTTTC TGGAGGGTGT CCAGGGCCTG TGGCAAAATC

ATGGCCCACA

CATTGGGTAA.TCTGACATTT

TGCAACTTTT

CGGAAAGAAC

CGCGCTGGAA CTTCACCGAC GAGAGTGGAT CGAGTCCATG TCTTCTTGGC CACGGTCGTG TTTTGTCCAA CTTCGGTTCA AAATAGCAGA GGCCTTCAAT CCGATTGTTT TAAGTTAATT AACATGGCGA TAATGAACTG

GGACGTTTTG

GCCCAGCAGC

AATGGTGCCA

ACCTCGCATC

GCCATGGGTG

CAATCAATCG

GCCAATCACA

AAAATAAAAC

ATGAAAGATG

GCTAGTGAAC

GAATACATCC

AAATTTCAGG

CTGTGTATTG

TTAGAGAAGG

AGCATGAAAC

GATTTCATTA

TCGGTGTTGA

CTCAATTTAC

GTACTTTGCA

TATATTGACC

TTCATGCACA

TGGGACTGCA

ATAGGCAATC

TCTAGTTTAT

CGTATTGCTC

CGAAATAAAT

GAGTTGGGTC

ACCCAGCTGG

AACAACAAGC

AACCACCAAG

GATGACACTG

GTATACCAGG

ATTTGCCCTA

TTATAGTACC

AATCAAGAAT

CCAGCACAAT

GTGCTGCAAC

AGGAACAAAG

ACCACGACAA

TTATGGTCTT

GAGGTGAGGA

TAAAAGGCAT

AATGGGTGTC

TGGTCAATAC

TGCTGAAAAG

TGATGGCCAT

TTGTGGCCTT

GAAGTTTTCG

TCATTTCGAT

TTATCATCTT

ACAAGGATCG

GCTTCATGAT

TGTATGTGGG

TTGTGGTTCT

CAGCCCCGAC

GTTTTAAGAA

TGACAAATCA

ATGACGAAAT

AGGTGGCCAT

CGAAGAA.ATC

ACAATAGACT

CCAGCATTAA

TAGCGATCGC

TGCCGATGAC

AGCCTACTiAT

CTCGTATACA

GACCAAAGAG

CAATGGTGGC

GGATTATGAA

TCCTTTTATC

AAATGATATc

CGATGACGAA

CGAAATCTTT

CTTTATTGTG

GATGTTTATG

TGGTAACTAT

GAGCCCGAAG

GTCTCTGCTG

TTTGCTTCGT

rATGGGCCGG

CATCTTTGCC

CTTCAAGGAC

rGTGTTCCGA

CGATGTCAGC

TAATCTTTTC

TGCCGACAAT

CTGGGTGAAA

P.ATAAGTGAC

CAI GGCGAT

TGGCGATGGC

k~AAATTCATG

GGAACATGAG

CTCATATQGT

1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 3240 3300 3360 3420 3480 3 540

AAAAGGGTAT

CGATACATGG

CGATGATTGG

GAGGTTTGTC

GAAGGGCGAG

CGATATGAAA

AAACTCAATA

CATACAGGAC

WO 98/28446 PCTUS97/24256 44 a

AGCCATAAGA

GGCGAGGAGA

GAGGCCGAGG

GACGAGATAA

CCGATCTTGG

AAAACTTTTC

ATGAGTAGCT

GATATACTGT

AAATGGTTGG

GTGATTGTCA

GCCGTGTTTA

TGGGAGGGTA

GTGCTATTGG

GCTGGAAAAT

CCGAATCGTA

GATCATGTAG

CAGATTATGA

AATATCTACA

AATCTGTTCA

TCATTAGAAA

GGCTCTAAAA

TTCGAAATAG

ATGTTTACCA

AAACTCAATG

TTACGATATC

TCCATCTTAG

CTCCGTGTGG

GGTATCCGGA

CTGTTGCTGT

GTCAAAGAGA

ATATTGCTGT

ATCGACCATT

AACGCGACGT

GCGATGAGGG

TCGACGACTA

CCGGCGACGA

AATTAATTGA

TAG CTTTGGC

ACTACATGGA

CCCTGGGCTT

TGCTATCGCT

GATCAATGCG

TGAAAGTTGT

TGTGTCTGAT

ATTTTAAGTG

ATGCCTGCAA

GTAATGCGTA

ACGATGCCAT

TGTATTTATA

TTGGTGTTAT

TGTTCATGAC

AACCATTAAA

TTACAGATAA

TGACCCTCGA

GGATATTCGT

ACTATTTCAA

GTCTTGTACT

TGAGAGTGGC

CGTTGCTGTT

TCTTGGTGAT

AGAGCGGCAT

TTCAGATGTC

CAAGGACGAG

CAGCAAAGAG

CCAGCTGGAT

TCCGGCCGAC

GGACTCGCCG

AAATAAATAT

CTTAGAAGAT

CAGGATATTT

TAAGGTTTAC

TATAAATTTG

CACACTGCGC

CGTGAATGCG

ATTTTGGCTT

TAAAGATGGT

AAGTGAAAAC

TCTCTGTCTA

TGATTCACGA

TTTCGTATTC

CATTGATAAT

AGAAGATCAG

AGCCATTCCA

AAAATTCGAT

TCGGTACGAC

AGTTATTTTC

AGAGCCATGG

CAGCGACATC

CAAAGTGGGT

CGCGTTAGCC

GTTCATCTTT

AAATGCTGTG

TACCTCAGCC

AGCCACAAGG

GACCTCGGCC

GGTGACATTA

TGTTTCCCCG

TTCTGGCAAG

TTTGAAACCG

GTTCATTTAC

ACGGTGATAT

TTCACCAATG

GTTGCCGTTT

GCCCTAAGGC

CTGGTTCAAG

ATTTTTGCCA

AATGACACTG

TACACCTGGG

TTTCAAGTGG

GAGGTGGACA

TTCATTATAT

TTTAATGAAC

AAAAAGTACT

AGACCGAGGT

ATAATCATTA

GCCTCCGAGG

AGTGGCGAAT

AATTTATTTG

ATTGAGAAGT

CGTGTCCTGC

ATGTCGTTGC

GCTATCTTTG

TATAATTTTA

GGTTGGGATG

GCAGCGCCGA

TCGACGAGGA

TCATTCATGC

ACTCGTACTA

GATGGGGCAA

CAGTTATCAC

CCGATCGACC

TCTTTTTGGA

CCTGGTGTTG

GGTCGGGCTT

CATT6CGTGC

CTATACCGTC

TTATGGGAGT

TGCTGAGCCA

AAAATTCGGC

CCACCTTTAA

AGCAGCCGAT

TTGGATCATT-

AAAAGAAGAA

ATAATGCTAT

GGCGACCACA

TGTTGTTCAT

CGTACAACAA

GTCTATTAAA.

ATGTAGTAGT

ATTTCGTATC

GTTTAGTCAA

CTGCCTTATT

GCATGTCCTT

AGACATTTGG

GTGTGTTAGA

GACCATCGAG

ACTGGACGAG

GCAAAACGAC

CAAGAAGtffTT

TTTACGACTG

TATGATTTTA

TGTCATGCAG

GATGTTGATC

GCTGGATTTC

AAATGATATA

TGTCTCTAGA

CATCTTCAAT

ACAGCTTTTT

TGAAATCATA

AATGAACTTC

GGGCTGGATC

CCGAGAAACC

TTTCACACTC

AGCTGGTGGA

GAAAAAGATG

AGCAATAGTA

TGGCTTAAAC

TGTCCTCGAC

AATATTCGCT

TGTCATTTTA

GCCGACACTG

GGGTGCCAAG

CAACATTTGT

CTTCATGCAT

CCAAAGTATG

TGCCATTATC

3600 3660 3 7:20 3780 3840 3900 3960 4020 4080 4140 4200 4260 4320 4380 4440 4500 4560 4620 4680 4740 4800 4860 4920 4980 5040 5100 5160 5220 5280 5340 5400 5460 AATGAGGAAG ATTGCGATCC. ACCCGACAAC GACAAGGGCT ATCCGGGCAA TTGTGGTTCA WO 98/28446 PTU9145 PCTIUS97/24256 45 GCGACTGTTG GAATTACGTT AATATGTACA TTGCTGTCAT GGTCTCACCG ACGACGATTA GGCACCCAGT ACATACGCTA CTGCAGATCC ACAAGCCGAA GGCGACATGA TGTACTGTGT AAGGGTAATC CGATCGAGGA GAGGGCTATG ATCCGGTGTC CTGATACAGA ATGCGTGGCG GAGGCGGCTG GTGGCGAAGA GGCGGCGGTG ATGATGGTGG CCCTCAGATC CAGATGCCGG GGCTGTGTTA GTGGCGGCAG TTTGTTACAA AAAACGGTCA AGGACGGCAG ATGTCTGA

TCTCCTTTCA

TCTCGAGAAC

CGATATGTAC

CGACCAGCTG

CAAGTACAAA

GGATATATTG

GACGGGTGAA

GTCAACACTG

GCGTTACAAG

TGGTGCTGAA

CTCAGCGACA

CGAAGCAGAT

TAATGGCCGC

TAAGGTTGTA

TATCTAGTTA

TATAGCCAGG

TACGAGATTT

TCCGAGTTTC

ATCATATCGA

GATGCCCTGA

ATTGGTGAGA

TGGCGCCAGC

AATGGCCCAC

GGCGGTGAGG

GGAGCAACGG

GGTGCCAGCG

CAAACGGCCG

TAAGCTTTTT

CTACGGAGGA

GGCAACAATT

TGGACGTGCT

TGGACATGCC

CCAAGGACTT

TAGCGGCGCG

GTGAGGAGTA

CCCAGGAGGG

GTGAAGGAGG

CGGCGGCGGG

TCGGCGGCCC

TACTGGTCGA

GATAGTTATT

TGTACAGGAG

CGATCCGGAG

GGAGCCGC5CG

GATATGTCGG

CTTTGCGCGC

ACCGGACACC

CTGCGCCAAG

TGATGAGGGC

CAGCGGCGGC

AGCCACATCA

CCTTAGTCCG

AAGCGATGGT

CATAACATCC

5520 5580 5640 5700 5760 5820 5880 5940 6000 6060 6120 6180 6240 6300 6318 ATACACTCGA GATCGCCGAG INFORMATION FOR SEQ ID NO:2: i)SEQUENCE CHARACTERISTICS: LENGTH: 6315 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

ATGACAGAAG

CGCGAATCAT

GAAAGAAAGA

GGTCCACAGC

AGCTTCCCGC

GTACTGACAT

GCAATGTGGC

CATCCCTTGT

ATTCCGACTC

TGTTACAAAT

GAGCCGCCGA

CGGATCCCAC

CGGAATTGGC

TTGTAGTAAT

TGCTCGATCC

TTTCGTTATT

GATATCTGAG

CGAACAACGT

AGGAGAGCAG

ACTTGAACAG

CTCCACTCCT

AAGTAAAGGA

ATTCAATCCG

CATTATCACC

GAAGAACGCA

ATCGCTGAAC

ATACGATATG

GGTGTGCCTA

CTCGAGGATA

AAGGATATTT

ATACGTCGTG

ACTATTCTAA

GTTTGTTCCG

ATGAAAAACA

ATGACGAGGA

TACCTGTTCG

TCGATCCCTT

TTCGTTTTTC

TAGCCATTTA

CTAATTGTAT

TCCCTTCACC

A7LAGGAGCTG

CGAAGATGAA

AATGCAGGGC

CTACAGTAAT

TGCCTCAAAA

TATTTTAGTG

TTTAATGATA

120 180 240 300 360 420 480 WO 98/28446 PCTIUS97/24256 46- ATGCCGACAA CGCCCACGGT CGAATCCACA GAGGTGATAT TCACCGGAAT CTACACATTT

GAATCAGCTG

GATGCATGGA

GATTTAGGTA

GCCATTGTGC

CGCGATGTGA

ATCTATATGG

GGCAATCTGA

GAGAACGATG

GAAGATTACG

GACTCATTCG

GATCTGTATC

ATCATCTTCC

TATGACGAAT

GAAGCTGAAG

GCTCAAGCGG

AAGAGTCCCA

GATGACAACA

AGCGTTATAC

AGCACGACTT

TCACACAAGT

AAGCCATTGG

TCGAATGCCG

TGTAATTTAG

TCACATGGTG

AGCAAATTGC

AGTAGTACGG

ATGGGTCAGG

GAGCCCGTCC

ATTGAACAAG

GATGGTCCCA

TGTGTATGGG

TTAAAGTGAT

ATTGGCTGGA

ATCTCGCAGC

CAGGTCTAAA

TAATTTTGAC

GTGTTCTAAC

CCGATGAAAA

GCGAGTCATA

TCTGCCTGCA

GTTGGGCTTT

AGCACGTGCT

TAGGTTCATT

TGCAAAAGAA

AAGCGGCAGC

CTCAGGATGC

CGTACTCTTG

ACAAGGAGAA

AAAGACAACC

CCTTATCCTT

ACACAATACG

TACTGCAAAC

TAACACCAAT

GTTCTAGACA

ATTTATTGGG

GCAGTCGCAA

CCGGTGGTGG

ATTATACAGA

AAACTCAAAC

CCGCTGGTCG

CATTCAAGGA

ACTGTTGTTG

GGCACGAGGT

CTTCGTAGTA

TTTGAGAACA

AACCATTGTC

AATGTTTTCC

ACAAAAGTGC

CTGGTTTCTA

TCCGGTGTGC

GGGCTTCGGC

CCTGTCGGCG

GCAAGCAGCT

CTATCTTGTG

GGCCGAAGAA

AGCCAAGGCG

AGCGGATGCC

CATTAGCTAT

GATGTCGATA

AGCACCTACC

ACCTGGTTCA

AAATGGGCGT

ATATCAGGAT

GTCCGAAGAG

TTCTTCATAT

TGGCATGGCG

CACACGCAAT

CTATCCCGAT

CGAAGCTGGC

AGTGGTAGAC

GCATAGTCGT

CATCGCCCTC

GGTGTGGTTA

TTCATTTTAT

ATAGCTTTAG

TTTAGGGTAC

GGTGCTGTCA

CTGTCGGTGT

ATTAAACGAT

CACAATAGCA

GGGAATGTAT

CCCAATCCCA

TTTCGTCTCA

GGACCCTGGC

AATTTGATTT

GAAGAGGCTG

GCCAAACTGG

GCTGCGGCAG

GAACTGTTTG

CGCAGCGTCG

ACAGCACCCG

CCATTTAACC

GGACGTTTTG

GCCCAGCAGC

AATGGTGCCA

ACCTCGCATC

GCCATGGGTG

CAATCAATCG

GCCAATCACA

AAAATAAAAC

ATGAAAGATG

GCTAGTGAAC

GAATATATCC

AAATTTCAGG

GCCCGTTTAC GTATCTTAGA CTTATGTGAC CATGGGCATA TGCGAGCTCT GAAAACCdSTA TTGAATCTGT AAAAAATCTA TCGCGCTGAT GGGCCTACAA TCCCCCTGGA CGGCAGTTGG ACAGTTCCAA TTGGTTTACG CCGGTGCGGG ACAATGCGGC ACTACGACTA CACCAGTTTC TGACCCAAGA TTTCTGGGAG ACATGTTGTT CTTTATAGTC TGGCCATTGT TGCCATGTCT CCGAGGAGGA GGCGATCCGA AGGAGCGGGC CAATGTAGCA CTCTGCATCC CGAGATGGCA TTGGCGGCGA GAAGGGCAAC

AAGTGGAATC-GGAGTCGGTG

CTACTAAAGT CCGTAAAGTT TACGCCGGGG ATCACGTAGT GTATACCAGG TAGCGATCGC 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2"400

ATTTGCCCTA

TTATAGTACC

AATCAAGAAT

CCAGCACAAT

GTGCTGCAAC

AGGAACAAAG

ACCACGACAA

TTATGGTCTT

GAGGTGAGGA

TAAAAGGCAT

AATGGGTCTC

TGCCGATGAC

AGCCTACTAT

CTCGTATACA

GACCAAAGAG

CAATGGTGGC

GGATTATGAA

TCCTTTTATC

AAATGATATC

CGATGACGAA

CGAAATCTTT

CTTTATTGTG

WO 98/28446 PCTIUS97/24256 47

C

C.

C

C C. C C

C.

TTCGATCCAT

GCCATGGATC

TTCTTCACGG

TACTACTTCC

GAATTGGGCC

GTATTCAAAT

ACAATGGGTG

GTGATGGGAA

CATGAATTAC

GTGCTGTGCG

TGTATACCCT

TTAGCTTTGC

GATACCAATA

CGTAATATTG

CAACCATCAG

GGCTTGATCA

ATGGAGTTCA

AACAACACAA

CTAAACCATA

AGCCATAAGA

GGCGAGGAGA

GAGGCCGAGG

GACGAGATAA

CCGATCTTGG

AAAACTTTTC

ATGAGTAGCT

GATATACTGT

AAATGGTTGG

GTGATTGTCA

GCCGTGTTTA

TGGGAGGGTA

GTGCTATTGG

TCGTGGAGCT

ATCACGACAT

CCACTTTTGC

AGGAAGGCTG

TGGAGGGTGT

TGGCAAAATC

CATTGGGTAA

TGCAACTTTT

CGCGCTGGAA

GAGAGTGGAT

TCTTCTTGGC

TTTTGTCCAA

AAATAGCAGA

CCGATTGTTT

AACATGGCGA

AAAAGGGTAT

CGATACATGG

CGATGATTGG

GAGGTTTGTC

ATCGACCATT

AACGCGACGT

GCGATGAGGG

TCGACGACTA

CCGGCGACGA

AATTAATTGA

TAGCTTTGGC

ACTACATGGA

CCCTGGGCTT

TGCTATCGCT

GATCAATGCG

TGAAAGTTGT

TGTGTCTGAT

CTTCATTACC

GAATCCGGAA

AATTGAGGCC

GAACATTTTC

CCAGGGCCTG

ATGGCCCACA

TCTGACATTT

CGGAAAGAAC

TTTCACCGAC

CGAGTCCATG

CACGGTCGTG

CTTCGGTTCA

GGCCTTCAAT

TAAGTTAATT

TAATGAACTG

GAAGGGCGAG

CGATATGAAA

AAACTCAATA

CATACAGGAC

CAAGGACGAG

CAGCAAAGAG

CCAGCTGGAT

TCCGGCCGAC

GGACTCGCCG

AAATAAATAT

CTTAGAAGAT

CAGGATATTT

TAAGGTCTAC

TATAAATTTG

CACACTGCGC

CGTGAATGCG

ATTTTGGCTT

CTGTGTATTG

TTGGAGAAGG

AGCATGAAAC

GATTTCATTA

TCGGTGTTGA

CTGAATTTAC

GTACTTTGCA

TATATTGACC

TTCATGCACA

TGGGACTGCA

ATCGGCAAkTT

TCTAGTTTAT

CGTATTGCTC

CGAAATAAAT

GAGTTGGGTC

ACCCAGCTGG

AACAACAAGC

AACCACCAAG

GATGACACTG

AGCCACAAGG

GACCTCGGCC

GGTGACATCA

TGTTTCCCCG

TTCTGGCAAG

TTTGAAACCG

GTTCATTTAC

ACGGTGATAT

TTCACCAATG

GTTGCCGTTT

GCCCTAAGGC

CTGGTTCAAG

ATTTTTGCCA

TGGTCAATAC

AATGTTCATG

TGCTGAAAAG

TGGTAACTAT

TGATGGCCAT

GAGCCCGAAG

TTGTGGCCTT GTCTCTGdTG GAAGTTTTCG

TTTGCTTCGT

TCATTTCGAT

TATGGGCCGG

TTATCATCTT

CATCTTTGCC

ACICAGGATCG

CTTCAAGGAC

GCTTCATGAT

TGTGTTCCGA

TGTATGTGGG

CGATGTCAGC

TTGTGGTTCT TAATCTTTTC CAGCCCCGAC

TGCCGACAAT

GTTTTAAGAA

CTGGGTGAAA

TGACAAATCA

AATAAGTGAC

ATGACGAAAT

CATGGGCGAT

AGGTGGCCAT

TGGCGATGGC

CCAAGAAATC

AAAATTCATA

ACAATAGACT

GGAACATGAG

CCAGCATTAA

CTCATATGGT

GCAGCGCCGA

GACCATCGAG

TCGACGAGGA

ACTGGACGAG

TCATTCATGC

CCAAAACGAC

ACTCGTACTA

CAAGAAGTTT

GATGGGGCAA

TTTACGACTG

CAGTTATCAC

TATGATTTTA

CCGATCGACC

TGTCATGCAG

TCTTTTTGGA

GATGTTGATC

CCTGGTGTTG

GCTGGATTTC

GGTCGGGCTT

AAATGATATA

CATTGCGTGC

TGTCTCTAGA

CTATACCGTC

CATCTTCAAT

TTATGGGAGT ACAGCTTTTT 2460 2520 2580 2640 2700 2760 .2 820 2880 2940 3000 3060 3120 3180 3240 3300 3360 3420 3480 3540 3600 3660 3720 3780 3840 3900 3960 4020 4080 4140 4200 4260 4320 WO 98/28446 PTU9145 PCTIUS97/24256 48 S. S

SS

GCTGGAAAAT

CCGAATCGTA

GATCATGTAG

CAGATTATGA

AATATCTACA

AATCTGTTCA

TCATTAGAAA

GGCTCTAAAA

TTCGAAJATAG

ATGTTTACCA

AAACTCAATG

TTACGATATC

TCCATCTTAG

CTCCGTGTGG

GGTATCCGGA

CTGTTGCTGT

GTCAAAGAGA

ATATTGCTGT

AATGAGGAAG

GCGACTGTTG

AATATGTACA

GGTCTCACCG

GGTACCCAGT

CTGCAGATCC

GGCGACATGA

AAGGGTAATC

GAGGGCTATG

CTGATACAGA

GAGGCGGCTG

GGCGGCGATG

GATGCCGGCG

ATTTTAAGTG

ATGCCTGCAA

GTAATGCGTA

ACGATGCCAT

TGTATTTATA

TTGGTGTTAT

TGTTCATGAC

AACCATTAAA

TTACAGATAA

TGACCCTCGA

GGATATTCGT

ACTATTTCAA

GTCTTGTACT

TGAGAGTGGC

CGTTGCTGTT

TCTTGGTGAT

AGAGCGGCAT

TTCAGATGTC

ATTGCGATCC

GAATTACGTT

TTGCTGTCAT

ACGACGACTA

ACATAAGATA

ACAAGCCGAA

TGTACTGTGT

CGATCGAGGA

ATCCGGTGTC

ATGCGTGGCG

GTGGCGAAGA

ATGATGGTGG

AAGCAGATGG

TAAAGATGGT

AAGTGAAAAC

TCTCTGTCTA

TGATTCACGA

TTTCGTATTC

CATTGATAAT

AGAAGATCAG

AGCCATTCCA

AAAATTCGAT

TCGGTACGAC

AGTTATTTTC

AGAGCCATGG

CAGCGACATC

CAAAGTGGGT

CGCGTTAGCC

GTTCATCTTT

AAATGCTGTG

TACCTCAGCC

ACCCGACAAC

TCTCCTTTCA

TCTCGAGAAC

TGATATGTAC

CGACCAGCTG

CAAGTACAAA

GGATATATTG

GACGGGTGAA

GTCGACACTG

GCGTTACAAG

TGGTGCTGAA

CTCAGCGACG

TGCCAGCGCC

AATGACACTG

TACACCTGGG

TTTCAAGTGG

GAGGTGGACA

TTCATTATAT

TTTAATGAAC

AAAAAGTACT

AGACCGAGGT

ATAATCATTA

GCCTCCGAGG

AGTGGCGAAT

AATTTATTTG

ATTGAGAAGT

CGTGTCCTGC

ATGTCGTTGC

GCTATCTTTG

TATAATTTTA

GGTTGGGATG

GACAAGGGCT

TATCTAGTTA

TATAGCCAGG

TACGAGATTT

TCCGAGTTCC

ATCATATCGA

GATGCCCTGA

ATTGGTGAGA

TGGCGCCAGC

AATGGCCCAC

GGCGGTGAGG

GCGGCGGGAG

GGCAATGGTG

TGCTGAGCCA

AAAATTCGGC

CCACCTTTAA

AGCAGCCGAT

TTGGATCATT

AAAAGAAGAA

ATAATGCTAT

GGCGACCACA

TGTTGTTCAT

CGTACAACAA

GTCTATTAAA

ATGTAGTAGT

ATTTCGTATC

GTTTAGTCAA

CTGCCTTATT

GCATGTCCTT

AGACATTTGG

GTGTGTTAGA-

ATCCGGGCAA

TAAGCTTTTT

CTACGGAGGA

GGCAACAATT

TGGACGTGCT

TGGACATGCC

CCAAGGACTT

TTGCGGCGCG

GTGAGGAGTA

CCCAGGAGGG

GTGAAGGCGG

CCACATCACC

GCGGCCCCCT

TGAAATCATA

AATGAACTTC

GGGCTGGATC

CCGAGAAICCC

TTTCACACTC

AGCAGGTGGA

GAAAAAGATG

AGCAATAGTA

tGGCTTAAAC

TGTCCTCGAC

AATATTCGCT

TGTCATTTTA

GCCGACACTG

GGGTGCCAAG

CAACATTTGT

CTTCATGCAT

CCAAAGTATG

TGCCATTATC

TTGTGGTTCA

GATAGTTATT

TGTACAGGAG

CGATCCGGAG

GGAGCCGCCG

GATATGTCGG

CTTTGCGCGC

ACCGGACACC

CTGCGCCAAG

TGATGAGGGC

CAGCGGCGGC

CACAGATCCA

TAGTCCGGGC

4380 4440 45 6o 4560 4620 4680 4740 4800 4860 4920 4980 5040 5100 5160 5220 5280 5340 5400 5460 5520 5580 5640 5700 57G0 5820 5880 5940 6000 6060 6120 6180 TGTGTTAGTG GCGGCAGTAA TGGCCGCCAA ACGGCCGTAC TGGTCGAAAGCG GGTT64 CGATGGTTTT WO 98/28446 WO 9828446PCTIUS97/24256 49 GTTACAAAAA ACGGTCATAA GGTTGTAATA CACTCGAGAT CGCCGAGCAT AACATCCAGG ACGGCAGATG TCTGA INFORMATION FOR SEQ ID NO:3: ()SEQUENCE CHARACTERISTICS: LENGTH: 2105 amino acids TYPE: amino acid STRANDEDNESS: not relevant TOPOLOGY: linear (ii) MOLECULE TYPE: protein 6300 6315 a.

a a a a.

(xi) SEQUENCE DE~ Met Thr Glu Asp Ser 1 5 Arg Pro Phe Thr Arg Glu His Giu Lys Gin 35 Giu Gin Ile Arg Tyr 50 Asp Pro Thr Leu Giu 65 Ser Phe Pro Pro Giu Phe Tyr Ser Asn Vai 100 Ile Phe Arg Phe Ser 115 Asn Pro Ile Arg Arg 130 Ser Leu Phe Ile Ile i4 5 Met Pro Thr Thr Pro 165 Ile Tyr Thr Phe Giu 180 Leu Cys Pro Phe Thr 195 3CRIPTION: Asp Ser Ile Ser Giu Giu Giu 10 Giu Lys Asp Gin 70 Leu Leu Ala Vai Thr 150 Thr Ser Tyr Leu Leu 40 Glu Val Ser Phe Lys 120 Ile Ile Giu Val Arg 200 Leu 25 Glu Asp Pro Thr Val 105 Ala Tyr Leu Ser Lys 185 Asp Gin Arg Glu Ile Pro 90 Val Met Ile Thr Thr 170 Val Ala Ile Lys Asp Pro 75 Leu Ile Trp Leu Asn 155 Giu Met Trp Giu Arg Giu Val Glu Ser Leu Vai 140 Cys Val Ala Asn Ile 220 Arg Gin Ala Gly Arg Asp Lys Leu 125 His Ile Ile Arg Trp 205 Ser Arg Ala Pro Met Ile Gly 110 Asp Pro Leu Phe Gly 190 Leu Leu Ile Giu Gin Gin Asp Lys Pro Leu Met Thr 175 Phe Asp Phe Ala Gly Pro Gly Pro Asp Phe Phe Ile 160 Gly Ile Phe SEQ ID NO:3: Val Val Ile Ala Leu Ala 210 Vai Thr Met Gly Asp Leu Gly Asn WO 98/28446 PCTIUS97/24256 50 Leu Ala Ala Leu Arg Thr 225 230 Phe Arg Vai Leu S. 5

S

S

S

Ala Ile Val Lys Vai Phe Lys Cys 290 Asp Giu 305 Giu Asn Gly Gin Pro Asn Ser Ala 370 His Val 385 Ile Ile Val Ala Ala Ala Lys Ala 450 Gin Asp 465 Lys Ser Glu Lys Vai Giu Pro Thr 530 Val Asn Ala 275 Ile Asn Asp Cys Tyr 355 Phe Leu Phe Met Giu 435 Ala Aia Pro Gly Val 515 Thr Pro Leu 260 Leu Lys Trp Gly Giy 340 Asp, Arg Gin Leu Ser 420 Giu Lys Ala Thr Asn 500 Glu Al a Gly 245 Arg Met Arg Phe Giu 325 Giu Tyr Leu Ala Gly 405 Tyr Giu Leu Asp Tyr 485 Asp Ser Pro Leu Asp Gly Phe Leu 310 Ser Asp Thr Met Ala 390 Ser Asp Aia Giu Ala 470 Ser Asp Giu Ala Lys Val Leu Pro 295 His Tyr Tyr Ser Thr 375 Gly Phe Giu Ile Glu 455 Ala Cys Asn Ser Thr 535 Thr Ile Gin 280 Leu Asn Pro Val Phe 360 Gin Pro Tyr Leu Arg 440 Arg Ala Ile Asn Val 520 Lys Ile Ile 265 Ile Asp Ser Val Cys 345 Asp Asp Trp Leu Gin 425 Giu Ala Ala Ser Lys 505 Ser Val Val 250 Leu Tyr Gly Asn Cys 330 Leu Ser Phe His Val 410 Lys Aia Asn Ala Tyr 490 Giu Val Arg Arg 235 Gly Thr Met Ser Ser 315 Gly Gin Phe Trp Met 395 Asn Lys Glu Val Leu 475 Glu Lys Ile Lys Ala Met Gly Trp 300 Ser Asn Giy Gly Glu 380 Leu Leu Ala Giu Ala 460 His Leu Met Gin Val 540 Val Ile Phe Ser 270 Val Leu 285 Gly Asn Asn Trp Val Ser Phe Gly 350 Trp Ala 365 Asp Leu Phe Phe Ile Leu Giu Giu 430 Ala Ala 445 Aia Gin Pro Glu Phe Val Ser Ile 510 Arg Gin 525 Ser Thr Ala Leu Lys Thr Val 240 Glu Ser 255 Leji Ser Thr Gin Leu Thr Phe Thr 320 Giy Ala 335 Pro Asn Phe Leu Tyr Gin Ile Val 400 Ala Ile 415 Giu Giu Ala Ala Ala Al1a Met Ala 480 Giy Gly 495 Arg Ser Pro Ala Thr Ser Leu 545 Ser Leu Pro Gly Ser Pro Phe Asn Leu 550 Arg Arg Gly Ser Arg Ser 555 560 WO 98/28446 PCT/US97/24256 51 Ser His Lys Tyr Thr Ile Arg Asn 565' Gly Gin Glu Ser 625 Ser Met Ile Pro Tyr 705 Glu Leu Glu Ala Cys 785 Phe Thr Lys Glu Glu 865 Glu Ser His Glu 610 Arg His Thr Gly Asp 690 Thr Pro Asn Arg Leu 770 Cys Asp Met Val Ala 850 Gly Leu Asp Leu 595 Asn His Gly Lys Ala 675 Ala Asp Val Asp Gly 755 Glu Trp Pro Phe Leu 835 Ser Trp Gly Arg 580 Pro Gly Ser Asp Glu 660 Ala Asn Glu Gin Ile 740 Glu Tyr Val Phe Met 820 Lys Met Asn Leu Lys Tyr Ala Ser Leu 645 Ser Thr His Ala Thr 725 Ile Asp Ile Trp Val 805 Ala Ser Lys Ile Glu 885 Pro Leu Ala Asp Ile Ile 615 Tyr Thr 630 Leu Gly Lys Leu Asn Gly Lys Glu 695 Gly Lys 710 Gin Thr Glu Gin Asp Asp Leu Lys 775 Leu Lys 790 Glu Leu Met Asp Gly Asn Leu Met 855 Phe Asp 870 Gly Val Val Asp 600 Val Ser Gly Arg Gly 680 Gin Ile Val Ala Glu 760 Gly Phe Phe His Tyr 840 Ala Phe Gin Gly Leu 585 Ser Pro His Met Ser 665 Ser Arg Lys Val Ala 745 Asp Ile Gin Ile His 825 Phe Met Ile Gly Gin Asn Ala Gin Ala 650 Arg Ser Asp His Asp 730 Gly Gly Glu Glu Thr 810 Asp Phe Ser Ile Leu 890 Thr Ala Tyr Ser 635 Ala Asn Thr Tyr His 715 Met Arg Pro Ile Trp 795 Leu Met Thr Pro Val 875 Ser Tyr Val Tyr 620 Arg Met Thr Ala Glu 700 Asp Lys His Thr Phe 780 Val Cys Asn Ala Lys 860 Ala Val Gin Thr 605 Cys Ile Gly Arg Gly 685 Met Asn Asp Ser Phe 765 Cys Ser Ile Pro Thr 845 Tyr Leu Leu Asp 590 Pro Asn Ser Ala Asn 670 Gly Gly Pro Val Arg 750 Lys Val Phe Val Glu 830 Phe Tyr Ser Arg Ala Met Leu Tyr Ser 655 Gin Gly Gin Phe Met 735 Ala Asp Trp Ile Val 815 Leu Ala Phe Leu Ser 895 Gin Ser Gly Thr 640 Thr Ser Tyr Asp Ile 720 Val Ser Ile Asp Val 800 Asn Glu Ile Gin Leu 880 Phe Arg Gly Arg Phe Gly Ile Pro 570 575 WO 98/28446 PCTIUS97/24256 -52 Arg Leu Leu Arg Val Phe Lys Leu Ala Lys Ser Trp Pro Thr Leu Asn 900 905 910 Leu Leu Ile Ser Ile Met Gly Arg Thr Met Gly Ala Leu Gly Asn Leu 915 920 925 Thr Phe Val Leu Cys Ile Ile Ile Phe Ile Phe Ala Vai Met Gf~y Met 930 935 940 Gin Leu Phe Gly Lys Asn Tyr Ile Asp His Lys Asp Arg Phe Lys Asp 945 950 955 960 His Giu Leu Pro Arg Trp Asn Phe Thr Asp Phe Met His Ser Phe Met 965 970 975 Ile Val Phe Arg Val Leu Cys Gly Glu Trp Ile Glu Ser Met Trp Asp 980 985 990 Cys Met Tyr Val Gly Asp Val Ser Cys Ile Pro Phe Phe Leu Ala Thr 995 1000 1005 Val Val Ile Gly Asn Leu Val Val Leu Asn Leu Phe Leu Ala Leu Leu 1010 1015 1020 Leu Ser Asn Phe Gly Ser Ser Ser Leu Ser Ala Pro Thr Ala Asp Asn 1025 1030 1035 1040 Asp Thr Asn Lys Ile Ala Giu Ala Phe Asn Arg Ile Ala Arg Phe Lys :::1045 1050 1055 *Asn Trp Val Lys Arg Asn Ile Ala Asp Cys Phe Lys Leu Ile Arg Asn 1060 1065 1070 *Lys Leu Thr Asn Gin Ile Ser Asp Gin Pro Ser Glu His Giy Asp Asn *1075 1080 1085 Glu Leu Glu Leu Gly His Asp Giu Ile Met Gly Asp Gly Leu Ile Lys 1090 1095 1100 :Lys Giy Met Lys Gly Giu Thr Gin Leu Giu Val Ala Ile Gly Asp Gly 1105 1110 1115 1120 Met Glu Phe Thr Ile His Gly Asp Met Lys Asn Asn Lys Pro Lys Lys 1125 1130 1135 Ser Lys Phe Met Asn Asn Thr Thr Met Ile Gly Asn Ser Ile Asn His 1140 1145 1150 *Gin Asp Asn Arg Leu Giu His Giu Leu Asn His Arg Gly Leu Ser Ile *.1155 1160 1165 Gin Asp Asp Asp Thr Ala Ser Ile Asn Ser Tyr Gly Ser His Lys Asn 1170 1175 1180 Arg Pro Phe Lys Asp Giu Ser His Lys Gly Ser Ala Giu Thr Ile Giu 1185 1190 1195 1200 Gly Glu Glu Lys Arg Asp Val Ser Lys Glu Asp Leu Gly Leu Asp Giu 1205 1210 1215 Glu Leu Asp Giu Giu Ala Glu Gly Asp Giu Gly Gin Leu Asp Gly Asp 1220 1225 1230 WO 98/28446 PCT/US97/24256 53 Ile Ile Ile His Ala Gin Asn Asp Asp Gu Ile Ile Asp Asp Tyr Pro 1235 1240 1245 Ala Asp Cys Phe Pro Asp Ser Tyr Tyr Lys Lys Phe Pro Ile Leu Ala 1250 1255 1260 Gly Asp Giu Asp Ser Pro Phe Trp Gin Gly Trp Gly Asn Leu Arg Leu 1265 1270 1275 1280 Lys Thr Phe Gin Leu Ile Giu Asn Lys Tyr Phe Glu Thr Ala Val Ile 1285 1290 1295 Thr Met Ile Leu Met Ser Ser Leu Ala Leu Ala Leu Giu Asp Vai His 1300 1305 1310 Leu Pro Asp Arg Pro Val Met Gin Asp Ile Leu Tyr Tyr Met Asp Arg 1315 1320 1325 Ile Phe Thr Val Ile Phe Phe Leu Giu Met Leu Ile Lys Trp, Leu Ala 1330 1335 1340 Leu Gly Phe Lys Val Tyr Phe Thr Asn Ala Trp, Cys Trp Leu Asp Phe 1345 1350 1355 1360 Val Ile Val Met Leu Ser Leu Ile Asn Leu Vai Ala Val Trp Ser Gly 1365 1370 1375 Leu Asn Asp Ile Ala Val Phe Arg Ser Met Arg Thr Leu Arg Ala Leu ***1380 1385 1390 *.*Arg Pro Leu Arg Ala Val Ser Arg Trp Giu Gly Met Lys Val Val Val 1395 1400 1405 Asn Ala Leu Val Gin Ala Ile Pro Ser Ile Phe Asn Vai Leu Leu Val 1410 1415 1420 Cys Leu Ile Phe Trp Leu Ile Phe Ala Ile Met Gly Val Gin Leu Phe 1425 1430 1435 1440 Gly Lys Tyr Phe Lys Cys Lys Asp Gly Asn Asp Thr Val Leu Ser 1445 1450 1455 His Glu Ile Ile Pro Asn Arg Asn Ala Cys Lys Ser Giu Asn Tyr Thr 1460 1465 1470 Trp Giu Asn Ser Ala Met Asn Phe Asp His Val Gly Asn Ala Tyr Leu 1475 1480 1485 Cys Leu Phe Gin Val Ala Thr Phe Lys Gly Trp Ile Gin Ile Met Asn *1490 1495 1500 Asp Ala Ile Asp Ser Arg Glu Val Asp Lys Gin Pro Ile Arg Giu Thr 1505 1510 1515 1520 Asn Ile Tyr Met Tyr Leu Tyr Phe Val Phe Phe Ile Ile Phe Gly Ser 1525 1530 1535 Phe Phe Thr Leu Asn Leu Phe Ile Gly Val Ile Ile Asp Asn Phe Asn 1540 1545 1550 Giu Gin Lys Lys Lys Ala Gly Gly Ser Leu Glu Met Phe Met Thr Giu 1555 1560 1565 WO 98/28446 PCTIUS97/24256 54- Asp Gin Lys Lys Tyr Tyr Asn Ala Met Lys Lys Met Gly Ser Lys Lys 1570 1575 1580 Pro Leu Lys Ala Ile Pro Arg Pro Arg Trp Arg Pro Gin Ala Ile Val 1585 1590 1595 1600 Phe Giu Ile Val Thr Asp Lys Lys Phe Asp Ile Ile Ile Met L(;u Phe 1605 1610 1615 Ile Gly Leu Asn Met Phe Thr Met Thr Leu Asp Arg Tyr Asp Ala Ser 1620 1625 1630 Glu Ala Tyr Asn Asn Val Leu Asp Lys Leu Asn Gly Ile Phe Val Val 1635 1640 1645 Ile Phe Ser Gly Giu Cys Leu Leu Lys Ile Phe Ala Leu Arg Tyr His 1650 1655 1660 Tyr Phe Lys Giu Pro Trp Asn Leu Phe Asp Val Val Val Val Ile Leu 1665 1670 1675 1680 Ser Ile Leu Gly Leu Val Leu Ser Asp Ile Ile Giu Lys Tyr Phe Val 1685 1690 1695 Ser Pro Thr Leu Leu Arg Val Val Arg Val Ala Lys Val Gly Arg Val 1700 1705 1710 *Leu Arg Leu Val Lys Gly Ala Lys Gly Ile Arg Thr Leu Leu Phe Ala *1715 1720 1725 .::Leu Ala Met Ser Leu Pro Ala Leu Phe Asn Ile Cys Leu Leu Leu Phe 1730 1735 1740 Leu Val Met Phe Ile Phe Ala Ile Phe Giy Met Ser Phe Phe met His *1745 1750 1755 -1760 .0 Val Lye Giu Lye Ser Gly Ile Asn Ala Val Tyr Asn Phe Lye Thr Phe 1765 1770 1775 S. ~Gly Gin Ser Met Ile Leu Leu Phe Gin Met Ser Thr Ser Ala Gly Trp :0*1780 1785 1790 Asp Gly Val Leu Asp Ala Ile Ile Asn Giu Giu Asp Cys Asp Pro Pro 1795 1800 1805 Asp Asn Asp Lye Gly Tyr Pro Gly Asn Cys Giy Ser Ala Thr Val Gly 1810 1815 1,820 00S 0 le Thr Phe Leu Leu Ser Tyr Leu Val Ile Ser Phe Leu Ile Val Ile *0 1825 1830 1835 1840 Asn Met Tyr Ile Ala Val Ile Leu Giu Asn Tyr Ser Gin Ala Thr Glu 1845 1850 1855 Asp Val Gin Glu Gly Leu Thr Asp Asp Asp Tyr Asp Met Tyr Tyr Glu 1860 1865 1870 Ile Trp Gin Gin Phe Asp Pro Glu Gly Thr Gin Tyr Ile Arg Tyr Asp 1875 1880 1885 Gin Leu Ser Glu Phe Leu Asp Vai Leu Giu Pro Pro Leu Gin Ile His 1890 1895 1900 WO 98/28446 PCTIUS97/24256 55 Lys Pro Asn Lys Tyr Lys Ile Ile Ser Met Asp Met Pro Ile Cys Arg 1905 1910 1915 1920 Gly Asp Met Met Tyr Cys Val Asp Ile Leu Asp Ala Leu Thr Lys Asp 1925 1930 1935 Phe Phe Ala Arg Lys Gly Asn Pro Ile Giu Giu Thr Gly Giu lIe Gly 1940 1945 1950 Giu Ile Ala Ala Arg Pro Asp Thr Giu Gly Tyr Asp Pro Val Ser Ser 1955 1960 1965 Thr Leu Trp Arg Gin Arg Giu Giu Tyr Cys Ala Lys Leu Ile Gin Asn 1970 1975 1980 Ala Trp Arg Arg Tyr Lys Asn Gly Pro Pro Gin Giu Gly Asp Giu Gly 1985 1990 1995 2000 Giu Ala Ala Gly Gly Giu Asp Gly Ala Giu Gly Gly Giu Gly Giu Gly 2005 2010 2015 Giy Ser Gly Gly Gly Gly Gly Asp Asp Gly Giy Ser Ala Thr Gly Ala 2020 2025 2030 Thr Ala Ala Ala Gly Ala Thr Ser Pro Ser Asp Pro Asp Ala Giy Glu 2035 2040 2045 Ala Asp Gly Ala Ser Val Gly Giy Pro Leu Ser Pro Gly Cys Val Ser *2050 2055 2060 Gly Gly Ser Asn Gly Arg Gin Thr Ala Val Leu Val Glu Ser Asp Gly 2065 2070 2075 2080 Phe Val Thr Lys Asn Gly His Lys Val Vai Ile His Ser Arg Ser Pro 02085 2090 2095 Ser Ile Thr Ser Arg Thr Ala Asp Val 2100 2105 INFORMATION FOR SEQ ID NO:4: i)SEQUENCE CHARACTERISTICS: 0 LENGTH: 2104 amino acids TYPE: amino acid STRANDEDNESS: not relevant TOPOLOGY: linear (ii)MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: Met Thr Giu Asp Ser Asp Ser Ile Ser Giu Giu Giu Arg Ser Leu Phe 1 5 10 Arg Pro Phe Thr Arg Giu Ser Leu Leu Gin Ile Giu Gin Arg Ile Ala 25 WO 98/28446 WO 9828446PCT/US97/24256 56 Giu Giu Asp Ser Phe Ile Asn Ser 145 Met Ile Leu Val Leu 225 Ala Val Val Lys Asp 305 Giu Gly His Gin Pro Phe Tyr Phe Pro 130 Leu Pro Tyr Cys Val 210 Ala Ile Lys Phe Cys 290 Giu Asn Gin Giu Ile Thr Pro S er Arg 115 Ile Phe Thr Thr Pro 195 Ile Ala Vai Asn 275 Ile Asn Asp Cys Lys Arg Leu Pro Asn 100 Phe Arg Ile Thr Phe 180 Phe Ala Leu Pro Leu 260 Leu Lys Trp Gly Gly 340 Gin Tyr Glu Glu Val Ser Arg Ile Pro 165 Giu Thr Leu Arg Gly 245 Arg Met Arg Phe Glu 325 Glu Lys Asp Gin 70 Leu Leu Ala Val Thr 150 Thr Ser Tyr Ala Thr 230 Leu Asp Gly Phe Leu 310 Ser Asp Leu 40 Giu Val Ser Phe Lys 120 Ile Ile Giu Val Arg 200 Val Arg Thr Ile Gin 280 Leu Asn Pro Val Giu Asp Pro Thr Val 105 Ala Tyr Leu Ser Lys 185 Asp Thr Val Ile Ile 265 Ile Asp Ser Val Cys 345 Arg Giu Ile Pro 90 Val Met Ile Thr Thr 170 Val Ala Met Leu Val 250 Leu Tyr Gly Asn Cys 330 Leu Arg Giu Val Giu Ser Leu Val 140 Cys Val Ala Asn Ile 220 Ala Ala Met Gly Trp 300 Ser Asn Gly Ala Gly Arg Asp Lys Leu 125 His Ile Ile Arg Trp 205 Asp Leu Val Phe Val 285 Gly Asn Vai Phe Ala Pro Met le Gly 110 Asp Pro Leu Phe Gly 190 Leu Leu Lys Ile Ser 270 Leu Asn Trp Ser Giy 350 Giu Gin Gln Asp Lys Pro Leu Met Thr 175 Phe Asp Gly Thr Giu 255 Leu Thr Leu Phe Gly 335 Pro Gly Pro Gly Pro Asp Phe Phe le 160 Gly Ile Phe Asn Val 240 Ser Ser Gin Thr Thr 320 Ala Asn Pro Asn Tyr 355 Asp Tyr Thr Ser Phe Asp Ser Phe Gly 360 Trp Ala Phe Leu 365 WO 98/28446 PCTIUS97124256 57 Ser His 385 Ile Val Ala Lys Gin 465 Lys Giu Val Pro Leu 545 Ser Gly Gin Glu Ser 625 Ser Met Ile Ala 370 Val Ile Ala Ala Ala, 450 Asp Ser Lys Giu Thr 530 Ser His Ser His Giu 610 Arg His Thr Giy Phe Leu Phe Met Giu 435 Ala Ala Pro Gly Val 515 Thr Leu Lys Asp Leu 595 Asn His Gly Lys Ala 675 Arg Gin Leu Ser 420 Giu Lys Ala Thr Asn 500 Glu Ala Pro Tyr Arg 580 Pro Giy Ser Asp Giu 660 Ala Leu Ala Gly 405 Tyr Glu Leu Asp Tyr 485 Asp Ser Pro Gly Thr 565 Lys Tyr Ala Ser Leu 645 Ser Thr Met Ala 390 Ser Asp Ala Glu Ala 470 Ser Asp Giu Ala Ser 550 Ile Pro Ala Ile Tyr 630 Leu Lys Asn Thr 375 Gly Phe Giu Ile Glu 455 Ala Cys Asn Ser Thr 535 Pro Arg Leu Asp Ile 615 Thr Giy Leu Gly Glu 695 Gin Pro Tyr Leu Arg 440 Arg Ala Ile Asn Val 520 Lys Phe Asn Val Asp 600 Val Ser Giy Arg Gly 680 Asp Trp Leu Gin 425 Giu Ala Ala Ser Lys 505 Ser Vai Asn Gly Leu 585 Ser Pro His Met Ser 665 Ser Phe Trp Glu 380 His Met Leu 395 Val Asn Leu 410 Lys Lys Ala Ala Glu Glu Asn Val Ala 460 Ala Leu His 475 Tyr Giu Leu 490 Giu Lys Met Val Ile Gin Arg Lys Val 540 Leu Arg Arg 555 Arg Gly Arg 570 Gin Thr Tyr Asn Ala Val Ala Tyr Tyr 620 Gin Ser Arg 635 Ala Ala Met 650 Arg Asn Thr Ser Thr Ala Phe Ile Giu Aia 445 Aia Pro Phe Ser Arg 525 Ser Gl y Phe Gin Thr 605 Cys Ile Gly Arg Gly 685 Phe Leu Giu 430 Aia Gin Giu Val Ile 510 Gin Thr Ser Gly Asp 590 Pro Asn Ser Aia Asn 670 Gly Ile Al7a 415 Giu Ala Ala Met Giy 495 Arg Pro Thr Arg Ile 575 Al a Met Leu Tyr Ser 655 Gli Gly Val 400 Ile Giu Ala Ala Ala 480 Gly Ser Ala Ser Ser 560 Pro Gin Ser Gly Thr 640 Thr Ser Tyr Asp Leu Tyr Gin Pro Asp 690 Ala Asn His Lys Gin Arg Asp Tyr Giu Met Gly Gin Asp WO 98/28446 PCT/US97/24256 58 Tyr 705 Glu Leu Glu Ala Cys 785 Phe Thr Lys Glu Glu 865 Glu Arg Leu Thr Gln 945 His Ile Cys Val Thr Asp Pro Val Asn Asp Arg Gly 755 Leu Glu 770 Cys Trp Asp Pro Met Phe Val Leu 835 Ala Ser 850 Gly Trp Leu Gly Leu Leu Leu Ile 915 Phe Val 930 Leu Phe Glu Leu Val Phe Met Tyr 995 Val Ile 1010 Glu Gin Ile 740 Glu Tyr Val Phe Met 820 Lys Met Asn Leu Arg 900 Ser Leu Gly Pro Arg 980 Val Gly Ala Thr 725 Ile Asp Ile Trp Val 805 Ala Ser Lys Ile Glu 885 Val Ile Cys Lys Arg 965 Val Gly Asn Gly 710 Gin Glu Asp Leu Leu 790 Glu Met Gly Leu Phe 870 Gly Phe Met Ile Asn 950 Trp Leu Asp Phe Lys Thr Gin Asp Lys 775 Lys Leu Asp Asn Met 855 Asp Val Lys Gly Ile 935 Tyr Asn Cys Val Val Ile Lys His His 715 Val Val Asp Met 730 Ala Ala Gly Arg 745 Glu Asp Gly Pro 760 Gly Ile Glu Ile Phe Gin Glu Trp 795 Phe Ile Thr Leu 810 His His Asp Met 825 Tyr Phe Phe Thr 840 Ala Met Ser Pro Phe Ile Ile Val 875 Gin Gly Leu Ser 890 Leu Ala Lys Ser 905 Arg Thr Met Gly 920 Ile Phe Ile Phe Ile Asp His Lys 955 Phe Thr Asp Phe 970 Gly Glu Trp Ile 985 Ser Cys Ile Pro 1000 Val Leu Asn Leu Lys His Thr Phe 780 Val Cys Asn Ala Lys 860 Ala Val Trp Ala Ala 940 Asp Met Glu Phe Asp Ser Phe 765 Cys Ser Ile Pro Thr 845 Tyr Leu Leu Pro Leu 925 Val Arg His Ser Phe Val Arg 750 Lys Val Phe Val Glu 830 Phe Tyr Ser Arg Thr 910 Gly Met Phe Ser Met 990 Leu Met 735 Al'a Asp Trp Ile Val 815 Leu Ala Phe Leu Ser 895 Leu Asn Gly Lys Phe 975 Trp Ala Val Ser Ile Asp Val 800 Asn Glu Ile Gin Leu 880 Phe Asn Leu Met Asp 960 Met Asp Thr Asp Asn Pro Phe Ile 720 1005 Phe Leu Ala Leu Leu 1020 1015 Leu Ser Asn 1025 Phe Gly Ser Ser Ser Leu Ser Ala Pro Thr Ala 1030 1035 Asp Asn 1040 WO 98/28446 PCT(US97I24256 59 Asp Thr Asn Lys Ile Ala Glu Ala Phe Asn Arg Ile Ala Arg Phe Lys 1045 1050 loss Asn Trp Val Lys Arg Asn Ile Ala Asp Cys Phe Lys Leu Ile Arg Asn 1060 1065 1070 Lys Leu Thr Asn Gin Ile Ser Asp Gin Pro Ser Glu His Gly Asp Asn 1075 1080 1085 Giu Leu Giu Leu Gly His Asp Giu Ile Met Gly Asp Gly Leu Ile Lys 1090 1095 1100 Lys Gly Met Lys Gly Glu Thr Gin Leu Giu Val Ala Ile Gly Asp Gly 1105 1110 1115 1120 Met Glu Phe Thr Ile His Gly Asp Met Lys Asn Asn Lys Pro Lys Lys 1125 1130 1135 Ser Lys Phe Ile Asn Asn Thr Thr Met Ile Gly Asn Ser Ile Asn His 1140 1145 1150 Gin Asp Asn Arg Leu Giu His Glu Leu Asn His Arg Gly Leu Ser Ile 1155 1160 1165 Gin Asp Asp Asp Thr Ala Ser Ile Asn Ser Tyr Gly Ser His Lys Asn 1170 1175 1180 Arg Pro Phe Lys Asp Glu Ser His Lys Gly Ser Ala Glu Thr Ile Giu 1185 1190 1195 1200 :::Gly Giu Giu Lys Arg Asp Val Ser Lys Giu Asp Leu Gly Leu Asp Glu 1205 1210 1215 :9Glu Leu Asp Glu Glu Ala Glu Gly Asp Giu Gly Gin Leu Asp Gly Asp *1220 1225 -1230 Ile Ile Ile His Ala Gin Asn Asp Asp Glu Ile Ile Asp Asp Tyr Pro 1235 1240 1245 :Ala Asp Cys Phe Pro Asp Ser Tyr Tyr Lys Lys Phe Pro Ile Leu Ala 1250 1255 1260 Gly Asp Giu Asp Ser Pro Phe Trp Gin Gly Trp Gly Asn Leu Arg Leu 1265 1270 1275 1280 *Lys Thr Phe Gin Leu Ile Giu Asn Lys Tyr Phe Glu Thr Ala Val Ile 1285 1290 1295 **Thr Met Ile Leu Met Ser Ser Leu Ala Leu Ala Leu Glu Asp Val His *:1300 1305 1310 Leu Pro Asp Arg Pro Val Met Gin Asp Ile Leu Tyr Tyr Met Asp Arg 1315 1320 1325 Ile Phe Thr Val Ile Phe Phe Leu Giu Met Leu Ile Lys Trp Leu Ala 1330 1335 1340 Leu Gly Phe Lys Val Tyr Phe Thr Asn Ala Trp Cys Trp Leu Asp Phe 1345 1350 1355 1360 Val Ile Val Met Leu Ser Leu Ile Asn Leu Val Ala Val Trp Ser Gly 1365 1370 1375 WO 98/28446 PCTIUS97/24256 Leu Asn Asp Ile Ala Val Phe Arg Ser Met Arg Thr Leu Arg Ala Leu 1380 .1385 1390 Arg Pro Leu Arg Ala Val Ser Arg Trp Giu Gly Met Lys Val Val Val 1395 1400 1405 Asn Ala Leu Val Gln Ala Ile Pro Ser Ile Phe Asn Val Leu Lgu Val 1410 1415 1420 Cys Leu Ile Phe Trp Leu Ile Phe Ala Ile Met Gly Vai Gin Leu Phe 1425 1430 1435 1440 Ala Gly Lys Tyr Phe Lys Cys Lys Asp Gly Asn Asp Thr Val Leu Ser 1445 1450 1455 His Giu Ile Ile Pro Asn Arg Asn Ala Cys Lys Ser Giu Asn Tyr Thr 1460 1465 1470 Trp, Giu Asn Ser Ala Met Asn Phe Asp His Val Gly Asn Ala Tyr Leu 1475 1480 1485 Cys Leu Phe Gin Val Ala Thr Phe Lys Gly Trp Ile Gin Ile Met Aen 1490 1495 1500 Asp Ala Ile Asp Ser Arg Giu Val Asp Lys Gin Pro Ile Arg Giu Thr 1505 1510 1515 1520 Asn Ile Tyr Met Tyr Leu Tyr Phe Val Phe Phe Ile Ile Phe Gly Ser 1525 1530 1535 :::Phe Phe Thr Leu Asn Leu Phe Ile Gly Val Ile Ile Asp Asn Phe Asn 1540 1545 1550 Giu Gin Lye Lys Lye Ala Gly Gly Ser Leu Giu Met Phe Met Thr Giu *1555 1560 1565 *Asp Gin Lys Lye Tyr Tyr Asn Ala Met Lye Lye Met Gly Ser Lye Lye 1570 1575 1580 :Pro Leu Lye Ala Ile Pro Arg Pro Arg Trp Arg Pro Gin Ala Ile Val 1585 1590 1595 1600 Phe Giu Ile Val Thr Asp Lye Lye Phe Asp Ile Ile Ile Met Leu Phe 1605 1610 1615 Ile Gly Leu Asn Met Phe Thr Met Thr Leu Asp Arg Tyr Asp Ala Ser 1620 1625 1630 **Glu Ala Tyr Asn Aen Val Leu Asp Lye Leu Aen Gly Ile Phe Val Val *:1635 1640 1645 Ile Phe Ser Gly Glu Cys Leu Leu Lye Ile Phe Ala Leu Arg Tyr His 1650 1655 1660 Tyr Phe Lye Giu Pro Trp Asn Leu Phe Asp Val Val Val Val Ile Leu 1665 1670 1675 1680 Ser Ile Leu Gly Let' Val Leu Ser Asp Ile Ile Git' Lye Tyr Phe Val 1685 1690 1695 Ser Pro Thr Let' Let' Arg Val Val'Arg Val Ala Lye Val Gly Arg Val 1700 1705 1710 WO 98/28446 PCTIUS97/24256 61- Leu Arg Leu Val Lys Gly Ala Lys Gly Ile Arg Thr Leu Leu Phe Ala 1715 1720 1725 Leu Ala Met Ser Leu Pro Ala Leu Phe Asn Ile Cys Leu Leu Leu Phe 1730 1735 1740 Leu Val Met Phe Ile Phe Ala Ile Phe Gly Met Ser Phe Phe Met His 1745 1750 1755 1760 Val Lys Giu Lys Ser Gly Ile.Asn Ala Val Tyr Asn Phe Lys Thr Phe 1765 1770 1775 Gly Gin Ser Met Ile Leu Leu Phe Gin Met Ser Thr Ser Ala Gly Trp 1780 1785 1790 Asp Gly Val Leu Asp Ala Ile Ile Asn Giu Glu Asp Cys Asp Pro Pro 1795 1800 1805 Asp Asn Asp Lys Gly Tyr Pro Gly Asn Cys Gly Ser Ala Thr Val Gly 1810 1815 1820 Ile Thr Phe Leu Leu Ser Tyr Leu Val Ile Ser Phe Leu Ile Val Ile 1825 1830 1835 1840 Asn Met Tyr Ile Ala Val Ile Leu Giu Asn Tyr Ser Gin Ala Thr Giu 1845 1850 1855 Asp Val Gin Giu Gly Leu Thr Asp Asp Asp Tyr Asp Met Tyr Tyr Glu 1860 1865 1870 I *le Trp, Gin Gin Phe Asp Pro Giu Gly Thr Gin Tyr Ile Arg Tyr Asp 1875 1880 1885 Gin Leu Ser Giu Phe Leu Asp Val Leu Giu Pro Pro Leu Gin le His 1890 1895 1900 Lys Pro Asn Lys Tyr Lys Ile Ile Ser Met Asp Met Pro Ile Cys Arg 1905 1910 1915 1920 :Giy Asp Met Met Tyr Cys Val Asp Ile Leu Asp Ala Leu Thr Lys Asp 1925 1930 1935 Phe Phe Ala Arg Lys Gly Asn Pro Ile Giu Giu Thr Gly Giu Ile Gly 1940 1945 1950 *Giu Ile Ala Ala Arg Pro Asp Thr Glu Gly Tyr Asp Pro Val Ser Ser 1955 1960 1965 ***Thr Leu Trp Arg Gin Arg Giu Giu Tyr Cys Ala Lys Leu Ile Gin Asn 1970 1975 1980 Ala Trp Arg Arg Tyr Lys Asn Giy Pro Pro Gin Giu Gly Asp Giu Gly 1985 1990 1995 2000 Giu Ala Ala Gly Gly Giu Asp Gly Ala Giu Gly Giy Giu Giy Giu Gly 2005 2010 2015 Gly Ser Giy Gly Gly Gly Asp Asp Asp Giy Gly Ser Ala Thr Ala Ala 2020 2025 2030 Gly Ala Thr Ser Pro Thr Asp Pro Asp Ala Gly Giu Ala Asp Gly Ala 2035 2040 2045 WO 98/28446 PCT/US97/24256 62 Ser Ala Gly Asn Gly Gly Gly Pro Leu Ser Pro Gly Cys Val Ser Gly 2050 2055 2060 Gly Ser Asn Gly Arg Gin Thr Ala Val Leu Val Glu Ser Asp Gly Phe 2065 2070 2075 2080 Val Thr Lys Asn Gly His Lys Val Val Ile His Ser Arg Ser Pro Ser 2085 2090 2095 Ile Thr Ser Arg Thr Ala Asp Val 2100 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID CGGTTGGGCT TTCCTGTC 18 INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 26 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: GGGAATTCCA ACATCTTCCA CCCCTC 26 INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 23 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA WO 98/28446 PCT/US97/24256 63 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: CCCGACGACA TCGACCCCTA CTA 23 INFORMATION FOR SEQ ID NO:8: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: CGTATCGCCT CCTCCTCG 18 INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: LENGTH: 29 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: GGGTCTAGAT CTTCGCCATC TTCGGCATG 29 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 27 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID GGGGAATTCC GGCTCCAACT GCTGCCA 27 WO 98/28446 PCT/US97/24256 64 INFORMATION FOR SEQ ID NO:11: SEQUENCE CHARACTERISTICS: LENGTH: 27 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: GGGTCTAGAC GACCACAACA ACTACTA 27 INFORMATION FOR SEQ ID NO:12: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: TCATACTTTG GCCCAATGTC INFORMATION FOR SEQ ID NO:13: A e SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: CCCGAATTAG AGAAGGTGCT G 21 INFORMATION FOR SEQ ID NO:14: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid WO 98/28446 PCT/US97/24256 65 STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: ACTATTGCTT GTGGTCGCCA C 21 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 25 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID CATCCTTCGC CGCCTAGACC ATGAC INFORMATION FOR SEQ ID NO:16: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear

S

(ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: GATTGAATGG ATCGAGCAGC C 21 INFORMATION FOR SEQ ID NO:17: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA WO 98/28446 66 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:i7: CGTTTCTCCT TTCATATCTA G INFORMATION FOR SEQ ID NO:18: ()SEQUENCE

CHARACTERISTICS:

LENGTH: 25 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: GGAGCGGCGG ccccGGCCCC GCTCA INFORMATION FOR SEQ ID NO:19: SEQUENCE CHARACTERISTICS: LENGTH: 2100 amino acids TYPE: amino acid STRANDEDNESS: not relevant TOPOLOGY: linear (ii) MOLECULE TYPE: protein PCT/US97/24256 (xi) SEQUENCE DESCRIPTION: Met Thr Giu Asp Ser Asp Ser Ile 1 5 Arg Pro Phe Thr Arg Giu Ser Leu 20 Ala Giu His Giu Lys Gin Lys Glu 35 40 Giu Val Pro Arg Tyr Gly Arg Lys 55 Asp Asp Giu Asp Giu Asp Giu Gly 70 Gin Gly Val Pro Ile Pro Val Arg Leu Ala Ser Thr Pro Leu Giu Asp 100 SEQ ID NO:19: Ser Glu Giu.Giu Arg 10 Val Gin Ile Giu Gin 25 Leu Glu Arg Lys Arg Lys Lys Gin Lys Giu Pro Gin Pro Asp Pro 75 Leu Gin Gly Ser Phe 90 Ile Asp Pro Tyr Tyr 105 Leu Ile Giu Arg Leu Pro Asn Phe Ala Gly Tyr Glu Glu Val WO 98/28446 PCT/US97/24256 67 Leu Thr Phe Val Val Val Ser Lys Gly Lys Asp Ile Phe Arg Phe Ser 115 120 125 Ala Ser Lys Ala Met Trp Met Leu Asp Pro Phe Asn Pro Ile Arg Arg 0 0.

0 00..

o. Val 145 Thr Thr Ser Tyr Al a 225 Thr Leu Asp Gly Phe 305 Tyr Ser Asp Thr Met 385 Ala Ser .j 3 Ala Thr Val Ala Leu 210 Tyr Phe Lys Val Leu 290 Pro His Phe Tyr Ser 370 Thr Gly Phe Ile Ile Glu Vai 195 Arg Val Arg Thr Ile 275 Gin Leu Asn Pro Val 355 Phe Gin Pro Tyr Tyr Leu Ser 180 Lys Asp Thr Val Ile 260 Ile Ile Asp Arg Leu 340 Cys Asp Asp Trp Leu 420 Ile Val Thr Val Al a Met Leu 245 Val Leu Tyr Gly Asn 325 Cys Leu Ser Phe His 405 Val Leu 150 Asn Glu Met Trp Giy 230 Arg Gly Thr Met Ser 310 Ser Gly Gin Phe Trp 390 Met Asn Val Cys Val Ala Asn 215 le Ala Ala Met Gly 295 Trp Ser Asn Gly Gly 375 Glu Leu Leu His Ile Ile Arg 200 Trp Asp Leu Val Phe 280 Val Gly Asn Ile Phe 360 Trp Asp Phe Ile Pro Leu Phe 185 Gly Leu Leu Lys Ile 265 Ser Leu Asn Trp Ser 345 Gly Ala Leu Phe Leu 425 Leu Met 170 Thr Phe Asp Gly Thr 250 Glu Leu Thr Leu Tyr 330 Gly Pro Phe Tyr Ile 410 Ala Phe Ile Gly Ile Phe Asn 235 Val Ser Ser Glu Thr 315 Ser Ala Asn Leu Gin 395 Val Ile Leu Pro Tyr Cys 205 Val Ala Ile Lys Phe 285 Cys Giu Asp Gin Asn 365 Ala Val Ile Ala Phe Thr Thr 190 Pro Ile Ala Val Asn 270 Ala Ile Asn Glu Cys 350 Tyr Phe Leu Phe Met 430 Ile Thr 175 Phe Phe Ala Leu Pro 255 Leu Leu Lys Trp Gly 335 Asp Gly Arg Arg Leu 415 Ser Ile 160 Pro Glu Thr Leu Arg 240 Gly Arg Met Lys Asp 320 Ile Asp Tyr Leu Ala 400 Gly Tyr Asp Glu Leu 435 Gin Arg Lys Ala Glu Glu Glu Giu Ala Ala Giu Glu Giu WO 98/28446 PTU9/45 PCTIUS97/24256 68 Ala Giu 465 Ala Ser Asp Glu Gin 545 Gly Thr Lys Tyr Al a 625 Ser Leu Ser Asn Asp 705 His Asp Gly Ile 450 Giu Glu Cys Asn Ser 530 Ala Ser Ile Pro Ala 610 Ile Tyr Leu Lys Gly 690 Tyr His Met Arg Arg Arg Giu Ile Asn 515 Val Thr Pro Arg Leu 595 Asp Ile Thr Giy Leu 675 Gly Glu Asp Lys His 755 Glu Al a Ala Ser 500 Lys Ser Lys Phe Asn 580 Val Asp Val Ser Gly 660 Arg Thr Ile Asn Asp 740 Ser Ala Asn Ala 485 Tyr Giu Val Val Asn 565 Gly Leu Ser Pro His 645 Met Asn Thr Giy Pro 725 Val Arg Giu Ala 470 Leu Giu Lys Ile Arg 550 Ile Arg Ser Asn Val 630 Gin Ala Arg Cys Leu 710 Phe Met Ala GlU 455 Gin His Leu Met Gin 535 Lys Arg Gly Thr Ala 615 Tyr Ser Val Asn Leu 695 Glu Ile Val Ser Ala Aia Pro Phe Ser 520 Arg Val Arg Arg Tyr 600 Val Tyr Arg Met Thr 680 Asp Cys Glu Leu Asp 760 Ala Gin Giu Val 505 Ile Gin Ser Gly Phe 585 Gin Thr Gly Ile Gly 665 Arg Thr Thr Pro Asn 745 Arg Ala Ala 475 Ala Giy Ser Ala Thr 555 Arg Ile Ala Met Leu 635 Tyr Ser Gin His Glu 715 Gin Ile Giu Lys 460 Ala Lys Giu Val Pro 540 Ser Ser Pro Gin Ser 620 Gly Thr Thr Ser Lys 700 Ala Thr Ile Asp le 780 Ala Asp Ser Lys Glu 525 Thr Leu Ser Gly Gin 605 Giu Ser Ser Met Val 685 Leu Gly Gin Giu Asp 765 Ala Ala Pro Gly 510 Val Thr Ser His Ser 590 His Giu Arg His Thr 670 Gly Asp Lys Thr Gin 750 Asp Lys Al a Thir 495 Asn Giu Ala Leu Lys 575 Asp Leu Asn His Gly 655 Lys Ala His Ile Val 735 Ala Glu Leu Ala 480 Tyr Asp Ser His Pro 560 Tyr Arg Pro Gly Ser 640 Asp Giu Thr Arg Lys 720 Val Ala Asp Gly Pro 770 Thr Phe Lys Asp Lys Ala Leu Giu Vai Leu Lys Gly Ile WO 98/28446 PCTIUS97/24256 -69 Asp Vai Phe Cys Val Trp Asp Cys Cys Trp Val Trp Leu Lys Phe Gin 785 790 795 800 Giu Trp Vai Ser Leu Ile Val Phe Asp Pro Phe Val Giu Leu Phe Ile 805 810 815 Thr Leu Cys Ile Vai Val Asn Thr Met Phe Met Ala Met Asp Hfis His 820 825 830 Asp Met Asn Lys Giu Met Giu Arg Val Leu Lys Ser Gly Asn Tyr Phe 835 840 845 Phe Thr Ala Thr Phe Ala Ile Giu Ala Thr Met Lys Leu Met Ala Met 850 855 860 Ser Pro Lys Tyr Tyr Phe Gin Glu Gly Trp Asn Ile Phe Asp Phe Ile 865 870 875 880 Ile Val Ala Leu Ser Leu Leu Giu Leu Gly Leu Giu Gly Vai Gin Gly 885 890 895 Leu Ser Vai Leu Arg Ser Phe Arg Leu Leu Arg Val Phe Lys Leu Ala 900 905 910 Lys Ser Trp Pro Thr Leu Asn Leu Leu Ile Ser Ile Met Gly Arg Thr :.:915 920 925 *Met Giy Ala Leu Gly Asn Leu Thr Phe Val Leu Cys Ile Ile Ile Phe 930 935 940 Ile Phe Ala Val Met Giy Met Gin Leu Phe Gly Lys Asn Tyr His Asp 945 950 955 960 Lys Asp Arg Phe Pro Asp Gly Asp Leu Pro Arg Trp Asn Phe Thr 965 970 975 :Asp Phe Met His Ser Phe Met Ile Val Phe Arg Val Leu Cys Gly Giu 980 985 990 Trp Ile Giu Ser Met Trp Asp Cys Met Tyr Vai Gly Asp Val Ser Cys 995 1000 1005 .le Pro Phe Phe Leu Ala Thr Val Vai Ile Gly Asn Leu Val Val Leu 1010 1015 1020 ***Asn Leu Phe Leu Ala Leu Leu Leu Ser Asn Phe Gly Ser Ser Ser Leu .;1025 1030 1035 1040 Ser Ala Pro Thr Ala Asp Asn Asp Thr Asn Lys Ile Ala Giu Ala Phe 1045 1050 1055 Asn Arg Ile Gly Arg Phe Lys Ser Trp Val Lys Arg Asn Ile Ala Asp 1060 1065 1070 Cys Phe Lys Leu Ile Arg Asn Lys Leu Thr Asn Gin Ile Ser Asp Gin 1075 1080 1085 Pro Ser Giu His Gly Asp Asn Giu Leu Giu Leu Gly His Asp Giu Ile 1090 1095 1100 Leu Ala Asp Gly Leu Ile Lys Lys Gly Ile Lys Giu Gin Thr Gin Leu 1105 1110 1115 1120 WO 98/28446 PCT/US97/24256 70 Glu Val Ala Ile Gly Asp Gly Met Glu Phe Thr Ile His Gly Asp Met 1125 1130 1135 Lys Asn Asn Lys Pro Lys Lys Ser Lys Tyr Leu Asn Asn Ala Thr Asp 1140 1145 1150 Asp Asp Thr Ala Ser Ile Asn Ser Tyr Gly Ser His Lys Asn Arg Pro 1155 1160 1165 Phe Lys Asp Glu Ser His Lys Gly Ser Ala Glu Thr Met Glu Gly Glu 1170 1175 1180 Glu Lys Arg Asp Ala Ser Lys Glu Asp Leu Gly Leu Asp Glu Glu Leu 1185 1190 1195 1200 Asp Glu Glu Gly Glu Cys Glu Glu Gly Pro Leu Asp Gly Asp Ile Ile 1205 1210 1215 Ile His Ala His Asp Glu Asp Ile Leu Asp Glu Tyr Pro Ala Asp Cys 1220 1225 1230 Cys Pro Asp Ser Tyr Tyr Lys Lys Phe Pro Ile Leu Ala Gly Asp Asp 1235 1240 1245 Asp Ser Pro Phe Trp Gin Gly Trp Gly Asn Leu Arg Leu Lys Thr Phe 1250 1255 1260 Arg Leu Ile Glu Asp Lys Tyr Phe Glu Thr Ala Val Ile Thr Met Ile S1265 1270 1275 1280 Leu Met Ser Ser Leu Ala Leu Ala Leu Glu Asp Val His Leu Pro Gin S1285 1290 1295 Arg Pro Ile Leu Gln Asp Ile Leu Tyr Tyr Met Asp Arg Ile Phe Thr 1300 1305 1310 Val Ile Phe Phe Leu Glu Met Leu Ile Lys Trp Leu Ala Leu Gly Phe 1315 1320 1325 Lys Val Tyr Leu Thr Asn Ala Trp Cys Trp Leu Asp Phe Val Ile Val 1330 1335 1340 S* Met Val Ser Leu Ile Asn Phe Val Ala Ser Leu Val Gly Ala Gly Gly 1345 1350 1355 1360 Ile Gin Ala Phe Lys Thr Met Arg Thr Leu Arg Ala Leu Arg Pro Leu 1365 1370 1375 Arg Ala Met Ser Arg Met Gln Gly Met Arg Val Val Val Asn Ala Leu 1380 1385 1390 Val Gin Ala Ile Pro Ser Ile Phe Asn Val Leu Leu Val Cys Leu Ile 1395 1400 1405 Phe Trp Leu Ile Phe Ala Ile Met Gly Val Gin Leu Phe Ala Gly Lys 1410 1415 1420 Tyr Phe Lys Cys Glu Asp Met Asn Gly Thr Lys Leu Ser His Glu Ile 1425 1430 1435 1440 Ile Pro Asn Arg Asn Ala Cys Glu Ser Glu Asn Tyr Thr Trp Val Asn 1445 1450 1455 WO 98/28446 PCTIUS97/24256 -71 Ser Ala Met Asn Phe Asp His Val Gly Asn Ala Tyr Leu Cys Leu Phe 1460 1465 1470 Gin Val Ala Thr Phe Lys Giy Trp Ile Gin Ile Met Asn Asp Ala Ile 1475 1480 1485 Asp Ser Arg Giu Val Asp Lys Gin Pro Ile Arg Glu Thr Asn ie Tyr 1490 1495 1500 Met Tyr Leu Tyr Phe Val Phe Phe Ile Ile Phe Giy Ser Phe Phe Thr 1505 1510 1515 1520 Leu Asn Leu Phe Ile Gly Vai Ile Ile Asp Asn Phe Aen Giu Gin Lys 1525 1530 1535 Lys Lys Ala Gly Gly Ser Leu Giu Met Phe Met Thr Glu Asp Gin Lys 1540 1545 1550 Lye Tyr Tyr Ser Ala Met Lys Lys Met Gly Ser Lys Lys Pro Leu Lys 1555 1560 1565 Ala Ile Pro Arg Pro Arg Trp Arg Pro Gin Ala Ile Val Phe Glu Ile 1570 1575 1580 Val Thr Asp Lys Lys Phe Asp Ile Ile Ile Met Leu Phe Ile Gly Leu .*1585 1590 1595 1600 ::.Asn Met Phe Thr Met Thr Leu Asp Arg Tyr Asp Ala Ser Asp Thr Tyr 1605 1610 1615 Asn Ala Val Leu Asp Tyr Leu Asn Aia Ile Phe Val Val Ile Phe Ser *1620 1625 1630 *Ser Glu Cys Leu Leu Lys Ile Phe Ala Leu Arg Tyr His Tyr Phe Ile 1635 1640 1-645 ~Glu Pro Trp Asn Leu Phe Asp Val Val Val Val Ile Leu Ser Ile Leu bob.* 1650 1655 1660 Gly Leu Val Leu Ser Asp Ile Ile Glu Lys Tyr Phe Val Ser Pro Thr 1665 1670 1675 1680 *Leu Leu Arg Val Vai Arg Val Ala Lys Val Gly Arg Val Leu Arg Leu 1685 1690 1695 Lys Gly Ala Lye Gly Ile Arg Thr Leu Leu Phe Ala Leu Ala Met a~:1700 1705 1710 Ser Leu Pro Ala Leu Phe Asn Ile Cys Leu Leu Leu Phe Leu Val Met 1715 1720 1725 Phe Ile Phe Ala Ile Phe Gly Met Ser Phe Phe Met His Val Lye Giu 1730 1735 1740 Lye Ser Gly Ile Asn Asp Val Tyr Asn Phe Lys Thr Phe Gly Gin Ser 1745 1750 1755 1760 Met Ile Leu Leu Phe Gi'n Met Ser Thr Ser Ala Gly Trp Asp Gly Val 1765 1770 1775 Leu Asp Ala Ile Ile Asn Giu Giu Ala Cys Asp Pro Pro Asp Asn Asp 1780 1785 1790 WO 98/28446 PCT[US97/24256 -72- Lys Gly Tyr Pro Gly Asn Cys Gly Ser Ala Thr Val Gly Ile Thr Phe 1795 1800 1805 Leu Leui Ser Tyr Leu Val Ile Ser Phe Leu Ile Val Ile Asn Met Tyr 1810 1815 1820 Ile Ala Vai Ile Leu Giu Asn Tyr Ser Gin Ala Thr Glu Asp Val Gin 1825 1830 1835 1840 Glu Gly Leu Thr Asp Asp Asp Tyr Asp Met Tyr Tyr Glu Ile Trp Gin 1845 1850 1855 Gin Phe Asp Pro Giu Giy Thr Gin Tyr Ile Arg Tyr Asp Gin Leu Ser 1860 1865 1870 Giu Phe Leu Asp Val Leu Giu Pro Pro Leu Gin Ile His Lys Pro Asn 1875 1880 1885 Lys Tyr Lys Ile Ile Ser Met Asp Ile Pro Ile Cys Arg Gly Asp Leu 1890 1895 1900 Met Tyr Cys Val Asp Ile Leu Asp Ala Leu Thr Lys Asp Phe Phe Ala 1905 1910 1915 1920 Arg Lys Gly Asn Pro Ile Glu Giu Thr Gly Giu Ile Gly Glu Ile Ala 1925 1930 1935 :::Ala Arg Pro Asp Thr Giu Gly Tyr Glu Pro Val Ser Ser Thr Leu Trp 1940 1945 1950 Arg Gin Arg Giu Glu Tyr Cys Ala Arg Leu Ile Gin His Ala Trp Arg *1955 1960 1965 Lys His Lys Ala Arg Gly Glu Gly Gly Gly Ser Phe Glu Pro Asp Thr 1970 1975 1980 His Gly Asp Giy Gly Asp Pro Asp Ala Gly Asp Pro Ala Pro Asp 1985 1990 1995 2000 Glu Ala Thr Asp Gly Asp Ala Pro Ala Gly Gly. Asp Gly Ser Val Asn 2005 2010 2015 *Gly Thr Ala Giu Gly Ala Ala Asp Ala Asp Glu Ser Asn Val Asn Ser 2020 2025 2030 *Pro Gly Glu Asp Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala *:2035 2040 2045 Ala Ala Gly Thr Thr Thr Ala Gly Set Pro Gly Ala Gly Ser Ala Gly 2050 2055 2060 Arg Gin Thr Ala Val Leu Val Glu Ser Asp Gly Phe Val Thr Lys Asn 2065 2070 2075 2080 Gly His Lys Val Val Ile His Ser Arg Ser Pro Ser Ile Thr Ser Arg 2085 2090 2095 Thr Ala Asp Val 2100

Claims (67)

1. An isolated nucleic acid molecule encoding a voltage-sensitive sodium channel of Musca domestica, wherein said voltage-sensitive sodium channel is capable of conferring sensitivity or resistance to an insecticide in Musca domestica.

2. The isolated nucleic acid molecule of claim 1 wherein said nucleic acid is deoxyribonucleic acid.

3. The isolated nucleic acid molecule of claim 2 wherein said deoxyribonucleic acid is cDNA.

4. The isolated nucleic acid molecule of claim 1 wherein said voltage-sensitive sodium channel confers susceptibility to an insecticide in Musca domestica.

5. The isolated nucleic acid molecule of claim 4 wherein said nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO:1.

6. The isolated nucleic acid molecule of claim 4 wherein said nucleic acid molecule encodes an amino acid sequence as shown in SEQ ID NO:3.

7. The isolated nucleic acid molecule of claim 1 wherein said voltage-sensitive sodium channel confers resistance to an insecticide in Musca domestica.

8. The isolated nucleic acid molecule of claim 7 wherein said nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO:2. WO 98/28446 PCT/US97/24256 74

9. The isolated nucleic acid molecule of claim 7 wherein said nucleic acid molecule encodes an amino acid sequence as shown in SEQ ID NO:4. The isolated nucleic acid molecule of claim 7 wherein said nucleic acid molecule has the nucleotide sequence of a second nucleic acid molecule with one or more mutations therein, wherein said second nucleic acid molecule encodes an insecticide sensitive voltage- sensitive sodium channel of Musca domestica, and wherein said one or more mutations in said second nucleic acid molecule render the resulting voltage-sensitive sodium channel resistant to an insecticide.

11. The isolated nucleic acid molecule of claim 10 wherein said nucleotide sequence of said second nucleic acid molecule encodes amino acid SEQ ID NO:3, and wherein said one or more mutations in said second nucleic acid molecule are selected from the group consisting of a substitution for amino acid residue 1014 of SEQ ID NO:3, a substitution for amino acid residue 1140 of SEQ ID NO:3, a substitution for amino acid residue 2023 of SEQ ID NO:3, a deletion of one or more of amino acid residues 2031-2034 S: of SEQ ID NO:3, a substitution for amino acid residue 2042 of SEQ ID NO:3, a substitution for amino acid residue 2054 of SEQ ID NO:3, and an insertion of one to three amino acid residues between amino acid residues 2055 and 2056 of SEQ ID NO:3.

12. The isolated nucleic acid molecule of claim 1 wherein said nucleic acid is ribonucleic acid.

13. The isolated nucleic acid molecule of claim 12 wherein said ribonucleic acid is mRNA. WO 98/28446 PCT/US97/24256 75

14. An antisense nucleic acid molecule complementary to at least a portion of the mRNA of claim 13. An expression vector comprising the antisense nucleic acid molecule of claim 14.

16. The expression vector of claim 15 wherein the expression vector is a baculovirus.

17. A method of decreasing expression of a voltage-sensitive sodium channel in an insect, said method comprising infecting an insect with the baculovirus vector of claim 16, wherein infection of said insect by said baculovirus results in incorporation of said antisense nucleic acid molecule into the genome of said insect, thereby blocking expression of voltage-sensitive sodium channels in said insect cell.

18. A ribozyme having a recognition sequence complementary to a portion of the mRNA of claim 13.

19. An expression vector comprising the o ribozyme of claim 18.

20. The expression vector of claim 19 wherein the expression vector is a baculovirus.

21. A method of decreasing expression of a voltage-sensitive sodium channel in an insect, said method comprising infecting an insect with the baculovirus vector of claim 20, wherein infection of said insect by said baculovirus results in expression of said ribozyme in said insect, thereby decreasing expression of voltage-sensitive sodium channels in said insect cell. WO 98/28446 PCT/US97/24256 76

22. A cell comprising the nucleic acid molecule of claim 1.

23. The cell of claim 22 wherein the cell is a Xenopus oocyte.

24. The cell of claim 22 wherein the cell is an insect cell line. The cell of claim 24 wherein said insect cell line is selected from the group consisting of a Drosophila Schneider cell line, a Drosophila Kc cell line, an Sf9 cell line, and a High Five® cell line.

26. An expression vector comprising the nucleic acid molecule of claim 1.

27. The expression vector of claim 26 wherein said expression vector is selected from the group consisting of a plasmid and a virus.

28. A cell comprising the expression vector of claim 26.

29. The cell of claim 28 wherein the cell is a Xenopus oocyte. The cell of claim 28 wherein the cell is an insect cell line.

31. The cell of claim 30 wherein said insect cell line is selected from the group consisting of a Drosophila Schneider cell line, a Drosophila K c cell line, an Sf9 cell line, and a High Five® cell line. WO 98/28446 PCT/US97/24256 77

32. The isolated nucleic acid molecule of claim 1 wherein said insecticide is selected from the group consisting of DDT, DDT analogs, and pyrethroids.

33. A method of producing a voltage-sensitive sodium channel, said method comprising: introducing the nucleic acid molecule of claim 1 into a cell; and allowing said cell to express said nucleic acid molecule resulting in the production of a voltage- sensitive sodium channel in said cell.

34. The method of claim 33 wherein the cell is a Xenopus oocyte.

35. The method of claim 33 wherein the cell is an insect cell line.

36. The method of claim 35 wherein said insect cell line is selected from the group consisting of a Drosophila Schneider cell line, a Drosophila K c cell line, an Sf9 cell line, and a High Five® cell line.

37. A method of producing a voltage-sensitive sodium channel, said method comprising: introducing the nucleic acid molecule of claim 1 and a second nucleic acid molecule encoding a tip E protein into a cell; and allowing said cell to coexpress said nucleic acid molecule and said second nucleic acid molecule, resulting in the production of a voltage- sensitive sodium channel in said cell.

38. The method of claim 37 wherein the cell is a Xenopus oocyte. WO 98/28446 PCT/US97/24256 78

39. The method of claim 37 wherein the cell is an insect cell line. The method of claim 39 wherein said insect cell line is selected from the group consisting of a Drosophila Schneider cell line, a Drosophila K c cell line, an Sf9 cell line, and a High Five® cell line.

41. A method of screening a chemical agent for the ability of the chemical agent to modify sodium channel function, said method comprising: introducing the nucleic acid molecule of claim 1 into a host cell; expressing said voltage-sensitive sodium channel encoded by said nucleic acid molecule in the host cell so as to result in the functional expression of a voltage-sensitive sodium channel in the host cell; exposing the cell to a chemical agent; and evaluating the exposed cell to determine if the chemical agent modifies the function of the voltage- sensitive sodium channel.

42. The method of claim 41 wherein the cell is a Xenopus oocyte. The method of claim 41 wherein the cell is an insect cell line.

44. The method of claim 43 wherein said insect cell line is selected from the group consisting of a Drosophila Schneider cell line, a Drosophila K C cell line, an Sf9 cell line, and a High Five® cell line. The method of claim 41 wherein said evaluation comprises monitoring sodium transport through said voltage-sensitive sodium channel. WO 98/28446 PCT/US97/24256 79

46. The method of claim 41 wherein said evaluation comprises monitoring quanidinium transport through said voltage-sensitive sodium channel.

47. A method of screening a chemical agent for the ability of the chemical agent to modify sodium channel function, said method comprising: introducing the nucleic acid molecule of claim 1 and a second nucleic acid molecule encoding a tip E protein into a host cell; allowing said host cell to coexpress said nucleic acid molecule and said second nucleic acid molecule so as to result in the functional expression of a voltage-sensitive sodium channel in the host cell; exposing the cell to a chemical agent; and evaluating the exposed cell to determine if the chemical agent modifies the function of the voltage- sensitive sodium channel.

48. The method of claim 47 wherein the cell is a Xenopus oocyte.

49. The method of claim 47 wherein the cell is an insect cell line.

50. The method of claim 49 wherein said insect cell line is selected from the group consisting of a Drosophila Schneider cell line, a Drosophila K, cell line, an Sf9 cell line, and a High Five® cell line.

51. The method of claim 47 wherein said evaluation comprises monitoring sodium transport through said voltage-sensitive sodium channel. WO 98/28446 PCT/US97/24256 80

52. The method of claim 47 wherein said evaluation comprises monitoring quanidinium transport through said voltage-sensitive sodium channel.

53. A method of obtaining DNA encoding a voltage-sensitive sodium channel, said method comprising: selecting a DNA molecule encoding a voltage-sensitive sodium channel of an insect, said DNA molecule having a nucleotide sequence selected from the group consisting of SEQ ID NO:1 and SEQ ID NO:2; designing an oligonucleotide probe for a voltage-sensitive sodium channel based on SEQ ID NO:1 or SEQ ID NO:2; probing a genomic or cDNA library of an insect with the oligonucleotide probe; and obtaining clones from said library that are recognized by said oligonucleotide probe, so as to obtain DNA encoding a voltage-sensitive sodium channel.

54. A method of obtaining DNA encoding a voltage-sensitive sodium channel, said method comprising: selecting a DNA molecule encoding a voltage-sensitive sodium channel of an insect, said DNA molecule having a nucleotide sequence selected from the group consisting of SEQ ID NO:1 and SEQ ID NO:2; designing degenerate oligonucleotide primers based on SEQ ID NO:1 or SEQ ID NO:2; and utilizing said oligonucleotide primers in a polymerase chain reaction on a DNA sample to identify homologous DNA encoding a voltage-sensitive sodium channel in said sample. An isolated nucleic acid molecule encoding a voltage-sensitive sodium channel of an insect, said nucleic acid molecule encoding a first amino acid sequence WO 98/28446 PCT/US97/24256 81 having at least 95% amino acid identity to a second amino acid sequence, said second amino acid sequence being as shown in SEQ ID NO:3.

56. An isolated nucleic acid molecule encoding a voltage-sensitive sodium channel of an insect, said nucleic acid molecule encoding a first amino acid sequence having at least .95% amino acid identity to a second amino acid sequence, said second amino acid sequence being as shown in SEQ ID NO:4.

57. An isolated voltage-sensitive sodium channel of Musca domestica, wherein said voltage-sensitive sodium channel is capable of conferring sensitivity or resistance to an insecticide in Musca domestica.

58. The voltage-sensitive sodium channel of claim 57 wherein said voltage-sensitive sodium channel confers susceptibility to an insecticide in Musca domestica. e.

59. The voltage-sensitive sodium channel of claim 58 wherein said voltage-sensitive sodium channel is encoded by a nucleotide sequence as shown.in SEQ ID NO:1.

60. The voltage-sensitive sodium channel of claim 58 wherein said voltage-sensitive sodium channel is comprised of a protein having an amino acid sequence as shown in SEQ ID NO:3.

61. The voltage-sensitive sodium channel of claim 57 wherein said voltage-sensitive sodium channel confers resistance to an insecticide in Musca domestica. WO 98/28446 PCT/US97/24256 82

62. The voltage-sensitive sodium channel of claim 61 wherein said voltage-sensitive sodium channel is encoded by a nucleotide sequence as shown in SEQ ID NO:2..

63. The voltage-sensitive sodium channel of claim 61 wherein said voltage-sensitive sodium channel is comprised of a protein having an amino acid sequence as shown in SEQ ID NO:4.

64. The voltage-sensitive sodium channel of claim 61 wherein said voltage-sensitive sodium channel is encoded by a nucleic acid molecule having the nucleotide sequence of a second nucleic acid molecule with one or more mutations therein, wherein said second nucleic acid molecule encodes an insecticide sensitive voltage- sensitive sodium channel of Musca domestica, and wherein said one or more mutations in said second nucleic acid molecule render the resulting voltage-sensitive sodium channel resistant to an insecticide.

65. The voltage-sensitive sodium channel of claim 64 wherein said nucleotide sequence of said second nucleic acid molecule encodes amino acid SEQ ID NO:3, and wherein said one or more mutations in said second nucleic acid molecule are selected from the group consisting of a substitution for amino acid residue 1014 of SEQ ID NO:3, a substitution for amino acid residue 1140 of SEQ ID NO:3, a substitution for amino acid residue 2023 of SEQ ID NO:3, a deletion of one or more of amino acid residues 2031-2034 of SEQ ID NO:3, a substitution for amino acid residue 2042 of SEQ ID NO:3, a substitution for amino acid residue 2054 of SEQ ID NO:3, and an insertion of one to three amino acid residues between amino acid residues 2055 and 2056 of SEQ ID NO:3. WO 98/28446 PCT/US97/24256 83

66. The voltage-sensitive sodium channel of claim 57 wherein said insecticide is selected from the group consisting of DDT, DDT analogs, and pyrethroids.

67. An antibody or fragment thereof specific for the voltage-sensitive sodium channel of claim 57.

68. The antibody of claim 67 wherein said antibody comprises a monoclonal antibody.

69. The antibody of claim 67 wherein said antibody comprises a polyclonal antibody. A method of detecting presence of a voltage-sensitive sodium channel in a sample, said method comprising: contacting a sample with the antibody or fragment thereof of claim 67, wherein said antibody or fragment thereof binds to any of said voltage-sensitive sodium channel present in said sample, forming a complex .i 'therewith; and detecting said complex, thereby detecting presence of a voltage-sensitive sodium channel in said sample.

71. An isolated voltage-sensitive sodium "channel of Musca domestica, wherein the voltage-sensitive sodium channel is comprised of a protein having a first amino acid sequence with at least 95% amino acid identity to a second amino acid sequence, said second amino acid sequence being as shown in SEQ ID NO:3.

72. An isolated voltage-sensitive sodium channel of Musca domestica, wherein the voltage-sensitive sodium channel is comprised of a protein having a first WO98/28446 PCT/US97/24256 84 amino acid sequence with at least 95% amino acid identity to a second amino acid sequence, said second amino acid sequence being as shown in SEQ ID NO:4.

73. A plasmid designated pPJI1 and deposited with the American Type Culture Collection under Accession No. 97831.

74. A KpnI/AatII restriction fragment of the plasmid designated pPJIl of claim 73, said restriction fragment being about 3620 bp. A plasmid designated pPJI2 and deposited with the American Type Culture Collection under Accession .No. 97832.

76. An AatII/SphII restriction fragment of the plasmid designated pPJI2 of claim 75, said restriction fragment being about 2700 bp.

77. An isolated nucleic acid molecule consisting of a KpnI/AatII restriction fragment of about 3620 bp of the plasmid designated pPJI1 ligated at the AatII site to the AatII site of an AatII/SphII restriction fragment of about 2700 bp of the plasmid designated pPJI2. DATED: 28 March 2002 PHILLIPS ORMONDE FIT PA CK Attorneys for: CORNELL RESEARCH FOUNDATION, INC.