DESCRIPTION

BACILLUS THURINGIENSIS GENES ENCODING NEMATODE-ACTIVE TOXINS

Cross-Reference to Related Applications

This application is a continuation-in-part of co-pending application Senal No.

08/485,568, filed June 7, 1995; which is a continuation-m-part of application Serial No.

08/310,197, filed September 21, 1994; which is a division of Senal No. 08/092,155, filed July 15, 1993, now U.S. Patent No. 5,350,577; which is a division of Senal No. 07/918,345, filed July

21, 1992, now U.S. Patent No. 5,270,448; which is a division of 07/558,738, filed July 27, 1990, now U.S. Patent No. 5,151,363. This application is also a continuation-in-part of co-pending application Senal No. 08/357,698, filed December 16, 1994; which is a division of Senal No.

08/176,403, filed December 30, 1993, now abandoned; which is a continuation-in-part of 07/999,053 , filed December 31 , 1992, now abandoned.

Background of the Invention The soil microbe Bacillus thuringiensis (B.t ) is a Gram-positive, spore-formmg bacterium charactenzed by parasporal crystalline protein inclusions. These inclusions often appear microscopically as distinctively shaped crystals. The proteins can be highly toxic to pests and specific in their toxic activity. Certain B t toxm genes have been isolated and sequenced, and recombinant DNA-based B.t. products have been produced and approved for use In addition, with the use of genetic engmeenng techniques, new approaches for dehvenng B.t endotoxins to agncultural environments are under development, including the use of plants genetically engineered with endotoxin genes for insect resistance and the use of stabilized intact microbial cells as B.t. endotoxin delivery vehicles (Gaertner and Kim, 1988). Thus, isolated B.t endotoxin genes are becoming commercially valuable.

Until the last ten years, commercial use of B.t. pesticides has been largely restncted to a narrow range of lepidopteran (caterpillar) pests. Preparations of the spores and crystals of B thuringiensis var. kurstaki have been used for many years as commercial insecticides for lepidopteran pests. For example, B. thuringiensis var. kurstaki HD-1 produces a crystal called a δ-endotoxm which is toxic to the larvae of a number of lepidopteran insects.

In recent years, however, investigators have discovered B. t pesticides with specificities for a much broader range of pests. For example, other species of B.t., namely B t var.

israelensis and B.t. var. tenebrionis (a.k.a. M-7, a.k.a. B.t. var. san diego), have been used commercially to control insects of the orders Diptera and Coleoptera, respectively (Gaertner, 1989). See also Couch, 1980 and Beegle, 1978. Kneg et al., 1983, describe Bacillus thuringiensis var. tenebrionis, which is reportedly active against two beetles in the order Coleoptera. These are the Colorado potato beetle, Leptinotarsa decemlineata, and the beetle

Agelastica alni.

Recently, new subspecies of B.t. have been identified, and genes responsible for active 6-endotoxin proteins have been isolated (Hδfte and Whiteley, 1989). Hδfte and Whiteley classified B.t. crystal protem genes into 4 major classes. The classes were Cryl (Lepidoptera- specific), Cryll (Lepidoptera- and Diptera-specific), Crylll (Coleoptera-specific), and Cry IV

(Diptera-specific). Prefontaine et ai, 1987, describe probes useful m classifying lepidopteran- active genes. The discovery of strains specifically toxic to other pests has been reported (Feitelson et α/., 1992).

The cloning and expression of a B.t. crystal protein gene in Escherichia coli has been descnbed in the published literature (Schnepf and Whiteley, 1981). U.S. Patent 4,448,885 and

U.S. Patent 4,467,036 both disclose the expression of B.t. crystal proteins in E. coli. U.S. Patents 4,797,276 and 4,853,331 disclose B. thuringiensis var. tenebrionis (a.k.a. B.t. san diego, a.k.a. M-7) which can be used to control coleopteran pests in vanous environments. U.S. Patent No. 4,918,006 discloses Bacillus thuringiensis var. israelensis toxins which are active against dipteran pests. This patent reports that a protein of about 27 kD, and fragments thereof, are responsible for the dipteran activity. U.S. Patent No. 4,849,217 discloses B.t. isolates which have activity against the alfalfa weevil. U.S. Patent No. 5,151,363 and U.S. Patent No. 4,948,734 disclose certain isolates of B.t. which have activity against nematodes.

The accepted methodology for control of nematodes has centered around the use of the drug benzimidazole and its congeners. The use of these drugs on a wide scale has led to many instances of resistance among nematode populations (Pnchard et al., 1980; Coles, 1986). There are more than 100,000 described species of nematodes.

A small number of research articles have been published concerning the effects of δ- endotoxins from B. thuringiensis species on the viability of nematode eggs. Bottjer et al. (1985) have reported that B.t. kurstaki and B.t. israelensis were toxic in vitro to eggs of the nematode

Trichostrongylus colubriformis. In addition, 28 other B.t. strains were tested with widely vanable toxicities. The most potent had LD _S0 values m the nanogram range. Ignoffo and Dropkm (1977) have reported that the thermostable toxin from Bacillus thuringiensis (beta exotoxm) was active against a free-living nematode, Panagrellus redivivus (Goodey); a plant-

parasitic nematode, Meloidogyne incognita (Chitwood); and a fungus-feeding nematode, Aphelenchus avena (Bastien). Beta exotoxin is a generalized cytotoxic agent with little or no specificity. Also, Ciordia and Bizzell (1961) gave a preliminary report on the effects of B. thuringiensis on some cattle nematodes. At the present time there is a need to have more effective means to control the many nematodes that cause considerable damage to susceptible hosts. Effective means would advantageously employ biological agents, such as B.t. pesticides. As a result of extensive research and investment of resources, many other patents have issued for new B.t. isolates and new uses of B.t. isolates. However, the discovery of new B.t. isolates and new uses of known B.t. isolates remains an empirical, unpredictable art.

Brief Summary of the Invention The subject invention concerns novel δ-endotoxin genes obtainable from B.t. isolates PS167P, PS80JJ1, PS158D5, PS169E, PS177F1, PS177G, PS204G4, and PS204G6, wherein the genes encode proteins which are active against nematode pests. These toxin genes can be transferred to suitable hosts as described herein.

Further aspects of the subject invention concern nematode-active toxins, and fragments thereof, encoded by the genes disclosed herein. Another embodiment of the subject invention concerns hosts transformed with the genes of the subject invention. In a preferred embodiment, the transformed hosts are plants.

Brief Description of the Drawings Figure 1 is a photograph of 9% SDS polyacrylamide gel electrophoresis showing alkali- soluble proteins of nematode active strains. Gel A: Lane (1) Protein standard, (2) PS17, (3) PS33F2, (4) PS52A1, (5) PS63B, (6),

PS69D1, (7) PS80JJ1, (8) PS177F1, (9) PS177G, (10) PS204G6, (11) Protein standard.

Gel B: Lane (1) Protein standard, (2) PS17, (3) PS33F2, (4) PS52A1, (5) PS63B, (6), PS69D1, (7) PS169E, (8) PS167P, (9) PS204G4, (10) PS158D5, (11) Protein standard.

Brief Description of the Sequences

SEQ ID NO. 1 is the nucleotide sequence of a "forward" oligonucleotide primer used for PCR amplification of the 80JJ1 and 167P genes.

SEQ FD NO. 2 is the nucleotide sequence of a "reverse" oligonucleotide primer used for PCR amplification of the 80JJ1 and 167P genes.

SEQ ID NO. 3 is the nucleotide sequence of the 80JJ1 toxin gene. SEQ ID NO. 4 is the amino acid sequence of the 80 J1 protein. SEQ ID NO. 5 is the nucleotide sequence of the 167P toxin gene. SEQ ID NO. 6 is the amino acid sequence of the 167P protein.

Detailed Disclosure of the Invention

The subject invention pertains to novel genes which encode nematode-active toxins. The toxins themselves are also an important aspect of the invention. A further embodiment of the subject invention is the transformation of suitable hosts to confer upon these hosts the ability to express nematode-active toxins.

The Bacillus thuringiensis isolates from which the genes of the subject invention can be obtained have been deposited in the permanent collection of the Agricultural Research Service Patent Culture Collection (NRRL), Northern Regional Research Center, 1915 North University Street, Peoria, Illinois 61604, USA. The accession numbers are as follows:

Culture Repository No. Deposit Date

B.t. strain PS80JJ1 NRRL B- 18679 July 17, 1990

B.t. strain PS 158D5 NRRL B-l 8680 July 17, 1990

B.t. strain PS 167P NRRL B-l 8681 July 17, 1990

B.t. strain PSI 69E NRRL B- 18682 July 17, 1990

B.t. strain PSI 77F1 NRRL B-18683 July 17, 1990

B.t. strain PS 177G NRRL B-l 8684 July 17, 1990

B.t. strain PS204G4 NRRL B-l 8685 July 17, 1990

B.t. strain PS204G6 NRRL B-l 8686 July 17, 1990

E. coli NM522(pMYC2379) NRRL B-21155 November 3, 1993

E. coli NM522(pMYC2382) NRRL B-21329 September 28, 1994

The subject cultures have been deposited under conditions that assure that access to the cultures will be available during the pendency of this patent application to one determined by the Commissioner of Patents and Trademarks to be entitled thereto under 37 CFR 1.14 and 35 USC 122. The deposits are available as required by foreign patent laws in countries wherein counterparts of the subject application, or its progeny, are filed. However, it should be understood that the availability of the deposits does not constitute a license to practice the subject invention in derogation of patent rights granted by governmental action.

Further, the subject culture deposits will be stored and made available to the public in accord with the provisions of the Budapest Treaty for the Deposit of Microorganisms, i.e., they

will be stored with all the care necessary to keep them viable and uncontaminated for a period of at least five years after the most recent request for the furnishing of a sample of a deposit, and in any case, for a period of at least 30 (thirty) years after the date of deposit or for the enforceable life of any patent which may issue disclosing the cultures. The depositor acknowledges the duty to replace the deposit(s) should the depository be unable to furnish a sample when requested, due to the condition of the deposit(s). All restrictions on the availability to the public of the subject culture deposits will be irrevocably removed upon the granting of a patent disclosing them.

Genes and toxins. The genes and toxins useful according to the subject invention include not only the full length sequences disclosed but also fragments of these sequences, variants, mutants, and fusion proteins which retain the characteristic pesticidal activity of the toxins specifically exemplified. In some instances, the fusion protein may contain, in addition to the characteristic pesticidal activity of the toxins specifically exemplified, another pesticidal activity contributed by the fusion process. As used herein, the terms "variants" or "variations" of genes refer to nucleotide sequences which encode the same toxins or which encode equivalent toxins having similar pesticidal activity. As used herein, the term "equivalent toxins" refers to toxins having the same or essentially the same biological activity against the target pests as the claimed toxins.

It should be apparent to a person skilled in this art that genes encoding nematode-active toxins can be identified and obtained through several means. The specific genes exemplified herein may be obtained from the isolates deposited at a culture depository as described above. These genes, or portions or variants thereof, may also be constructed synthetically, for example, by use of a gene synthesizer. Variations of genes may be readily constructed using standard techniques for making point mutations. Also, fragments of these genes can be made using commercially available exonucleases or endonucleases according to standard procedures. For example, enzymes such as Bal3\ or site-directed mutagenesis can be used to systematically cut off nucleotides from the ends of these genes. Also, genes which encode active fragments may be obtained using a variety of restriction enzymes. Proteases may be used to directly obtain active fragments of these toxins. Equivalent toxins and/or genes encoding these equivalent toxins can be derived from

B.t. isolates and/or DNA libraries using the teachings provided herein. There are a number of methods for obtaining the pesticidal toxins of the instant invention. For example, antibodies to the pesticidal toxins disclosed and claimed herein can be used to identify and isolate other toxins from a mixture of proteins. Specifically, antibodies may be raised to the portions of the toxins

which are most constant and most distinct from other B.t toxins. These antibodies can then be used to specifically identify equivalent toxins with the charactenstic activity by immunoprecipitation, enzyme linked immunosorbent assay (ELISA), or Western blotting. Antibodies to the toxins disclosed herem, or to equivalent toxins, or fragments of these toxins, can readily be prepared using standard procedures m this art. The genes which encode these toxins can then be obtained from the microorganism.

Fragments and equivalents which retain the pesticidal activity of the exemplified toxins would be within the scope of the subject invention. Also, because of the redundancy of the genetic code, a vanety of different DNA sequences can encode the amino acid sequences disclosed herein It is well within the skill of a person trained in the art to create these alternative DNA sequences encoding the same, or essentially the same, toxins. These variant DNA sequences are within the scope of the subject invention As used herein, reference to "essentially the same" amino acid sequence refers to sequences which have amino acid substitutions, deletions, additions, or insertions which do not matenally affect the pesticidal activity of the protem

A further method for identifying the toxms and genes of the subject mvention is through the use of oligonucleotide probes. These probes are nucleotide sequences having a means for detection As is well known in the art, if the probe molecule and nucleic acid sample hybndize by forming a strong bond between the two molecules, it can be reasonably assumed that the probe and sample have substantial homology The probe's means of detection provides a means for determining in a known manner whether hybndization has occurred. Such a probe analysis provides a rapid method for identifying toxin-encoding genes of the subject invention The nucleotide segments which are used as probes according to the invention can be synthesized by use of DNA synthesizers using standard procedures. These nucleotide sequences can also be used as PCR pnmers to amplify genes of the subject invention.

Certain toxins of the subject invention have been specifically exemplified herein. Since these toxms are merely exemplary of the toxins of the subject invention, it should be readily apparent that the subject invention further compnses vanant or equivalent toxins (and nucleotide sequences coding for equivalent toxins) having the same or essentially the same pesticidal activity of the exemplified toxins. These equivalent toxins can have ammo acid homology with an exemplified toxin. This amino acid homology will typically be greater than 75%, preferably be greater than 90%, and most preferably be greater than 95%. The ammo acid homology will be highest m certain cntical regions of the toxin which account for biological activity or are involved in the determination of three-dimensional configuration which ultimately is responsible

for the biological activity. In this regard, certain amino acid substitutions are acceptable and can be expected if these substitutions are in regions which are not critical to activity or are conservative amino acid substitutions which do not affect the three-dimensional configuration of the molecule. For example, amino acids may be placed in the following classes: non-polar, uncharged polar, basic, and acidic. Conservative substitutions whereby an amino acid of one class is replaced with another amino acid of the same type fall within the scope of the subject invention so long as the substitution does not materially alter the biological activity of the compound. Table 1 provides a listing of examples of amino acids belonging to each class.

Table 1

Class of Amino Acid Examples of Amino Acids

Nonpolar Ala, Val, Leu, He, Pro, Met, Phe, Trp

Uncharged Polar Gly, Ser, Thr, Cys, Tyr, Asn, Gin

Acidic Asp, Glu

Basic Lys, Arg, His

In some instances, non-conservative substitutions can also be made. The critical factor is that these substitutions must not significantly detract from the biological activity of the toxin. The toxins of the subject invention can also be characterized in terms of the shape and location of toxin inclusions.

Following is a table which provides characteristics of certain isolates useful according to the subject invention.

Table 2. Descnption of B.t. strains toxic to nematodes

Culture Crystal Approx. MW (kDa) Serotype NRRL Deposit

Descnption Deposit Date

PS80JJ1 multiple 130, 90, 47, 37 4a4b, sotto B-l 8679 7-17-90 attached

PS158D5 attached 80 novel B-l 8680 7-17-90 amorphic

PS167P attached 120 novel B-l 8681 7-17-90 amorphic

PS169E attached 150, 128, 33 non-motile B-l 8682 7-17-90 amorphic

PS177F1 multiple 140, 116, 103, 62 non-motile B-l 8683 7-17-90 attached

PS177G multiple 135, 125, 107, 98, 62 non-motile B-l 8684 7-17-90 attached

PS204G4 multiple 105, 98, 90, 60, 44, 37 non-motile B-l 8685 7-17-90 attached

PS204G6 long 23, 21 wuhanensis B-l 8686 7-17-90 amorphic

N.D. = not determined

Recombinant hosts. The toxm-encodmg genes harbored by the isolates of the subject invention can be introduced into a wide vanety of microbial or plant hosts. Expression of the toxin gene results, directly or indirectly, in the intracellular production and maintenance of the pesticide With suitable microbial hosts, e.g , Pseudomonas, the microbes can be applied to the situs of the pest, where they will proliferate and be ingested by the pest. The result is control of the pest. Alternatively, the microbe hosting the toxin gene can be treated under conditions that prolong the activity of the toxin and stabilize the cell The treated cell, which retains the toxic activity, then can be applied to the environment of the target pest.

Where the B t toxin gene is introduced via a suitable vector into a microbial host, and said host is applied to the environment in a living state, it is advantageous to use certain host microbes. For example, microorganism hosts can be selected which are known to occupy the pest's habitat. Microorganism hosts may also live symbiotically with a specific species of pest. These microorganisms are selected so as to be capable of successfully competing m the particular environment with the wild-type microorganisms, provide for stable maintenance and expression of the gene expressing the polypeptide pesticide, and, desirably, provide for improved protection of the pesticide from environmental degradation and inactivation

A large number of microorganisms are known to inhabit the habitat of pests. These microorganisms include bacteria, algae, and fungi. Of particular interest are microorganisms, such as bacteria, e.g. , genera Pseudomonas, Erwinia, Serratia, Klebsiella, Xanthomonas, Streptomyces, Rhizobium, Rhodopseudomonas, Methy lophilius, Agrobacterium, Acetobacter, Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc, and Alcaligenes; fungi, e.g., genera

Metarhizium, Bavaria, Saccharomyces, Cryptococcus, Kluyveromyces, Sporobolomyces, Rhodotorula, and Aureobasidium. Of particular interest are such bacterial species as Pseudomonas syringae, Pseudomonas fluorescens, Serratia marcescens, Acetobacter xylinum, Agrobacterium tumefaciens, Rhodopseudomonas spheroides, Xanthomonas campestris, Rhizobium melioti, Alcaligenes entrophus, and Azotobacter vinlandii; and yeast species such as Rhodotorula rubra, R. glutinis, R. marina, R. aurantiaca, Cryptococcus albidus, C. diffluens, C. laurentii, Saccharomyces rosei, S. pretoriensis, S. cerevisiae, Sporobolomyces roseus, S. odorus, Kluyveromyces veronae, and Aureobasidium pollulans. Of particular interest are the pigmented microorganisms. A wide variety of ways are available for introducing a B.t. gene encoding a toxin into a microorganism host under conditions which allow for stable maintenance and expression of the gene. These methods are well known to those skilled in the art and are described, for example, in United States Patent No. 5,135,867, which is incorporated herein by reference.

Control of nematodes using the isolates, toxins, and genes of the subject invention can be accomplished by a variety of methods known to those skilled in the art. These methods include, for example, the application of B.t. isolates to the pests (or their location), the application of recombinant microbes to the pests (or their locations), and the transformation of plants with genes which encode the pesticidal toxins of the subject invention. Recombinant microbes may be, for example, a B.t., E. coli, or Pseudomonas. Transformations can be made by those skilled in the art using standard techniques. Materials necessary for these transformations are disclosed herein or are otherwise readily available to the skilled artisan. For example, the gene encoding the 167P toxin is provided herein as SEQ ID NO. 5. The deduced amino acid sequence for the 167P toxin is provided in SEQ ID NO. 6.

Treatment of cells. As mentioned above, B.t. or recombinant cells expressing a B.t. toxin can be treated to prolong the toxin activity and stabilize the cell. The pesticide microcapsule that is formed comprises the B.t. toxin within a cellular structure that has been stabilized and will protect the toxin when the microcapsule is applied to the environment of the target pest. Suitable host cells may include either prokaryotes or eukaryotes, normally being limited to those cells which do not produce substances toxic to higher organisms, such

as mammals. However, organisms which produce substances toxic to higher organisms could be used, where the toxic substances are unstable or the level of application sufficiently low as to avoid any possibility of toxicity to a mammalian host. As hosts, of particular interest will be the prokaryotes and the lower eukaryotes, such as fungi. The cell will usually be intact and be substantially in the proliferative form when treated, rather than in a spore form, although in some instances spores may be employed.

Treatment of the microbial cell, e.g., a microbe containing the B.t. toxin gene, can be by chemical or physical means, or by a combination of chemical and/or physical means, so long as the technique does not deleteriously affect the properties of the toxin, nor diminish the cellular capability of protecting the toxin. Examples of chemical reagents are halogenating agents, particularly halogens of atomic no. 17-80. More particularly, iodine can be used under mild conditions and for sufficient time to achieve the desired results. Other suitable techniques include treatment with aldehydes, such as glutaraldehyde; anti-infectives, such as zephiran chloride and cetylpyridinium chloride; alcohols, such as isopropyl and ethanol; various histologic fixatives, such as Lugol iodine, Bouin's fixative, various acids, and Helly's fixative (See: Humason, 1967); or a combination of physical (heat) and chemical agents that preserve and prolong the activity of the toxin produced in the cell when the cell is administered to the host animal. Examples of physical means are short wavelength radiation such as gamma-radiation and X-radiation, freezing, UV irradiation, lyophilization, and the like. Methods for treatment of microbial cells are disclosed in United States Patent Nos.

4,695,455 and 4,695,462, which are incoφorated herein by reference.

The cells generally will have enhanced structural stability which will enhance resistance to environmental conditions. Where the pesticide is in a proform, the method of cell treatment should be selected so as not to inhibit processing of the proform to the mature form of the pesticide by the target pest pathogen. For example, formaldehyde will crosslink proteins and could inhibit processing of the proform of a polypeptide pesticide. The method of cell treatment retains at least a substantial portion of the bio-availability or bioactivity of the toxin.

Characteristics of particular interest in selecting a host cell for purposes of production include ease of introducing the B.t. gene into the host, availability of expression systems, efficiency of expression, stability of the pesticide in the host, and the presence of auxiliary genetic capabilities. Characteristics of interest for use as a pesticide microcapsule include protective qualities for the pesticide, such as thick cell walls, pigmentation, and intracellular packaging or formation of inclusion bodies; survival in aqueous environments; lack of

mammalian toxicity; attractiveness to pests for ingestion; ease of killing and fixing without damage to the toxin; and the like. Other considerations include ease of formulation and handling, economics, storage stability, and the like.

Growth of cells. The cellular host containing the B.t. insecticidal gene may be grown in any convenient nutrient medium, where the DNA construct provides a selective advantage, providing for a selective medium so that substantially all or all of the cells retain the B.t. gene.

These cells may then be harvested in accordance with conventional ways. Alternatively, the cells can be treated prior to harvesting.

The B.t. cells of the invention can be cultured using standard art media and fermentation techniques. Upon completion of the fermentation cycle the bacteria can be harvested by first separating the B.t. spores and crystals from the fermentation broth by means well known in the art. The recovered B.t. spores and crystals can be formulated into a wettable powder, liquid concentrate, granules or other formulations by the addition of surfactants, dispersants, inert carriers, and other components to facilitate handling and application for particular target pests. These formulations and application procedures are all well known in the art and are used with commercial strains of B. thuringiensis (HD-1) active against Lepidoptera, e.g., caterpillars. The B.t. isolates (spores and crystals) of the subject invention can be used to control nematode pests.

The B.t. toxins of the invention can be administered orally in a unit dosage form such as a capsule, bolus or tablet, or as a liquid drench when used as an anthelmintic in mammals.

The drench is normally a solution, suspension or dispersion of the active ingredient, usually in water, together with a suspending agent such as bentonite and a wetting agent or like excipient. Generally, the drenches also contain an antifoaming agent. Drench formulations generally contain from about 0.001 to 0.5% by weight of the active compound. Preferred drench formulations may contain from 0.01 to 0.1% by weight, the capsules and boluses comprise the active ingredient admixed with a carrier vehicle such as starch, talc, magnesium stearate, or dicalcium phosphate.

Where it is desired to administer the toxin compounds in a dry, solid unit dosage form, capsules, boluses or tablets containing the desired amount of active compound usually are employed. These dosage forms are prepared by intimately and uniformly mixing the active ingredient with suitable finely divided diluents, fillers, disintegrating agents and/or binders such as starch, lactose, talc, magnesium stearate, vegetable gums and the like. Such unit dosage formulations may be varied widely with respect to their total weight and content of the

nematode-active agent, depending upon the factors such as the type of host animal to be treated, the seventy and type of infection and the weight of the host.

When the active compound is to be administered via an animal feedstuff, it is intimately dispersed in the feed or used as a top dressing or the form of pellets which may then be added to the finished feed or, optionally, fed separately. Alternatively, the compounds may be administered to animals parenterally, for example, by intraluminal, intramuscular, lntratracheal, or subcutaneous injection, in which event the active ingredient is dissolved or dispersed in a liquid earner vehicle. For parenteral administration, the active matenal is suitably admixed with an acceptable vehicle, preferably of the vegetable oil vanety, such as peanut oil, cotton seed oil and the like. Other parenteral vehicles, such as organic preparations using solketal, glycerol, formal and aqueous parenteral formulations, are also used. The active compound or compounds are dissolved or suspended in the parenteral formulation for administration; such formulations generally contain from 0.005 to 5% by weight of the active compound. When the toxins are administered as a component of the feed of the animals, or dissolved or suspended in the dnnkmg water, compositions are provided in which the active compound or compounds are intimately dispersed m an inert earner or diluent. By inert carrier is meant one that will not react with the nematode-active agent and one that may be administered safely to animals. Preferably, a carrier for feed administration is one that is, or may be, an ingredient of the animal ration.

Suitable compositions include feed premixes or supplements which the active ingredient is present in relatively large amounts and which are suitable for direct feeding to the animal or for addition to the feed either directly or after an intermediate dilution or blending step. Typical earners or diluents suitable for such compositions include, for example, distillers' dned grains, corn meal, citrus meal, fermentation residues, ground oyster shells, wheat shorts, molasses solubles, corn cob meal, edible bean mill feed, soya gnts, crushed limestone and the like.

In addition to having anthelminthic activity within the digestive tract of mammals, spores from nematicidal B.t. isolates will pass through the animals' digestive tract, germinate and multiply in the feces, and thereby provide additional control of nematode larva which hatch and multiply therein.

As would be appreciated by a person skilled in the art, the pesticidal concentration will vary widely depending upon the nature of the particular formulation, particularly whether it is a concentrate or to be used directly. The pesticide will be present in at least 1% by weight

and may be 100% by weight. The dry formulations will have from about 1-95% by weight of the pesticide while the liquid formulations will generally be from about 1-60% by weight of the solids in the liquid phase. The formulations will generally have from about IO ² to about IO ⁴ cells/mg. These formulations will be administered at about 50 mg (liquid or dry) to 1 kg or more per hectare.

The formulations can be applied to the environment of the nematode pests, e.g., plants, soil, or water by spraying, dusting, sprinkling, or the like.

Mutants. Mutants of the B.t. isolates of the subject invention can be made by procedures well known in the art. For example, an asporogenous mutant can be obtained through ethylmethane sulfonate (EMS) mutagenesis. The mutants can be made using ultraviolet light and nitrosoguanidine by procedures well known in the art.

A smaller percentage of the asporogenous mutants will remain intact and not lyse for extended fermentation periods; these strains are designated lysis minus (-). Lysis minus strains can be identified by screening asporogenous mutants in shake flask media and selecting those mutants that are still intact and contain toxin crystals at the end of the fermentation. Lysis minus strains are suitable for a cell treatment process that will yield a protected, encapsulated toxin protein.

To prepare a phage resistant variant of said asporogenous mutant, an aliquot of the phage lysate is spread onto nutrient agar and allowed to dry. An aliquot of the phage sensitive bacterial strain is then plated directly over the dried lysate and allowed to dry. The plates are incubated at 30 °C. The plates are incubated for 2 days and, at that time, numerous colonies could be seen growing on the agar. Some of these colonies are picked and subcultured onto nutrient agar plates. These apparent resistant cultures are tested for resistance by cross streaking with the phage lysate. A line of the phage lysate is streaked on the plate and allowed to dry. The presumptive resistant cultures are then streaked across the phage line. Resistant bacterial cultures show no lysis anywhere in the streak across the phage line after overnight incubation at 30 °C. The resistance to phage is then reconfirmed by plating a lawn of the resistant culture onto a nutrient agar plate. The sensitive strain is also plated in the same manner to serve as the positive control. After drying, a drop of the phage lysate is plated in the center of the plate and allowed to dry. Resistant cultures showed no lysis in the area where the phage lysate has been placed after incubation at 30 °C for 24 hours.

Following are examples which illustrate procedures, including the best mode, for practicing the invention. These examples should not be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.

Example 1 - Culturing .t. Strains

A subculture of a B.t. strain can be used to inoculate the following medium, a peptone, glucose, salts medium.

Bacto Peptone 7.5 g/1

Glucose 1.0 g/1 KH ₂ P0 ₄ 3.4 g/1

K ₂ HP0 ₄ 4.35 g/1

Salt Solution 5.0 ml/1

CaCl ₂ Solution 5.0 ml/1

Salts Solution (100 ml) MgS0 ₄ -7H ₂ 0 2.46 g

MnS0 ₄ -H ₂ 0 0.04 g

ZnS0 ₄ -7H ₂ 0 0.28 g

FeS0 ₄ -7H ₂ 0 0.40 g

CaC12 Solution (100 ml) CaCl ₂ -2H ₂ 0 3.66 g pH 7.2 The salts solution and CaCl ₂ solution are filter-sterilized and added to the autoclaved and cooked broth at the time of inoculation. Flasks are incubated at 30 °C on a rotary shaker at 200 m for 64 hr. The above procedure can be readily scaled up to large fermentors by procedures well known in the art.

The B.t. spores and crystals, obtained in the above fermentation, can be isolated by procedures well known in the art. A frequently-used procedure is to subject the harvested fermentation broth to separation techniques, e.g., centrifugation.

Example 2 - Activity of Bacillus thuringiensis Isolates Against Panagrellus redivivus

Worms were collected in a tube and allowed to settle for about 15 minutes. The water was decanted and replaced with fresh water three or four times until the water remained clear. 250 μl rinsed nematodes (20-30 worms), and 100 μl of a spore/crystal suspension were added

to 650 μl water in each well of a multi-well tray. Nematodes were counted and the numbers recorded. After four days, the live worms were counted and percent mortality calculated.

Table 3. Bioassay results U.S. Patent No. 4,948,734 Mortality

B.t. strain No.

PS17 90%

PS33F2 30%

PS52A1 100% PS63B 92%

PS69D1 100% Novel B.t. strain No. PS80JJ1 99% PS158D5 99% PS167P 96%

PS169E 100% PS177F1 96% PS177G 100% PS204G4 100% PS204G6 100%

Control 0%

Tables 4 and 5 show the molecular mass of the alkali-soluble proteins in each novel nematode-active strain, as compared to previously known B.t. strains.

Table 4. Previously known nematode-active strains

B.t. Strain Approximate Molecular Mass of Proteins (kDa)

PS17 155, 145, 135

PS33F2 140, 94, 86, 68, 65, 62

PS52A1 57, 45

PS63B 84, 82, 78

PS69D1 135, 46, 32

Table 5. New Nematode-Active Strains

Novel B.t. Strain Approximate Molecular Mass of Proteins (kDa)

PS80JJ1 130, 90, 47, 37

PS158D5 80 PS167P 120

PS169E 150, 128, 33

PS177F1 140, 116, 103, 62

PS177G 135, 125, 107, 98, 62

PS204G4 105 , 98, 90, 60, 44, 37 PS204G6 23, 21

Example 3 - Cloning and Expression of a of Novel Toxin Gene from Bacillus thuringiensis Strain PS80JJ1 Total cellular DNA was prepared from Bacillus thuringiensis (B. t. ) cells grown to an optical density, at 600 nm, of 1.0. Cells were pelleted by centrifugation and resuspended in protoplast buffer (20 mg/ml lysozyme in 0.3M sucrose, 25mM Tris-Cl (pH 8.0), 25mM EDTA). After incubation at 37°C for 1 hour, protoplasts were lysed by two cycles of freezing and thawing. Nine volumes of a solution of 0.1 M NaCl, 0.1% SDS, 0.1 M Tris-Cl were added to complete lysis. The cleared lystate was extracted twice with phenol.chloroform

(1:1). Nucleic acids were precipitated with two volumes of ethanol and pelleted by centrifugation. The pellet was resuspended in TE buffer and RNase was added to a final concentration of 50 μg/ml. After incubation at 37°C for 1 hour, the solution was extracted once each with phenol hloroform (1:1) and TE-saturated chloroform. DNA was precipitated from the aqueous phase by the addition of one-tenth volume of 3M NaOAc and two volumes of ethanol. DNA was pelleted by centrifugation, washed with 70% ethanol, dried, and resuspended in TE buffer.

An approximately 700-800 bp DNA fragment from a novel PS80JJ1 130 kDa toxin gene was obtained by polymerase chain reaction (PCR) amplification using PS80JJ1 cellular DNA and the following primers:

"Forward": 5' GGACCAGGATTTACAGG(TA)GG(AG)(AG)A 3 '

(SEQ ID NO. 1)

"Reverse": 5' TAACGTGTAT(AT)CG(CG)TTTTAATTT(TA)GA(CT)TC 3'

(SEQ ID NO. 2). The DNA fragment was cloned into pBluescript S/K (Stratagene, LaJolla, CA) and partially sequenced by dideoxynucleotide DNA sequencing methodology (Sanger et al, 1977) using Sequenase (US Biochemicals, Cleveland, OH). DNA sequences unique to at least one

PS80JJ1 toxin gene were identified by computer comparison with other known δ-endotoxin genes.

The 700-800 bp DNA fragment was radiolabelled with ³² P and used in standard hybridizations of Southern blots of PS80JJ1 total cellular DNA. Hybridizing bands included an approximately 1.8 kbp £cøRI fragment and an approximately 9.5 kbp Hindlll fragment.

These hybridizing DNA bands contain toxin genes or restriction fragments of toxin genes from PS80JJl.

A gene library was constructed from PS80JJ1 DNA partially digested with NdeU. Partial restriction digests were fractionated by agarose gel electrophoresis. DNA fragments 9.3 to 23 kbp in size were excised from the gel, electroeluted from the gel slice, purified on an Elutip-D ion exchange column (Schleicher and Schuell, Keene, NΗ), and recovered by ethanol precipitation. The NdeU inserts were ligated into 5α ΗI-digested LambdaGem- 11 (Promega, Madison,WI). Recombinant phage were packaged and plated on E. coli KW251 cells. Plaques were screened by hybridization with the probe described above. Hybridizing phage were plaque-purified and used to infect liquid cultures of E. coli KW251 cells for isolation of DNA by standard procedures (Maniatis et ai).

For subcloning the gene encoding the PS80JJ1 130 kDa toxin, preparative amounts of phage DNA were digested with Xhol and electrophoresed on an agarose gel. The approximately 12 kbp band containing the toxin gene was excised from the gel, electroeluted from the gel slice, and purified by ion exchange chromatography as described above. The purified DNA insert was ligated into pHTBluell (an E. colilB. thuringiensis shuttle vector comprised of pBluescript S/K [Stratagene, La Jolla, CA] and the replication origin from a resident B.t. plasmid [Lereclus et al.]). The ligation mix was used to transform frozen, competent E. coli NM522 cells (ATCC 47000). β-galactosidase- transformants were screened by restriction digestion of alkaline lysate plasmid minipreps as above. The desired plasmid construct, pMYC2379, contains a toxin gene that is novel compared to other toxin genes containing insecticidal proteins.

The PS80JJ1 toxin gene encoded by pMYC2379 was sequenced using the ABI373 automated sequencing system and associated software. Sequence analysis of the toxin gene

revealed that it encodes a protein of approximately 130,000 daltons, deduced from the DNA sequence. The nucleotide and deduced amino acid sequences are shown in SEQ ID NOS. 3 and 4, respectively. pMYC2379 was introduced into the acrystalliferous (Cry ^" ) B.t. host, CryB (A. Aronson, Purdue University, West Lafayette, IN), by electroporation. Expression of the

130kDa toxin was demonstrated by SDS-PAGE analysis.

A subculture of E. coli NM522 containing plasmid pMYC2379 was deposited in the permanent collection of the Patent Culture Collection (NRRL), Regional Research Center, 1815 North University Street, Peoria, Illinois 61604 USA or November 3, 1991. The accession number is NRRL B-21155.

Example 4 - Cloning and Expression of a Novel Toxin Gene from Bacillus thuringiensis PS167P

Total cellular DNA was prepared as in Example 3. An approximately 700-800 bp DNA fragment from novel PS167P 130 kDa toxin genes was obtained by polymerase chain reaction (PCR) amplification using PS167P cellular DNA and SEQ ID NOS. 1 and 2. This DNA fragment was cloned into pBluescript S/K (Stratagene, La Jolla, CA) and partially sequenced by dideoxynucleotide DNA sequencing methodology (Sanger et al., 1977) using Sequenase (U.S. Biochemicals, Cleveland, OH). DNA sequences unique to at least two PS167P toxin genes were identified by computer comparison with other known δ-endotoxin genes.

The 700-800 bp DNA fragment was radiolabelled with ³² P and used in standard hybridizations of Southern blots of PS167P total cellular DNA. Hybridizing bands included approximately 1.8 kbp and 2.3 kbp £coRI fragments and approximately 5.5 kbp and 8.0 kbp H/πdlll fragments. These DNA fragments contain toxin genes or restriction fragments of toxin genes unique to PS167P.

A gene library was constructed from PS167P DNA partially digested with NdeU. Partial restriction digests were fractionated by agarose gel electrophoresis. DNA fragments 9.3 to 23 kbp in size were excised from gel, electroeluted from the gel slice, purified on an Elutip-D ion exchange column (Schleicher and Schuell, Keene, NΗ), and recovered by ethanol precipitation. The NdeU inserts were ligated into 5αmΗI-digested LambdaGem-11 (Promega, Madison, WI). Recombinant phage were packaged and plated on E. coli KW251 cells. Plaques were screened by hybridization with the probe described above. Hybridizing phage

were plaque-purified and used to infect liquid cultures of E. coli KW251 cells for isolation of DNA by standard procedures (Maniatis et al., 1989).

Southern blot analysis revealed that one of the recombinant phage isolates contained an approximately 5 kbp Sail band that hybridized to the PS 167P toxin gene probe. One of the Sail sites flanking the PS167P toxin gene resides in the phage vector DNA sequence, while the other flanking Sail site is located within the PS167P DNA insert. This Sail fragment was subcloned by standard methods into pBluescript S/K (Stratagene, San Diego, CA) for DNA sequence analysis. The DNA insert was subcloned further as a Sacl-Kpnl fragment into pHTBluell (an E. colilB. thuringiensis shuttle vector comprised of pBluescript S/K and the replication origin from a resident B.t. plasmid [Lereclus et al, 1989] to yield pMYC2382. To test expression of the PS167P toxin gene in B.t., pMYC2382 was introduced into the acrystalliferous (Cry-) B.t. host, CryB (A. Aronson, Purdue University, West Lafayette, IN) by electroporation. Expression of the approximately 130 kDa PS167P toxin encoded by pMYC2382 was demonstrated by SDS-PAGE analysis. The PS167P toxin gene encoded by pMYC2382 was sequenced using the ABB 73 automated sequenceing system and associated software. The PS167P toxin nucleotide (SEQ ID NO. 5) and deduced amino acid (SEQ ID NO. 6) sequences are novel compared to other toxin genes encoding pesticidal proteins.

A subculture of E. coli NM522 containing plasmid pMYC2382 was deposited in the permanent collection of the Patent Culture Collection (NRRL), Regional Research Center,

1815 North University Street, Peoria, Illinois 61604 USA on September 28, 1994. The accession number is NRRL B-21329.

Example 5 - Insertion of Toxin Genes Into Plants One aspect of the subject invention is the transformation of plants with genes encoding a toxin active against nematode pests. The transformed plants are resistant to attack by nematodes.

Genes encoding pesticidal toxins, as disclosed herein, can be modified for optimum expression in plant, linked to a plant selectable marker gene, and inserted into a genome of plant cell using a variety of techniques which are well known to those skilled in the art. Any plant may be used in accordance with this invention, including angiosperms, gymnosperms, monocotyledons and dicotyledons. Preferred plants include soybean, sunflower, cotton, potato, alfalfa, maize, rice and wheat. The transformation method itself is not critical to the invention but may include transformation with T-DNA using Agrobacterium tumefaciens or

A. rhizogenes as the transformation agent, liposome fusion, microinjection, microprojectile bombardment, chemical agent (PEG or calcium chlonde)-assιsted DNA uptake, or electroporation, as well as other possible methods. Reference may be made to the literature for full details of the known methods, especially Holsters et al., 1978; Fromm et al , 1985; Horsch et al., 1985; Herrera-Estrella et al , 1983; Crossway et al., 1986; Lin, 1966, and

Steinkiss and Stabel, 1983.

Use of a plant selectable marker in transformation allows for selection of transformed cells rather than cells that do not contain the inserted DNA Vanous markers exist for use in plant cells and generally provide resistance to a biocide or antibiotic, including but not limited to, kanamycin, G418, hygromycin, and phosphinothncin. Visual markers including but not limited to b-glucuronidase, b-galactosidase, B-peru protem, green fluorescent protein, and luciferase may also be used. After transformation, those cells that have the DNA insert can be selected for by growth in a defined medium and assayed for marker expression, whether by resistance or visualization Cells containing the DNA insert can be regenerated into plants. As long as stably transformed plants are obtained, the method used for regeneration will depend on the plant tissue and transformation method used and is not cntical to the invention. However, for example, where cell suspensions have been used for transformation, transformed cells can be induced to produce cal and the calli subsequently induced to form shoots, which may then be transferred to an appropnate nutrient medium to regenerate plants. Alternatively, explants such as hypocotyl tissue or embryos may be transformed and regenerated by shoot induction in the appropnate media, followed by root and whole plant formation Whatever regeneration method is used, the result will be stably transformed plants that can vegetatively and sexually transmit the transformed traιt(s) to progeny, so that, if necessary, the transformed plant can be crossed with untransformed plants m order to transfer the trait to more appropnate germplasm for breeding puφoses.

Example 6 - Cloning of Novel B t Genes Into Insect Viruses

A number of viruses are known to mfect insects. These viruses mclude, for example, baculoviruses and entomopoxviruses. In one embodiment of the subject invention, nematode- active genes, as descnbed herein, can be placed with the genome of the insect virus, thus enhancing the pathogemcity of the virus. Methods for constructing insect viruses which comprise B.t toxin genes are well known and readily practiced by those skilled in the art. These procedures are described, for example, in Merryweather et al (1990) and Martens et al. (1990)

It should be understood that the examples and embodiments described herein are for illustrative puφoses only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and the scope of the appended claims.

References

U.S. Patents

U.S. Patent No. 4,448,885. U.S. Patent No. 4,467,036. U.S. Patent No. 4,695,455 U.S. Patent No. 4,695,462. U.S. Patent No. 4,797,276. U.S. Patent No. 4,849,217. U.S. Patent No. 4,853,331. U.S. Patent No. 4,918,006. U.S. Patent No. 4,948,734. U.S. Patent No. 5,135,867. U.S. Patent No. 5,151,363.

Foreign Patent Documents

EP 120 516.

Other References

An et al. (1985) EMBOJ. 4:277-287.

Beegle, CC, (1978) "Use of Entomogenous Bacteria in Agroecosystems," Developments in ndustrial Microbiology 20:97-104.

Bottjer, Bone, and Gill (1985) Experimental Parasitology 60:239-244.

Ciordia, H., W.E. Bizzell (1961) Jour, of Parasitology 47:41 [abstract].

Coles, G.C. (1986) "Anthelmintic resistance in sheep," In Veterinary Clinics of North America: Food Animal Practice, Vol 2:423-432 (Herd, R.P., eds.) W.B. Saunders, New York.

Couch, T.L. (1980) "Mosquito Pathogenicity of Bacillus thuringiensis var. israelensis," Developments in Industrial Microbiology 22:61-76.

Feitelson, J.S., J. Payne, L. Kim (1992) Bio/Technology 10:271-275.

Fraley et al. (1985) Crit. Rev. Plant Sci. 4:1-46.

Gaertner, F.H. (1989) "Cellular Delivery Systems for Insecticidal Proteins: Living and Non-Living Microorganisms," in Controlled Delivery of Crop Protection Agents, R.M. Wilkins, ed., Taylor and Francis, New York and London, 1990, pp. 245-255.

Gaertner, F.H., L. Kim (1988) TfBTECH 6:S4-S7.

Hoekema (1985) In: 77ιe Binary Plant Vector System, Offset-durkkerij Kanters B.V., Alblasserdam, Chapter 5.

Hόfte, H., H.R. Whiteley (1989) Microbiological Reviews 52(2):242-255.

Holsters et al. (1978) Mol. Gen. Genet. 163:181-187.

Humason, Gretchen L., Animal Tissue Techniques, W.H. Freeman and Company, 1967.

Ignoffo, CM. and Dropkin, V.H. (1977) J. Kans. Entomol. Soc. 50:394-398.

Krieg, A., A.M. Huger, G.A. Langenbruch, W. Schnetter (1983) Z. ang. Ent. 96:500-508.

Lereclus, D. et al. (1989) FEMS Microbiology Letters 60:211-218.

Maniatis, T., E.F. Fritsch, J. Sambrook (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

Martens, J.W.M., G. Honee, D. Zuidema, J.W.M. van Lent, B. Visser, J.M. Vlak (1990) Appl. Environmental Microbiol. 5t5(9):2764-2770.

Merryweather, A.T., U. Weyer, M.P.G. Harris, M. Hirst, T. Booth, R.D. Possee (1990) J. Gen. Virol. 77:1535-1544.

Prefontaine, G., P. Fast, P.C.K. Lau, M.A. Hefford, Z. Hanna, R. Brosseau (1987) Appl. Environ. Microbiol. 53(12):2808-2814.

Prichard, R.K. et al. (1980) "The problem of anthelmintic resistance in nematodes," Austr. Vet. J. 56:239-251.

Sanger et al. [1977] Proc. Natl. Acad. Sci. USA 74:5463-5467.

Schnepf, H.E., H.R. Whiteley (1981) Proc. Natl. Acad. Sci. USA 78:2893-2897.

SEQUENCE LISTING (1) GENERAL INFORMATION:

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(ii) TITLE OF INVENTION: Novel Bacillus thuringiensis Genes Encoding Nematode-Active Toxins

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(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (352) 375-8100

(B) TELEFAX: (352) 372-5800

(2) INFORMATION FOR SEQ ID NO:1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 bases

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1 : GGACCAGGAT TTACAGGWGG RRA 23

(2) INFORMATION FOR SEQ ID NO:2 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 29 bases

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2 : TAACGTGTAT WCGSTTTTAA TTTWGAYTC 29

(2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 3561 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3 :

ATGGATTGTA ATTTACAATC ACAACAAAAT ATTCCTTATA ATGTATTAGC AATACCAGTA 60

TCTAATGTTA ATGCGTTGGT TGATACAGCT GGAGATTTAA AAAAAGCATG GGAAGAATTT 120

CAAAAAACTG GTTCTTTTTC ATTAACAGCT TTACAACAAG GATTTTCTGC CTCACAAGGA 180

GGAGCATTCA ATTATTTAAC ATTATTACAA TCAGGAATAT CATTAGCTGG TTCTTTTGTC 240

CCTGGAGGTA CTTTTGTAGC ACCCATTGTT AATATGGTTA TTGGTTGGTT ATGGCCACAT 300

AAAAACAAGA CAGCGGATAC AGAAAATTTA ATAAAATTAA TTGATGAAGA AATTCAAAAA 360

CAATTAAACA AAGCCTTATT AGACCAAGAT AGAAACAATT GGACCTCTTT TTTAGAAAGT 420

ATATTTGATA CTTCAGCTAC AGTAAGTAAT GCAATTATAG ATGCACAGTG GTCAGGTACT 480

GTAGATACTA CAAATAGACA ACAAAAAACT CCAACAACAT CAGATTATCT AAATGTTGTT 540

GGAAAATTTG ATTCAGCGGA TTCTTCAATT ATAACTAATG AAAATCAAAT AATGAATGGC 600

AACTTTGACG TAGCTGCAGC ACCCTATTTT GTTATAGGAG CAACATTACG TCTTTCATTA 660

TATCAATCTT ATATTAAATT TTGTAATAGT TGGATTGATG CAGTTGGATT TAGTACAAAT 720

GATGCTAATA CACAAAAAGC TAATTTAGCT CGTACGAAAT TAACTATGCG TACTACAATT 780

AATGAATATA CACAAAGAGT TATGAAAGTT TTTAAAGATT CCAAGAATAT GCCTACAATA 840

GGTACTAATA AATTTAGTGT TGATGCTTAT AATGTATATG TTAAAGGAAT GACATTAAAT 900

GTTTTAGATA TGGTAGCAAT ATGGTCTTCA TTATATCCAA ATGATTATAC TTCACAAACA 960

GCCATAGAAC AAACACGTGT CACTTTTTCA AATATGGTTG GACAAGAAGA AGGTACAGAT 1020

GGAACCCTAA AAATTTACAA TACTTTTGAT TCTCTTAGTT ATCAACATAG CCTAATACCT 1080

AATAATAATG TTAATTTAAT TTCTTATTAT ACTGATGAAT TGCAAAATCT AGAATTAGCA 1140

GTATATACTC CTAAAGGTGG AAGTGGATAC GCTTATCCTT ATGGATTTAT TTTAAATTAT 1200

GCAAACAGCA ACTACAAATA TGGTGATAAT GATCCAACAG GCAAACCATT AAATAAACAA 1260

GATGGACCTA TACAACAAAT AAATGCAGCA ACTCAAAACA GTAAATATCT AGATGGAGAA 1320

ACAATAAATG GAATAGGGGC ATCCTTACCT GGTTATTGTA CTACAGGATG TTCAGCAACA 1380

GAACAACCTT TTAGTTGTAC TTCTACTGCT AATAGCTATA AAGCAAGCTG TAATCCTTCA 1440

GATACTAATC AAAAAATTAA TGCTTTATAT GCTTTTACAC AAACTAATGT AAAGGGAAGC 1500

ACGGGGAAAT TAGGAGTACT GGCAAGTCTT GTTCCATATG ATTTAAATCC TAAAAATGTA 1560

TTTGGTGAAT TAGATTCAGA TACAAATAAT GTTATCTTAA AAGGAATTCC TGCAGAAAAA 1620

GGGTATTTTC CTAATAATGC GCGACCTACT GTTGTAAAAG AATGGATTAA TGGTGCAAGT 1680

GCTGTACCAT TTTATTCAGG AAATACTTTA TTTATGACGG CTACGAATTT AACAGCTACT 1740

CAATATAAAA TTAGAATACG TTATGCAAAT CCAAATTCAG ATACTCAAAT CGGTGTACTA 1800

ATTACGCAAA ATGGTTCTCA AATTTCCAAT AGTAATCTAA CACTTTATAG TACTACTGAT 1860

TCAAGTATGA GTAGTAATTT ACCACAAAAT GTATATGTCA CAGGGGAAAA TGGAAATTAT 1920

ACACTTCTAG ATTTATATAG TACTACTAAT GTTTTATCAA CAGGAGATAT TACATTAAAA 1980

CTTACAGGAG GAAATCAAAA AATATTTATT GATCGAATAG AATTTATTCC TACTATGCCT 2040

GTACCTGCTC CTACTAATAA CACTAATAAC AATAACGGCG ATAACGGCAA TAACAATCCC 2100

CCACACCACG GTTGTGCAAT AGCTGGTACA CAACAACTTT GTTCTGGACC ACCTAAGTTT 2160

GAACAAGTAA GTGATTTAGA AAAAATTACA ACGCAAGTAT ATATGTTATT CAAATCTTCT 2220

TCGTATGAAG AATTAGCTCT AAAAGTTTCT AGCTATCAAA TTAATCAAGT GGCATTGAAA 2280

GTTATGGCAC TATCTGATGA AAAGTTTTGT GAAGAAAAAA GATTGTTACG AAAATTAGTC 2340

AATAAAGCAA ACCAATTACT AGAAGCACGT AACTTACTAG TAGGTGGAAA TTTTGAAACA 2400

ACTCAAAATT GGGTACTTGG AACAAATGCT TATATAAATT ATGATTCGTT TTTATTTAAT 2460

GGAAATTATT TATCCTTACA ACCAGCAAGT GGATTTTTCA CATCTTATGC TTATCAAAAA 2520

ATAGATGAGT CAACATTAAA ACCATATACA CGATATAAAG TTTCTGGATT CATTGGGCAA 2580

AGTAATCAAG TAGAACTTAT TATTTCTCGT TATGGAAAAG AAATTGATAA AATATTAAAT 2640

GTTCCATATG CAGGGCCTCT TCCTATTACT GCTGATGCAT CGATAACTTG TTGTGCACCA 2700

GAAATAGACC AATGTGATGG GGGGCAATCT GATTCTCATT TCTTCAACTA TAGCATCGAT 2760

GTAGGTGCAC TTCACCCAGA ATTAAACCCT GGCATTGAAA TTGGTCTTAA AATTGTGCAA 2820

TCAAATGGTT ATATAACAAT TAGTAATCTA GAAATTATTG AAGAACGTCC ACTTACAGAA 2880

ATGGAAATTC AAGCAGTCAA TCGAAAAGAT CACAAATGGA AAAGAGAAAA ACTTCTAGAA 2940

TGTGCAAGTG TTAGTGAACT TTTACAACCA ATCATTAATC AAATCGATTC ATTGTTCAAA 3000

GATGCAAACT GGTATAATGA TATTCTTCCT CATGTCACAT ATCAAACTCT AAAAAATATT 3060

ATAGTACCCG ATTTACCAAA ATTAAAACAT TGGTTCATAG ATCATCTCCC AGGTGAATAT 3120

CATGAAATTG AACAACAAAT GAAAGAAGCT CTAAAACATG CATTTACACA ATTAGACGAG 3180

AAAAATTTAA TCCACAATGG TCACTTTGCA ACTAACTTAA TAGATTGGCA AGTAGAAGGT 3240

GATGCTCGAA TGAAAGTATT AGAAAATAAT GCTTTGGCAT TACAACTTTC CAATTGGGAT 3300

TCTAGTGTTT CACAATCTAT TGATATATTA GAATTTGATG AAGATAAAGC ATATAAACTT 3360

CGCGTATATG CTCAAGGAAG CGGAACAATC CAATTTGGAA ACTGTGAAGA TGAAGCCATC 3420

CAATTTAATA CAAACTCATT CGTATATAAA GAAAAAATAA TCTATTTCGA TACCCCATCA 3480

ATTAACTTAC ACATACAATC AGAAGGTTCT GAATTCGTTG TAAGTAGTAT CGACCTCGTT 3540

GAATTATCAG ACGACGAATA A 3561

(2) INFORMATION FOR SEQ ID NO:4 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1186 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

Met Asp Cys Asn Leu Gin Ser Gin Gin Asn lie Pro Tyr Asn Val Leu 1 5 10 15

Ala lie Pro Val Ser Asn Val Asn Ala Leu Val Asp Thr Ala Gly Asp 20 25 30

Leu Lys Lys Ala Trp Glu Glu Phe Gin Lys Thr Gly Ser Phe Ser Leu 35 40 45

Thr Ala Leu Gin Gin Gly Phe Ser Ala Ser Gin Gly Gly Ala Phe Asn 50 55 60

Tyr Leu Thr Leu Leu Gin Ser Gly lie Ser Leu Ala Gly Ser Phe Val 65 70 75 80

Pro Gly Gly Thr Phe Val Ala Pro lie Val Asn Met Val lie Gly Trp 85 90 95

Leu Trp Pro His Lys Asn Lys Thr Ala Asp Thr Glu Asn Leu lie Lys 100 105 110

Leu lie Asp Glu Glu lie Gin Lys Gin Leu Asn Lys Ala Leu Leu Asp 115 120 125

Gin Asp Arg Asn Asn Trp Thr Ser Phe Leu Glu Ser lie Phe Asp Thr 130 135 140

Ser Ala Thr Val Ser Asn Ala lie lie Asp Ala Gin Trp Ser Gly Thr 145 150 155 160

Val Asp Thr Thr Asn Arg Gin Gin Lys Thr Pro Thr Thr Ser Asp Tyr 165 170 175

Leu Asn Val Val Gly Lys Phe Asp Ser Ala Asp Ser Ser lie lie Thr 180 185 190

Asn Glu Asn Gin lie Met Asn Gly Asn Phe Asp Val Ala Ala Ala Pro 195 200 205

Tyr Phe Val lie Gly Ala Thr Leu Arg Leu Ser Leu Tyr Gin Ser Tyr

210 215 220 lie Lys Phe Cys Asn Ser Trp lie Asp Ala Val Gly Phe Ser Thr Asn 225 230 235 240

Asp Ala Asn Thr Gin Lys Ala Asn Leu Ala Arg Thr Lys Leu Thr Met 245 250 255

Arg Thr Thr lie Asn Glu Tyr Thr Gin Arg Val Met Lys Val Phe Lys 260 265 270

Asp Ser Lys Asn Met Pro Thr lie Gly Thr Asn Lys Phe Ser Val Asp 275 280 285

Ala Tyr Asn Val Tyr Val Lys Gly Met Thr Leu Asn Val Leu Asp Met 290 295 300

Val Ala lie Trp Ser Ser Leu Tyr Pro Asn Asp Tyr Thr Ser Gin Thr 305 310 315 320

Ala lie Glu Gin Thr Arg Val Thr Phe Ser Asn Met Val Gly Gin Glu 325 330 335

Glu Gly Thr Asp Gly Thr Leu Lys lie Tyr Asn Thr Phe Asp Ser Leu 340 345 350

Ser Tyr Gin His Ser Leu lie Pro Asn Asn Asn Val Asn Leu lie Ser 355 360 365

Tyr Tyr Thr Asp Glu Leu Gin Asn Leu Glu Leu Ala Val Tyr Thr Pro 370 375 380

Lys Gly Gly Ser Gly Tyr Ala Tyr Pro Tyr Gly Phe lie Leu Asn Tyr 385 390 395 400

Ala Asn Ser Asn Tyr Lys Tyr Gly Asp Asn Asp Pro Thr Gly Lys Pro 405 410 415

Leu Asn Lys Gin Asp Gly Pro He Gin Gin He Asn Ala Ala Thr Gin 420 425 430

Asn Ser Lys Tyr Leu Asp Gly Glu Thr He Asn Gly He Gly Ala Ser 435 440 445

Leu Pro Gly Tyr Cys Thr Thr Gly Cys Ser Ala Thr Glu Gin Pro Phe 450 455 460

Ser Cys Thr Ser Thr Ala Asn Ser Tyr Lys Ala Ser Cys Asn Pro Ser 465 470 475 480

Asp Thr Asn Gin Lys He Asn Ala Leu Tyr Ala Phe Thr Gin Thr Asn 485 490 495

Val Lys Gly Ser Thr Gly Lys Leu Gly Val Leu Ala Ser Leu Val Pro 500 505 510

Tyr Asp Leu Asn Pro Lys Asn Val Phe Gly Glu Leu Asp Ser Asp Thr 515 520 525

Asn Asn Val He Leu Lys Gly He Pro Ala Glu Lys Gly Tyr Phe Pro 530 535 540

Asn Asn Ala Arg Pro Thr Val Val Lys Glu Trp He Asn Gly Ala Ser 545 550 555 560

Ala Val Pro Phe Tyr Ser Gly Asn Thr Leu Phe Met Thr Ala Thr Asn 565 570 575

Leu Thr Ala Thr Gin Tyr Lys He Arg He Arg Tyr Ala Asn Pro Asn 580 585 590

Ser Asp Thr Gin He Gly Val Leu He Thr Gin Asn Gly Ser Gin He 595 600 605

Ser Asn Ser Asn Leu Thr Leu Tyr Ser Thr Thr Asp Ser Ser Met Ser 610 615 620

Ser Asn Leu Pro Gin Asn Val Tyr Val Thr Gly Glu Asn Gly Asn Tyr 625 630 635 640

Thr Leu Leu Asp Leu Tyr Ser Thr Thr Asn Val Leu Ser Thr Gly Asp 645 650 655

He Thr Leu Lys Leu Thr Gly Gly Asn Gin Lys He Phe He Asp Arg 660 665 670

He Glu Phe He Pro Thr Met Pro Val Pro Ala Pro Thr Asn Asn Thr 675 680 685

Asn Asn Asn Asn Gly Asp Asn Gly Asn Asn Asn Pro Pro His His Gly 690 695 700

Cys Ala He Ala Gly Thr Gin Gin Leu Cys Ser Gly Pro Pro Lys Phe 705 710 715 720

Glu Gin Val Ser Asp Leu Glu Lys He Thr Thr Gin Val Tyr Met Leu 725 730 735

Phe Lys Ser Ser Ser Tyr Glu Glu Leu Ala Leu Lys Val Ser Ser Tyr 740 745 750

Gin He Asn Gin Val Ala Leu Lys Val Met Ala Leu Ser Asp Glu Lys 755 760 765

Phe Cys Glu Glu Lys Arg Leu Leu Arg Lys Leu Val Asn Lys Ala Asn 770 775 780

Gin Leu Leu Glu Ala Arg Asn Leu Leu Val Gly Gly Asn Phe Glu Thr 785 790 795 800

Thr Gin Asn Trp Val Leu Gly Thr Asn Ala Tyr He Asn Tyr Asp Ser 805 810 815

Phe Leu Phe Asn Gly Asn Tyr Leu Ser Leu Gin Pro Ala Ser Gly Phe 820 825 830

Phe Thr Ser Tyr Ala Tyr Gin Lys He Asp Glu Ser Thr Leu Lys Pro 835 840 845

Tyr Thr Arg Tyr Lys Val Ser Gly Phe He Gly Gin Ser Asn Gin Val 850 855 860

Glu Leu He He Ser Arg Tyr Gly Lys Glu He Asp Lys He Leu Asn 865 870 875 880

Val Pro Tyr Ala Gly Pro Leu Pro He Thr Ala Asp Ala Ser He Thr 885 890 895

Cys Cys Ala Pro Glu He Asp Gin Cys Asp Gly Gly Gin Ser Asp Ser 900 905 910

His Phe Phe Asn Tyr Ser He Asp Val Gly Ala Leu His Pro Glu Leu 915 920 925

Asn Pro Gly He Glu He Gly Leu Lys He Val Gin Ser Asn Gly Tyr 930 935 940

He Thr He Ser Asn Leu Glu He He Glu Glu Arg Pro Leu Thr Glu 945 950 955 960

Met Glu He Gin Ala Val Asn Arg Lys Asp His Lys Trp Lys Arg Glu 965 970 975

Lys Leu Leu Glu Cys Ala Ser Val Ser Glu Leu Leu Gin Pro He He 980 985 990

Asn Gin He Asp Ser Leu Phe Lys Asp Ala Asn Trp Tyr Asn Asp He 995 1000 1005

Leu Pro His Val Thr Tyr Gin Thr Leu Lys Asn He He Val Pro Asp 1010 1015 1020

Leu Pro Lys Leu Lys His Trp Phe He Asp His Leu Pro Gly Glu Tyr 1025 1030 1035 1040

His Glu He Glu Gin Gin Met Lys Glu Ala Leu Lys His Ala Phe Thr 1045 1050 1055

Gin Leu Asp Glu Lys Asn Leu He His Asn Gly His Phe Ala Thr Asn 1060 1065 1070

Leu He Asp Trp Gin Val Glu Gly Asp Ala Arg Met Lys Val Leu Glu 1075 1080 1085

Asn Asn Ala Leu Ala Leu Gin Leu Ser Asn Trp Asp Ser Ser Val Ser 1090 1095 1100

Gin Ser He Asp He Leu Glu Phe Asp Glu Asp Lys Ala Tyr Lys Leu 1105 1110 1115 1120

Arg Val Tyr Ala Gin Gly Ser Gly Thr He Gin Phe Gly Asn Cys Glu 1125 1130 1135

Asp Glu Ala He Gin Phe Asn Thr Asn Ser Phe Val Tyr Lys Glu Lys 1140 1145 1150

He He Tyr Phe Asp Thr Pro Ser He Asn Leu His He Gin Ser Glu 1155 1160 1165

Gly Ser Glu Phe Val Val Ser Ser He Asp Leu Val Glu Leu Ser Asp 1170 1175 1180

Asp Glu 1185

(2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 3504 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5

ATGACAAATC CAACTATACT ATATCCTAGT TACCATAATG TATTAGCTCA TCCGATTAGA 60

TTAGATTCTT TTTTTGATCC ATTTGTAGAG ACATTTAAGG ATTTAAAAGG GGCTTGGGAA 120

GAATTCGGAA AAACGGGATA TATGGACCCC TTAAAACAAC ACCTTCAAAT CGCATGGGAT 180

ACTAGTCAAA ATGGAACAGT GGATTATTTA GCATTAACAA AAGCATCTAT ATCTCTCATA 240

GGTTTAATTC CTGGTGCAGA CGCTGTAGTC CCTTTTATTA ATATGTTTGT AGACTTTATT 300

TTTCCGAAAT TATTTGGAAG AGGTTCTCAA CAAAATGCTC AAGCTCAATT TTTCGAACTA 360

ATCATAGAAA AAGTTAAAGA ACTTGTTGAT GAAGATTTTA GAAACTTTAC CCTTAATAAT 420

CTACTCAATT ACCTTGATGG TATGCAAACA GCCTTATCAC ATTTCCAAAA CGATGTACAA 480

ATTGCTATTT GTCAAGGAGA ACAACCAGGA CTTATGCTAG ATCAAACACC AACGGCTTGT 540

ACTCCTACTA CAGACCATTT AATTTCTGTA AGAGAATCTT TTAAAGATGC TCGAACTACA 600

ATTGAAACAG CTTTACCACA TTTTAAAAAT CCTATGCTAT CCACAAATGA TAACACTCCA 660

GATTTTAATA GCGACACTGT CTTATTAACA TTACCAATGT ATACAACAGG AGCGACTTTA 720

AATCTTATAT TACATCAAGG GTATATTCAA TTCGCAGAAA GATGGAAATC TGTAAATTAT 780

GATGAAAGTT TTATAAATCA AACAAAAGTT GATTTGCAAC GTCGTATTCA GGACTATTCT 840

ACTACTGTAT CTACCACTTT TGAAAAATTC AAACCTACTC TAAATCCATC AAATAAAGAA 900

TCTGTTAATA AGTATAATAG ATATGTTCGT TCCATGACTC TTCAATCTTT AGACATTGCT 960

GCAACATGGC CTACTTTAGA TAATGTTAAT TACCCTTCCA ATGTAGATAT TCAATTGGAT 1020

CAAACTCGCT TAGTATTTTC AGATGTTGCA GGACCTTGGG AAGGTAATGA TAATATAACT 1080

TCGAATATTA TAGATGTATT AACACCAATA AATACAGGGA TAGGATTTCA AGAAAGTTCA 1140

GATCTTAGAA AATTCACTTA TCCACGAATA GAATTACAAA GCATGCAATT CCATGGACAA 1200

TATGTAAACT CAAAAAGTGT AGAACATTGT TATAGCGATG GTCTTAAATT AAATTATAAA 1260

AATAAAACTA TAACTGCAGG TGTAAGTAAT ATTGATGAAA GTAATCAAAA TAATAAACAT 1320

AACTATGGTC CTGTAATAAA TAGTCCTATT ACTGATATCA ACGTAAATTC CCAAAATTCT 1380

CAATATTTAG ATTTAAATTC AGTCATGGTA AATGGTGGTC AAAAAGTAAC CGGGTGTTCA 1440

CCACTTAGTT CAAATGGTAA TTCTAATAAT GCTGCTTTAC CTAATCAAAA AATAAATGTT 1500

ATTTATTCAG TACAATCAAA TGATAAACCA GAAAAACATG CAGACACTTA TAGAAAATGG 1560

GGATATATGA GCAGTCATAT TCCTTATGAT CTTGTTCCAG AAAATGTAAT TGGAGATATA 1620

GATCCGGATA CTAAACAACC GTCATTGCTT CTTAAAGGGT TTCCGGCAGA AAAAGGATAT 1680

GGTGACTCAA TTGCATATGT ATCAGAACCT TTAAATGGTG CGAATGCAGT TAAACTTACT 1740

TCATATCAAG TTCTCCAAAT GGAAGTTACA AATCAAACAA CTCAAAAATA TCGTATTCGC 1800

ATACGTTATG CTACAGGTGG AGATACAGCT GCTTCTATAT GGTTTCATAT TATTGGTCCA 1860

TCTGGAAATG ATTTAACAAA CGAAGGCCAT AACTTCTCTA GTGTATCTTC TAGAAATAAA 1920

ATGTTTGTTC AGGGTAATAA CGGAAAATAT GTATTGAACA TCCTTACAGA TTCAATAGAA 1980

TTACCATCAG GACAACAAAC TATTCTTATT CAAAATACTA ATTCTCAAGA TCTTTTTTTA 2040

GATCGTATTG AATTTATTTC TCTCCCTTCT ACTTCTACTC CTACTTCTAC TAATTTTGTA 2100

GAACCTGAAT CATTAGAAAA GATCATAAAC CAAGTTAATC AATTATTTAG CTCCTCATCT 2160

CAAACTGAAT TGGCTCACAC TGTAAGCGAT TATAAAATTG ATCAAGTAGT GCTAAAAGTA 2220

AATGCCTTAT CCGACGATGT ATTTGGTGTA GAGAAAAAAG CATTACGTAA ACTTGTGAAT 2280

CAGGCCAAAC AACTCAGTAA AGCACGAAAT GTATTGGTCG GTGGAAACTT TGAAAAAGGT 2340

CATGAATGGG CACTAAGCCG TGAAGCAACA ATGGTCGCAA ATCATGAGTT ATTCAAAGGG 2400

GATCATTTAT TATTACCACC ACCAACCCTA TATCCATCGT ATGCATATCA AAAAATTGAT 2460

GAATCGAAAT TAAAATCCAA TACACGTTAT ACTGTTTCCG GCTTTATTGC GCAAAGTGAA 2520

CATCTAGAAG TCGTTGTGTC TCGATACGGG AAAGAAGTAC ATGACATGTT AGATATCCCG 2580

TATGAAGAAG CCTTACCAAT TTCTTCTGAT GAGAGTCCAA ATTGTTGCAA ACCAGCTGCT 2640

TGTCAGTGTT CATCTTGTGA TGGTAGTCAA TCAGATTCTC ATTTCTTTAG CTATAGTATC 2700

GATGTTGGTT CCCTACAATC AGATGTAAAT CTCGGCATTG AATTCGGTCT TCGTATTGCG 2760

AAACCAAACG GATTTGCGAA AATCAGTAAT CTAGAAATTA AAGAAGATCG TCCATTAACA 2820

GAAAAAGAAA TCAAAAAAGT ACAACGTAAA GAACAAAAAT GGAAAAAAGC ATTTAACCAA 2880

GAACAAGCCG AAGTAGCGAC AACACTCCAA CCAACGTTAG ATCAAATCAA TGCTTTGTAT 2940

CAAAATGAAG ATTGGAACGG TTCCGTTCAC CCGGCCAGTG ACTATCAACA TCTGTCCGCT 3000

GTTGTTGTAC CAACGTTACC AAAACAAAGA CATTGGTTTA TGGAGGGTCG AGAAGGCGAA 3060

CATGTTGTTC TGACGCAACA ATTCCAACAA GCATTGGATC GTGCGTTCCA ACAAATCGAA 3120

GAACAAAACT TAATCCACAA TGGTAATTTG GCGAATGGAT TAACAGATTG GACTGTCACA 3180

GGAGATGCAC AACTTACGAT CTTTGACGAA GATCCAGTAT TAGAACTAGC GCATTGGGAT 3240

GCAAGTATCT CTCAAACCAT TGAAATTATG GATTTTGAAG GAAGACACAG AATACAAACT 3300

GCGTGTACGT GGAAAAGGCA AAGGAACAGT TACCGTTCAA CATGGAGGAA GAGATTAGAA 3360

ACGATGACAT TCAATACAAC GAGTTTTACA ACACAAGAAC AAACCTTCTA CTTCGAAGGA 3420

GATACAGTGG ACGTACATGT TCAATCAGAG AATAACACAT TCCTGATAGA TAGTGTGGAA 3480

CTCATTGAAA TCATAGAAGA GTAA 3504

(2) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1167 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: εingle

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:

Met Thr Asn Pro Thr He Leu Tyr Pro Ser Tyr His Asn Val Leu Ala 1 5 10 15

His Pro He Arg Leu Asp Ser Phe Phe Asp Pro Phe Val Glu Thr Phe 20 25 30

Lys Asp Leu Lys Gly Ala Trp Glu Glu Phe Gly Lys Thr Gly Tyr Met 35 40 45

Asp Pro Leu Lys Gin His Leu Gin He Ala Trp Asp Thr Ser Gin Asn 50 55 60

Gly Thr Val Asp Tyr Leu Ala Leu Thr Lys Ala Ser He Ser Leu He 65 70 75 80

Gly Leu He Pro Gly Ala Asp Ala Val Val Pro Phe He Asn Met Phe 85 90 95

Val Asp Phe He Phe Pro Lys Leu Phe Gly Arg Gly Ser Gin Gin Asn 100 105 110

Ala Gin Ala Gin Phe Phe Glu Leu He He Glu Lys Val Lys Glu Leu 115 120 125

Val Asp Glu Asp Phe Arg Asn Phe Thr Leu Asn Asn Leu Leu Asn Tyr 130 135 140

Leu Asp Gly Met Gin Thr Ala Leu Ser His Phe Gin Asn Asp Val Gin 145 150 155 160

He Ala He Cys Gin Gly Glu Gin Pro Gly Leu Met Leu Asp Gin Thr 165 170 175

Pro Thr Ala Cys Thr Pro Thr Thr Asp His Leu He Ser Val Arg Glu 180 185 190

Ser Phe Lys Asp Ala Arg Thr Thr He Glu Thr Ala Leu Pro His Phe 195 200 205

Lys Asn Pro Met Leu Ser Thr Asn Asp Asn Thr Pro Asp Phe Asn Ser 210 215 220

Asp Thr Val Leu Leu Thr Leu Pro Met Tyr Thr Thr Gly Ala Thr Leu 225 230 235 240

Asn Leu He Leu His Gin Gly Tyr He Gin Phe Ala Glu Arg Trp Lys 245 250 255

Ser Val Asn Tyr Asp Glu Ser Phe He Asn Gin Thr Lys Val Asp Leu 260 265 270

Gin Arg Arg He Gin Asp Tyr Ser Thr Thr Val Ser Thr Thr Phe Glu 275 280 285

Lys Phe Lys Pro Thr Leu Asn Pro Ser Asn Lys Glu Ser Val Asn Lys 290 295 300

Tyr Asn Arg Tyr Val Arg Ser Met Thr Leu Gin Ser Leu Asp He Ala 305 310 315 320

Ala Thr Trp Pro Thr Leu Asp Asn Val Asn Tyr Pro Ser Asn Val Asp 325 330 335

He Gin Leu Asp Gin Thr Arg Leu Val Phe Ser Asp Val Ala Gly Pro 340 345 350

Trp Glu Gly Asn Asp Asn He Thr Ser Asn He He Asp Val Leu Thr 355 360 365

Pro He Asn Thr Gly He Gly Phe Gin Glu Ser Ser Asp Leu Arg Lys 370 375 380

Phe Thr Tyr Pro Arg He Glu Leu Gin Ser Met Gin Phe His Gly Gin 385 390 395 400

Tyr Val Asn Ser Lys Ser Val Glu His Cys Tyr Ser Asp Gly Leu Lys 405 410 415

Leu Asn Tyr Lys Asn Lys Thr He Thr Ala Gly Val Ser Asn He Asp 420 425 430

Glu Ser Asn Gin Asn Asn Lys His Asn Tyr Gly Pro Val He Asn Ser 435 440 445

Pro He Thr Asp He Asn Val Asn Ser Gin Asn Ser Gin Tyr Leu Asp 450 455 460

Leu Asn Ser Val Met Val Asn Gly Gly Gin Lys Val Thr Gly Cys Ser 465 470 475 480

Pro Leu Ser Ser Asn Gly Asn Ser Asn Asn Ala Ala Leu Pro Asn Gin 485 490 495

Lys He Asn Val He Tyr Ser Val Gin Ser Asn Asp Lys Pro Glu Lys 500 505 510

His Ala Asp Thr Tyr Arg Lys Trp Gly Tyr Met Ser Ser His He Pro 515 520 525

Tyr Asp Leu Val Pro Glu Asn Val He Gly Asp He Asp Pro Asp Thr 530 535 540

Lys Gin Pro Ser Leu Leu Leu Lys Gly Phe Pro Ala Glu Lys Gly Tyr 545 550 555 560

Gly Asp Ser He Ala Tyr Val Ser Glu Pro Leu Asn Gly Ala Asn Ala 565 570 575

Val Lys Leu Thr Ser Tyr Gin Val Leu Gin Met Glu Val Thr Asn Gin 580 585 590

Thr Thr Gin Lys Tyr Arg He Arg He Arg Tyr Ala Thr Gly Gly Asp 595 600 605

Thr Ala Ala Ser He Trp Phe His He He Gly Pro Ser Gly Asn Asp 610 615 620

Leu Thr Asn Glu Gly His Asn Phe Ser Ser Val Ser Ser Arg Asn Lys 625 630 635 640

Met Phe Val Gin Gly Asn Asn Gly Lys Tyr Val Leu Asn He Leu Thr 645 650 655

Asp Ser He Glu Leu Pro Ser Gly Gin Gin Thr He Leu He Gin Asn 660 665 670

Thr Asn Ser Gin Asp Leu Phe Leu Asp Arg He Glu Phe He Ser Leu 675 680 685

Pro Ser Thr Ser Thr Pro Thr Ser Thr Asn Phe Val Glu Pro Glu Ser 690 695 700

Leu Glu Lys He He Asn Gin Val Asn Gin Leu Phe Ser Ser Ser Ser 705 710 715 720

Gin Thr Glu Leu Ala His Thr Val Ser Asp Tyr Lys He Asp Gin Val 725 730 735

Val Leu Lys Val Asn Ala Leu Ser Asp Asp Val Phe Gly Val Glu Lys 740 745 750

Lys Ala Leu Arg Lys Leu Val Asn Gin Ala Lys Gin Leu Ser Lys Ala 755 760 765

Arg Asn Val Leu Val Gly Gly Asn Phe Glu Lys Gly His Glu Trp Ala 770 775 780

Leu Ser Arg Glu Ala Thr Met Val Ala Asn His Glu Leu Phe Lys Gly 785 790 795 800

Asp His Leu Leu Leu Pro Pro Pro Thr Leu Tyr Pro Ser Tyr Ala Tyr 805 810 815

Gin Lys He Asp Glu Ser Lys Leu Lys Ser Asn Thr Arg Tyr Thr Val 820 825 830

Ser Gly Phe He Ala Gin Ser Glu His Leu Glu Val Val Val Ser Arg 835 840 845

Tyr Gly Lys Glu Val His Asp Met Leu Asp He Pro Tyr Glu Glu Ala 850 855 860

Leu Pro He Ser Ser Asp Glu Ser Pro Asn Cys Cys Lys Pro Ala Ala 865 870 875 880

Cys Gin Cys Ser Ser Cys Asp Gly Ser Gin Ser Asp Ser His Phe Phe 885 890 895

Ser Tyr Ser He Asp Val Gly Ser Leu Gin Ser Asp Val Asn Leu Gly 900 905 910

He Glu Phe Gly Leu Arg He Ala Lys Pro Asn Gly Phe Ala Lys He 915 920 925

Ser Asn Leu Glu He Lys Glu Asp Arg Pro Leu Thr Glu Lys Glu He 930 935 940

Lys Lys Val Gin Arg Lys Glu Gin Lys Trp Lys Lys Ala Phe Asn Gin 945 950 955 960

Glu Gin Ala Glu Val Ala Thr Thr Leu Gin Pro Thr Leu Asp Gin He 965 970 975

Asn Ala Leu Tyr Gin Asn Glu Asp Trp Asn Gly Ser Val His Pro Ala 980 985 990

Ser Asp Tyr Gin His Leu Ser Ala Val Val Val Pro Thr Leu Pro Lys 995 1000 1005

Gin Arg His Trp Phe Met Glu Gly Arg Glu Gly Glu His Val Val Leu 1010 1015 1020

Thr Gin Gin Phe Gin Gin Ala Leu Asp Arg Ala Phe Gin Gin He Glu 1025 1030 1035 1040

Glu Gin Asn Leu He His Asn Gly Asn Leu Ala Asn Gly Leu Thr Asp 1045 1050 1055

Trp Thr Val Thr Gly Asp Ala Gin Leu Thr He Phe Asp Glu Asp Pro 1060 1065 1070

Val Leu Glu Leu Ala His Trp Asp Ala Ser He Ser Gin Thr He Glu

1075 1080 1085

He Met Asp Phe Glu Gly Arg His Arg He Gin Thr Ala Cys Thr Trp 1090 1095 1100

Lys Arg Gin Arg Asn Ser Tyr Arg Ser Thr Trp Arg Lys Arg Leu Glu 1105 1110 1115 1120

Thr Met Thr Phe Asn Thr Thr Ser Phe Thr Thr Gin Glu Gin Thr Phe 1125 1130 1135

Tyr Phe Glu Gly Asp Thr Val Asp Val His Val Gin Ser Glu Asn Asn 1140 1145 1150

Thr Phe Leu He Asp Ser Val Glu Leu He Glu He He Glu Glu

1155 1160 1165