WO1996017937A2 - PESTICIDAL COMPOSITION COMPRISING CryIF CHIMERIC AND CryIA(c) CHIMERIC BACILLUS THURINGIENSIS DELTA-ENDOTOXIN - Google Patents

Abstract

Disclosed are compositions and processes for controlling lepidopteran pests. These compositions comprise synergistic combinations of a CryIF chimeric and CryIA(c) chimeric Bacillus thuringiensis δ-endotoxin. These compositions have been found to exhibit excellent activity against lepidopteran pests.

Description

PESΗCIDAL COMPOSITION COMPRISING CiylF CHIMERIC AND CιyΙA(c) CHIMERIC BACIL¬ LUS THURINGIENSIS DELTA-ENDOTOXIN

Background of the Invention

The soil microbe Bacillus thuringiensis (B.t.) is a Gram-positive, spore-forming bacterium characterized 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. toxin 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 engineering techniques, new approaches for delivering these B.t. endotoxins to agricultural 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, F.H., L. Kim [1988] T1BTECH 6:S -S7). Thus, isolated B.t. endotoxin genes are becoming commercially valuable. Until the last ten years, commercial use of B.t. pesticides has been largely restricted to a narrow range of lepidopteran (caterpillar) pests. Preparations of the spores and crystals of B. thuringiensis subsp. 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 -endotoxin which is toxic to die 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 israelensis and tenebrionis (a.k.a. B.t. M-7, a.k.a. B.t. san diego), have been used commercially to control insects of the orders Diptera and Coleoptera, respectively (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). See also Couch,

T.L. (1980) "Mosquito Pathogenicity of Bacillus thuringiensis var. israelensis " Developments in Industrial Microbiology 22:61-76; Beegle, C.C, (1978) "Use of Entomogenous Bacteria in Agroecosystems," Developments in Industrial Microbiology 20:97-104. Krieg, A-, A.M. Huger, G.A. Langenbruch, W. Schnetter (1983) Z ang. Ent. 96:500-508, 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 Agelastica alni.

Recently, new subspecies of B.t. have been identified, and genes responsible for active δ- endotoxin proteins have been isolated (Hόfte, H., H.R. Whitelev [1989] Microbiological Reviews 52(2):242-255). Hόfte and Whitelev classified B.t. crystal protein genes into 4 major classes. The classes were Cryl (Lepidoptera-specific), CiyE. (Lepidoptera- and Diptera-specific), Crylll (Coleoptera-specific), and Cry-V (Diptera-specific). The discovery of strains specifically toxic to other pests has been reported. (Feitelson. J.S.. J. Payne. L. Kim [1992] Bio/Technology 10:271-275). The cloning and expression of a B.t. crystal protein gene in Escherichia coli has been described in the published literature (Schnepf, H.E., H.R. Whiteley [1981] Proc. Natl. Acad. Sci. USA 78:2893-

2897). U.S. Patent No. 4,448,885 and U.S. Patent No. 4,467,036 both disclose the expression of a B.t. crystal protein in E. coli. Hybrid B.t. crystal protein genes have been constructed that exhibit increased toxicity and display an expanded host range to a target pest. See U.S. Patent Nos. 5,128,130 and

5,055,294. U.S. Patent Nos. 4,797,276 and 4,853,331 disclose B. thuringiensis strain san diego (a.k.a.

B.t. tenebrionis, a.k.a. M-7) which can be used to control coleopteran pests in various environments.

U.S. Patent No. 4,918,006 discloses B.t. toxins having activity against dipterans. 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.

As a result of extensive research and investment of resources, 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. A majority of Bacillus thuringiensis δ-endotoxin crystal protein molecules are composed of two functional segments. The protease -resistant core toxin is the first segment and corresponds to about the first half of the protein molecule. The three-dimensional structure of a core segment of a crylllA B.t. δ- endotoxin is known and it is proposed that all related toxins have that same overall structure (Li, J., J.

Carroll, D.J. Ellar [1991] Nature 353:815-821). The second half of the molecule is the second segment. For purposes of this application, this second segment will be referred to herein as the "protoxin segment."

The protoxin segment is believed to participate in toxin crystal formation (Arvidson, H., P.E. Dunn, S.

Strand. Al. Aronson [19&9] Molecular Microbiology 3:1533-1534; Choma, C.T., W.K. Surewicz, P.R.

Carey. M. Pozsgay, T. Raynor. H. Kaplan [1990] Eur. J. Biochem. 189:523-527). The full 130 kDa toxin molecule is rapidly processed to the resistant core segment by protease in the insect gut. The protoxin segment may thus convey a partial insect specificity for the toxin by limiting the accessibility of the core to the insect by reducing the protease processing of the toxin molecule (Haider, M.Z., B.H.

Knowles, D.J. Ellar [1986] Eur. J. Biochem. 156:531-540) or by reducing toxin solubility (Aronson,

A.I., E S. Han, W. McGaughey, D. Johnson [1991] Appl. Environ. Microbiol. 57:981-986).

Chimeric proteins joined within the toxin domains have been reported between CrylC and CryΙA(b) (Honee, G.. D. Convents, J. Van Rie. S. Jansens, M. Perferoen, B. Visser [1991] Mot.

Microbiol. 5:2799-2806); however, the activity of these chimeric proteins was either much less, or undetectable. when compared to CrylC on a relevant insect.

Honee et al. (Honee, G, W. Vriezen. B. Visser [1990] Appl. Environ. Microbiol. 56:823-825) also reported making a chimeric fusion protein by linking tandem toxin domains of CrylC and CryIA(b). The resulting protein had an increased spectrum of activity equivalent to the combined activities of the individual toxins: however, the activity of the chimeric was not increased toward any one of the target insects. When toxins or biologically active agents are blended together, the biological activity of the resulting mixture can be affected in several ways. The resultant biological activity can be the sum of the activity of each of the toxins. Biological activity of the mixture may be less than the sum of the activity of each of the agents, or the resultant activity may be greater than the sum of the activity of each of the agents.

A nucleotide β-exotoxin produced by a particular B.t. strain was found to act in synergy with the protein δ-endotoxins in B.t. var. kurstaki to yield increased activity against the lepidopteran pest Spodoptera exigua (Moar, W.J., W.L.A. Osbrink, J.T> Trumble [1986] J. Econ. Entomol. 79:1443- 1446). Enhanced toxicity to mosquito larvae occurs with the mixture of the 27 kDa and the 65 or 130 kDa proteins from B.t. var. israelensis (Chilcott, C.N., D.J. Ellar [1988] J. Gen. Microbiology

132:2551-2558; Yuetal, 1987; Wu, D., F.N. Chang [1985] FEBS 190(2):232-236). The CrylVA and CrylVB toxins from B.t. var. israelensis have also been used together (Angsuthanasomat, C, N. Crickmore, D.J. Ellar [1992] FEMS Microbiol. Lett. 94:63-68).

Brief Summary of the Invention

The subject invention concerns the discovery of advantageous increased activity against lepidopteran pests achieved by the combination of two Bacillus thuringiensis (B.t.) δ -endotoxin proteins. More specifically, a CryEF chimeric toxin combined with a CryΙA(c) chimeric toxin act in synergy to yield unexpected enhanced toxicity to lepidopteran pests. The synergistic effect of the subject invention may be achieved by combining, as in a mixture, isolates that each produce one of d e toxin proteins. Recombinant hosts engineered to express both of die toxins of the subject invention can also be used to achieve the synergistic effect. Suitable recombinant hosts include prokaryotes and lower eukaryotes, as well as plants.

Chimeric CryEF genes useful according to the subject invention can be assembled d at substitute a heterologous protoxin segment for all or part of the native crylF protoxin segment. In particular, all or part of the protoxin-encoding region of a cryIA(b) gene can be used in place of all or part of the region which encodes the protoxin for a native crylF toxin. Similarly, a chimeric gene can be constructed wherein the region encoding all or part of the protoxin of a crylF toxin is replaced by DNA encoding all or part of the protoxin of a c_yIA(c)/cryIA(b) chimeric gene. In a specific embodiment, the cryIA(c)/cryIA(b) chimeric gene is that which has been denoted 436 and which is described in U.S. Patent

No. 5, 128, 130. This gene can be obtained from the plasmid in P. βuorescens MR436.

The chimeric gene can be introduced into a wide variety of microbial or plant hosts. A transformed host expressing the chimeric gene can be used to produce the lepidopteran-active toxins of the subject invention. Transformed hosts can be used to produce the insecticidal toxins or, in the case of a plant cell transformed to produce die toxins, die plant will become resistant to insect attack. The subject invention further pertains to the use of the chimeric toxins, or hosts containing the genes encoding die chimeric toxins, in med ods for controlling lepidopteran pests. Still further, die invention includes combinations of substantially intact treated B t cells, or recombinant cells expressmg the genes and producmg the toxins of the invention The cells can be treated to prolong the pesucidal activity when the substantially intact cells are applied to the environment of the target pest Such treatment can be by chemical or physical means, or a combination of chemical and physical means, so long as die technique does not deleteπously affect the synergistic properties of the pesticides, nor diminish the cellular capability m protectmg the pesticides The treated cell acts as a protective coatmg for die pesϋcidal toxins The toxins become available to act as such upon ingestion by a target pest

Bnef Description of the Drawings Figure 1 - The BamUl site is removed from pMYC1050 by a fill-in reaction with Klenow polymerase to give plasmid pMYC1050Δ_tømHI To facilitate cloning, an Nstl DNA fragment that contains most of the toxin open reading frame is cloned into pGEM5 The resulting plasmid is called pGEMtox C=C/αI, H=_ ./.dIII

Figure 2 - BamRl or Pvul cloning sites were introduced into toxin DNA by the technique of Splice Overlap Extension (SOE) DNA fragments with die new sites are used to replace homologous

DNA fragments in pGEMtox The resulting plasmids are pGEMtoxδαmHl or pGEMtox/VuI The letters A through G below the arrows correspond to oligonucleotide primers in die text Letters above vertical lines correspond to restnction enzyme sites

Figure 3 - The DNA fragment containing d e BamW mutation is used to replace the homologous fragment in pGEMtoxPvwI The resulting plasmid which contains both clomng sites is pGEMtox-BαmHI/Pvt-I To construct an expression plasmid, d e toxin-containing Nsil fragment is excised for clomng into die pTJS260 broad host-range vector B=_3omHI, C=Clal, H=/_ wdin, ?=Pvul Figure 4 - The Nsil toxin-containing fragment with die new restriction sites is ligated to d e vector-containing DNA from pMYC 1050Δ_tømHI to give pMYC2244 A BamHl-Pvul PCR-derived D A fragment containing die crylF toxin is exchanged for the equivalent fragment in pMYC2244 The resulting chimera is called pMYC2239 B=βαmHI, C=C/αI, H=_/mdIII, N=N_;I, ?=Pvul

Figure 5 - The small Apal DΝA fragment of pMYC2047 is substituted for the homologous region of pMYC2239 to give plasmid pMYC2244 This chimera consists of crylF in the toxin region and cr IA(b) m d e protoxin

Figure 6 - Silent codon changes are introduced mto die cry IF toxin by SOE The Spel-Kpnl

PCR DΝA fragment wid the changes is substituted for the homologous toxin-containing fragment in pMYC2047 The resulting plasmid is pMYC2243 Letters H through K below the arrow s correspond to oligonucleotide primers in die text

Figure 7 - Silent codon changes are introduced mto pMYC2244 bv substitution of the homologous fragment with the small Apal DΝA fragment of pMYC2243 The final plasmid is pMYC2523 ?=Pvul Figure 8 - A chimeric toxin containing the 436 protoxin is constructed by substituting a PCR- generated _°vuI-B-tEII protoxin DNA for the homologous fragment in pMYC2523. The final plasmid is pMYC2254. Letters F and M below die arrows correspond to oligonucleotide primers in die text.

Figure 9 - A CryIF/C-y-A(b) chimeric protein sequence and residue-by-residue substitutions. The 'Cons' line shows a CryIF/CryIA(b) chimeric sequence. The 'Alt' lines show residue-by-residue substitutions found in the 436 protein, CryIA(b) variant proteins and Cry F protoxins.

Brief Description of the Sequences

SEQ ID NO. 1 is oligonucleotide primer "A" SEQ ID NO. 2 is oligonucleotide primer "B"

SEQ ID NO. 3 is oligonucleotide primer "C"

SEQ ID NO.4 is oligonucleotide primer "D"

SEQ ID NO. 5 is oligonucleotide primer "E"

SEQ ID NO. 6 is oligonucleotide primer T" SEQ ID NO. 7 is oligonucleotide primer "G"

SEQ ID NO. 8 is oligonucleotide primer "L"

SEQ ID NO. 9 is oligonucleotide primer "N"

SEQ ID NO. 10 is oligonucleotide primer "O"

SEQ ID NO. 11 is oligonucleotide primer "H" SEQ ID NO. 12 is oligonucleotide primer "I"

SEQ ID NO. 13 is oligonucleotide primer T

SEQ ID NO. 14 is oligonucleotide primer "K"

SEQ ID NO. 15 is oligonucleotide primer "P"

SEQ ID NO. 16 is oligonucleotide primer "Q" SEQ ID NO. 17 is oligonucleotide primer "M"

SEQ ID NO. 18 shows die toxin-encoding DNA sequence of pMYC2224.

SEQ ID NO. 19 shows die predicted amino acid sequence of the toxin encoded by pMYC2224.

SEQ ID NO. 20 shows the toxin-encoding DNA sequence of pMYC2239.

SEQ ID NO. 21 shows the predicted amino acid sequence of die toxin encoded by pMYC2239 SEQ ID NO. 22 shows die toxin-encoding DNA sequence of pMYC2244, which encodes a crylF/cry IA(b) chimeric toxin.

SEQ ID NO.23 shows the predicted amino acid sequence of die cryIF/cryIA(b) chimeric toxin encoded by pMYC2244.

SEQ ID NO. 24 shows the toxin-encoding DNA sequence of pMYC2243. SEQ ID NO.25 shows the predicted amino acid sequence of d e toxin encoded by pMYC2243. SEQ ID NO. 26 shows the toxin-encoding DNA sequence of pMYC2523, which encodes a cryIF/cryIA(b) chimenc toxm with codon rework

SEQ ID NO.27 shows the predicted ammo acid sequence of the toxm encoded by pMYC2523 SEQ ID NO. 28 shows the toxin-encoding DNA sequence of pMYC2254, which encodes a cryEF/436 chimenc toxm

SEQ ID NO.29 shows d e predicted ammo acid sequence of d e toxin encoded by pMYC2254 SEQ ID NO.30 is a characteπs tic sequence of cry I toxins This sequence ends at residue 601 of SEQ ID NO 23

SEQ ID NO. 31 is die eight ammo acids preceding ammo acid 1043 m SEQ ED NO 23 SEQ ID NO.32 shows die ammo acid sequence of a native cryIF/cryIA(b) toxin

SEQ ID NO.33 shows the ammo acid sequence of a native cryIA(b) toxin SEQ ID NO. 34 shows die ammo acid sequence of a cryIA(c)/cryIA(b) toxm

Detailed Disclosure of the Invention The subject invention concerns the unexpected enhanced pesticidal activity resulting from the combination of a CryEF chimeπc toxm and a CryIA(c) chimenc toxm The combmation surpπsmgly has increased activity against lepidopteran pests Preparations of combinations of isolates that produce the two chimenc toxins can be used to practice the subject invention Pseudomonas fluorescens cells transformed idi B t genes can serve as one source of the toxins of die subject invention For example, a lactose-inducible P fluorescens strain compnsmg a gene encodmg a Cry IF/CryIA(b) toxm, and P fluorescens MR436, which comprises a gene encodmg a CryIA(c)/CryIA(b) chimenc toxin, can be used to practice die subject invention These two Pseudomonas strains can be combmed m a physical blend diat exhibits adv antageous enhanced pesticidal activity Genes encodmg die toxins of the v ention can be used to transform smtable hosts so that a smgle host will produce d e two toxins providmg d e advantageous effect

Bactena harboring plasnuds useful accordmg to the subject invention are the following

Culture Repository No U S Patent No

P fluorescens (pM3, 130-7) NRRL B-18332 5,055,294

P fluorescens MR436 NRRL B- 18292 5.128,130 (pM2.16-l l. aka pMYC436)

E coh NM522 (pMYC 1603) NRRL B-18517 5,188,960

It should be understood that die availability of a deposit does not constitute a license to practice die subject v ention in derogation of patent nghts granted by governmental action

In accordance ith die subject invention, it has been discovered diat products compnsmg die two chimenc toxins have been discovered to require a lower total protem content for product application, d us providmg die user greater economy Insects which are less susceptible to die action of a smgle toxm will be more greatlv affected bv die combination of toxins of die subject invention, rendering a product containing the two toxins more efficacious tiian products containing a single toxin. Additionally, pests are less likely to develop a rapid resistance to a product containing the two toxins, than to products containing a single toxin.

Combinations of die toxins described in die invention can be used to control lepidopteran pests. Adult lepidopterans, i.e., butterflies and modis, primarily feed on flower nectar and are a significant effector of pollination- The larvae, i.e., caterpillars, nearly all feed on plants, and many are serious pests. Caterpillars feed on or inside foliage or on the roots or stem of a plant, depriving die plant of nutrients and often destroying die plant's physical support structure. Additionally, caterpillars feed on fruit, fabrics, and stored grains and flours, ruining diese products for sale or severely diminishing their value. As used herein, reference to lepidopteran pests refers to various life stages of the pest, including larval stages.

The chimeric toxins of the subject invention comprise a full core N-teπninal toxin portion of a

B.t. toxin and, at some point past die end of die toxin portion, die protein has a transition to a heterologous protoxin sequence. The N-terminal toxin portion of a B.t. toxin is refererred to herein as the "core" toxin. The transition to die heterologous protoxin segment can occur at approximately the toxin/ protoxin junction or, in die alternative, a portion of the native protoxin (extending past the toxin portion) can be retained widi die transition to die heterologous protoxin occurring downstream. As an example, one chimeric toxin of die subject invention has die full toxin portion of crylF (amino acids 1- 601) and a heterologous protoxin (amino acids 602 to die C-terminus). In a preferred embodiment, die heterologous portion of the protoxin is derived from a cryIA(b) or 436 toxin. A person skilled in diis art will appreciate that B.t. toxins, even within a certain class such as crylF, will vary to some extent in lengdi and die precise location of the transition from toxin portion to protoxin portion. Typically, die cryIA(b) and crylF toxins are about 1150 to about 1200 amino acids in lengdi. The transition from toxin portion to protoxin portion will typically occur at between about 50% to about 60% of the full length toxin. The chimeric toxin of the subject invention will include d e fidl expanse of iis core N-terminal toxin portion- Thus, the chimeric toxin will comprise at least about 50% of d e full lengdi cryEF B.t. toxin. This will typically be at least about 590 amino acids. Widi regard to die protoxin portion, die full expanse of die cryIA(b) protoxin portion extends from die end of die toxin portion to the C-terminus of die molecule. It is die last about 100 to 150 amino acids of diis portion which are most critical to include in die chimeric toxin of the subject invention. In a chimeric toxin specifically exemplified herein, at least amino acids 1043 (of SEQ ID NO. 23) to die C-terminus of the cryIA(b) molecule are utilized. Amino acid 1043 in SEQ ID NO. 23 is preceded by die sequence TVT Pro Asn Asn Thr Val Thr Cys (SEQ ID NO. 31). This amino acid sequence marks the location in die protoxin segment of die molecule beyond which heterologous amino acids will always occur in the chimeric toxin. In another example, d e peptide shown as SEQ ID NO. 31 occurs at amino acids 1061 to 1068. In this case, amino acids 1069 to die C-terminus are preferably heterologous (SEQ ID NO. 29).

The peptide shown in SEQ ID NO. 31 can be found at positions 1061 to 1068 in Figure 9. Thus, it is at least the last approximately 5 to 10% of d e overall B.t. protein which should comprise heterologous DNA (compared to the cryEF core N-terminal toxm portion) m the chimenc toxin of the subject invention In die specific examples contained herem, heterologous protoxm sequences occur from ammo acid 640 to the C-terminus

Thus, a preferred embodiment of the subject invention is a chimenc B t toxm of about 1150 to about 1200 ammo acids in length, wherein the chimenc toxm compnses a cry F core N-terminal toxm portion of at least about 50 to 60% of a full cryEF molecule, but no more dian about 90 to 95% of d e full molecule The chimenc toxm further compnses a cryIA(b) or a 436 protoxm C-teπmnal portion which compnses at least about 5 to 10% of die cryEA ) or 436 molecule The transition from crylF to cryIA(b) or 436 sequence thus occurs within the protoxm segment (or at the junction of the toxm and protoxm segments) between about 50% and about 95% of the wav through the molecule In die specific examples provided herem, the transitions from the crvIF sequence to die heterologous proto m sequences occur pπor to the end of die peptide sequence shown in SEQ ID NO 31

A specific embodiment of die subject invention is the chimenc toxin shown m Figure 9 Other constructs mav be made and used by tiiose skilled in tius art havmg the benefit of die teachmgs provided herem The core toxm segment of cryl proteins characteristically ends widi the sequence Val/Leu Tyr/Ile

De Asp Arg/Lys Ile/Phe Glu Ile/Phe/Leu Ile/Leu/Val Pro/Leu Ala/Val Glu/Thr/Asp (SEQ ID NO 30), which ends at residue 601 of SEQ ID NO 23 Additionally, the protoxm segments of d e cryl toxins (which follow residue 601) bear more sequence similanty dian die toxm segments Because of dus sequence similanty, the transition pomt m die protoxm segment for making a chimenc protem between the crvEF sequence and die crvIA(b) or 436 sequence can be readilv determined bv one skilled in die art

From studies of data regarding the partial proteolysis of Cryl genes, the heterogeneity and least-conserved amino acid regions are found after the conserved cryl protoxin sequence, positions 1061 - 1068 of Figure 9

Therefore a chimenc toxm of the subject invention can compnse the full cr IF toxin and a portion of d e crvIF protoxin, transiQoning to the corresponding cr IA(b) or 436 sequence at any position between die end of die toxm segment (as defined above) and die end of d e peptide sequence shown in SEQ ED NO 31 Preferabl , the ammo acid sequence of die C-terminus of die chimenc tox compnses a crvIA(b) sequence or a sequence from die 436 gene or an equivalent of one of these sequences

are well known in the art CrvIF genes and toxins hav e been descnbed m. for example Chambers et al (1991) J Bacteriol 173 3966 CrvIA(b) genes and toxins have been descnbed m, for example. Hofte et al (1986) Eur J Biochem 161 273 Geiser et al (1986) Gene 48 109, and Haider et al (1988) Nucleic Acids Res 16 10927 The skilled artisan havmg d e benefit of die teachmgs contained herem could readilv identify and use DNA which encodes die toxm N-terminal portion of a cr IF molecule and die C-teπrunal protoxm portion of d e crvIA(b) toxins

Figure 9 provides examples of ammo acid substitutions which can be used in die to ins of d e subject invention It is also well known m the art that vaπous mutations can be made m a toxm sequence without changing die activity of a toxm Furthermore, due to d e degeneracy of die genetic code, a vanety of DNA sequences can be used to encode a particular toxm These alternative DNA and ammo acid sequences can be used accordmg to the subject invention by a person skilled m this art

The protoxm substitution techniques of d e subject invention can be used widi other classes of B t endotoxins to enhance expression of the toxin The technique would be most applicable to other B t toxins which have die charactenstic sequence shown in SEQ ID NO 30

The flow charts of Figures 1-8 provide a general overview of vector construction diat can be earned out accordmg to the subject invention BamHl and Pvul clomng sites can be introduced mto a cryIA(c)/cryIA(b) chimenc toxm gene by mutagenesis usmg die PCR technique of Splice Overlap Extension (SOE) (Horton, R M , H D Hunt, S N Ho, J K Pullen, L R Pease [1989] Gene 11 61-68) to give plasmid pMYC2224 A region of the crylF gene from a crv/ -contaimng plasmid such as pMYC1260 can be generated by PCR and substituted for the BamHl-Pvul cryIA(c)/ctyIA(b) gene fragment of pMYC2224 The new plasmid, which we designated pMYC2239, consisted of a short segment of cryΙA(c) followed by cry IF to the toxin/protoxin segment junction Thus, die protoxm segment was now deπved from cryl A(b) (pMYC 1050) An Apal fragment denved from the cry IF clone

(pMYC2047) was substituted for the Apal fragment m pMYC2239 The resulting clone (pMYC2244) consisted of cryEF from the initiator methiomne to die toxin/protoxin segment junction and cryIA(b) to the end of die coding region Clone pMYC2243 was constructed by SOE to introduce sdent codon changes m a limited region The Apal fragment from pMYC2243 that contained the silent changes was substituted for the Apal fragment m pMYC2244 to give clone pMYC2523 The chimeπc pMYC2523 showed an expression improvement over pMYC2243, which contains unchanged crylF protem sequence

A crvIF/436 chimera can be assembled by substituting die A -.I--__.EII protem segment- containing fragment of pMYC2523 with an eqmvalent fragment generated by PCR from a plasmid containing a cryIA(c)/cryIA(b) gene One such gene is die 436 gene (e g , pMYC467. as disclosed m

U S Patent Nos 5,055,294 and 5,169.760) This construction also results m improved expression compared to the native crylF protem sequence

Genes and toxins The genes and toxins useful accordmg to the subject invention mclude not onlv d e full lengdi sequences disclosed but also fragments of these sequences, aπants mutants and fusion proteins which retain die charactenstic pesticidal activity of die to ins specificalh exemplified herem As used herein, the terms "vanants" or "vaπations" of genes refer to nucleotide sequences which encode die same toxins or which encode eqmvalent toxins havmg pesticidal activitv As used herein, the term "eqmvalent toxins" refers to toxins havmg die same or essentially the same biological activity against the target pests as the claimed toxins It should be apparent to a person skilled m tins art diat genes encodmg active toxins can be identified and obtamed dirough several means The specific genes or gene portions exemplified herem mav be obtamed from die isolates deposited at a culture depository as descnbed abov e 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 BaB 1 or site-directed mutagenesis can be used to systematically cut off nucleotides from die ends of these genes. Also, genes which encode active fragments may be obtained using a variety of restriction enzymes. Proteases may be used to direcdy obtain active fragments of tiiese toxins.

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

A further method for identifying die toxins and gene portions useful according to die subject invention is dirough die use of oligonucleotide probes. These probes are detectable nucleotide sequences. These sequences may be detectable by virtue of an appropriate label or may be made inherently fluorescent as described in International Application No. WO93/16094. As is well known in die art, if the probe molecule and nucleic acid sample hybridize by forming a strong bond between the two molecules, it can be reasonably assumed diat the probe and sample have substantial homology . Preferably . hybridization is conducted under stringent conditions by techniques well-known in die art, as described, for example, in Keller, G.H., M.M. Manak (1987) DNA Probes, Stockton Press, New York, NY., pp. 169-170. Detection of die probe provides a means for determining in a known manner whether hybridization 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 die invention can be syndiesized using DNA syndiesizer and standard procedures. These nucleotide sequences can also be used as PCR primers to amplify- genes of die subject invention.

Certain toxins of die subject invention have been specifically exemplified herein. Since these toxins are merely exemplary of the toxins of die subject invention, it should be readily apparent that the subject invention comprises variant or equivalent toxins (and nucleotide sequences coding for equivalent toxins) having the same or similar pesticidal activity of the exemplified toxin. Equivalent toxins will have amino acid homology widi an exemplified toxi - This amino acid homology will typicall be greater dian 75%. preferably be greater than 90%, and most preferably be greater than 95%. The amino acid homology will be highest in critical regions of die toxin which account for biological activity or are involved in the determination of three-dimensional configuration which ultimately is responsible for the biological activit '. In this regard, certain amino acid substitutions are acceptable and can be expected if tiiese substitutions are m regions which are not cπϋcal to activity or are conservative ammo acid substitutions which do not affect die tiiree-dimensional configuration of the molecule For example, ammo acids may be placed m die following classes non-polar, uncharged polar, basic, and acidic Conservative substitutions whereby an ammo acid of one class is replaced widi another amino acid of the same type fall within the scope of die subject invention so long as the substitution does not materially alter the biological activity of die compound Table 1 provides a listing of examples of ammo acids belonging to each class

Table 1.

Class of Ammo Acid Examples of Ammo 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 cntical factor is that tiiese substitutions must not significa ly detract from die biological activity of the toxm

Recombinant hosts The genes encodmg die toxins of die subject invention can be introduced mto a wide vaπety of microbial or plant hosts Expression of the toxm gene results, directly or lndirettly, m the intracellular production and mamtenance of die pesticide Conjugal transfer and recombinant transfer can be used to create a B t strain that e presses both toxins of the subject invention Other host organisms mav also be transformed with one or both of die toxin genes en used to accomplish die svnergistic effect Widi suitable microbial hosts, e , Pseudomonas, the microbes can be applied to die situs of die pest, where they will proliferate and be mgested The result is control of the pest Alternativel , die microbe hosting die toxm gene can be treated under conditions diat prolong die activity of the toxm and stabilize die cell The treated cell, which retams the toxic activity, then can be applied to the environment of the target pest

Where the B t toxm gene is introduced v la a smtable vector mto a microbial host, and said host is applied to die environment m a living state, it is essential diat certain host microbes be used Microorganism hosts are selected which are known to occupy die "phytosphere" (phv lloplane, phvllosphere. rhizosphere, and/or rhizoplane) of one or more crops of mterest These microorganisms are selected so as to be capable of successfully competing m die particular environment (crop and odier msect habitats) widi die wild-type πucroorgamsms, provide for stable mamtenance and expression of the gene expressing the polypeptide pesticide, and, desirably, provide for unproved protection of the pesticide from environmental degradation and inactivation

A large number of microorganisms are known to inhabit the phylloplane (the surface of the plant leaves) and/or die rhizosphere (die soil surrounding plant roots) of a wide vanety of important crops These microorganisms include bacteria, algae, and fungi Of particular interest are πucroorgamsms, such as bactena, e g , genera Pseudomonas, Erwinia, Serratia, Klebsiella, Xanthomonas, Streptomyces, Rhizobium, Rhodopseudomonas, Methylophilius, Agrobactenum, Acetobacter, Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc, and Alca genes, fungi, particularly yeast, e g , genera Saccharomyces, Cryptococcus, Kluyveromyces, Sporobolomvces, Rhodotorula, and Aureobasidium Of particular mterest are such phytosphere bactenal species as Pseudomonas synngae, Pseudomonas fluorescens, Serratia marcescens, Acetobacter xylinum, Agrobactenum tumefaciens, Rhodopseudomonas spheroides, Xanthomonas campestris, Rhizobium me oti, Alca genes entrophus, and Azotobacter vinlandit, and phytosphere veast species such as Rhodotorula rubra. R gluttnis, R marina. R aurantiaca, Cryptococcus albidus, C diflluens, C laurentn, Saccharomyces rosei, S pretonensis, S cerevisiae, Sporobolomvces roseus, S odorus, Kluyveromyces veronae, and

Aureobasidium pollulans Of particular mterest are the pigmented microorganisms

A wide vanety of wavs are available for introducing a B t gene encodmg a toxm mto a microorganism host under conditions which allow for stable mamtenance and expression of the gene These methods are well known to diose skilled m die art and are descnbed, for example, m United States Patent No 5,135,867, which is incorporated herem bv reference

Treatment of cells Bacillus thuringiensis or recombinant cells expressing the B t toxins can be treated to prolong the toxm activity and stabilize the cell The pesticide microcapsule diat is formed compnses die B t toxm or toxins within a cellular structure that has been stabilized and will protect die toxin when die microcapsule is applied to the environment of the target pest Smtable host cells mav include either prokaryotes or eukaryotes, normally bemg limited to tiiose 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 die toxic substances are unstable or die level of application sufficientlv low as to avoid am possibility of toxicity to a mammalian host As hosts of particular mterest will be die prokarvotes and die lo er eukaryotes, such as fiingi The cell will usuallv be intact and be substantially m die proliferative form when treated radier dian m a spore form although m some instances spores mav be employed

Treatment of the microbial cell, e . a microbe containing the B t toxm gene or genes can be bv chemical or physical means, or by a combmation of chemical and/or physical means so long as the techmque does not deletenouslv affect die properties of the toxm nor diminish die cellular capability of protectmg the toxm Examples of chemical reagents are halogenatmg agents particularly halogens of atomic no 17-80 More particularly iodine can be used under mild conditions and for sufficient time to achiev e die desired results Other suitable techniques mclude treatment with aldehydes, such as glutaraldeh de, anti-infectives, such as zephiran chlonde and cetylpyndinium chlonde. alcohols, such as isopropyl and etiianol, vanous histologic fixatives, such as Lugol iodine, Bourn's fixative, vanous acids and Helly's fixative (See Humason, Gretchen L., Animal Tissue Techniques, W H Freeman and Company, 1967), or a combmation of physical (heat) and chemical agents that preserve and prolong the activity of the toxm produced in the cell when the cell is administered to the host environment Examples of physical means are short wavelengtii radiation such as gamma-radiation and X -radiation, freezing, UV irradiation, lyophilization, and die like Metiiods for treatment of microbial cells are disclosed United States Patent Nos 4,695,455 and 4,695,462, which are incorporated herem by reference

The cells generally will have enhanced structural stability which will enhance resistance to environmental conditions Where the pesticide is m a proform, the metiiod of cell treatment should be selected so as not to inhibit processing of the proform to the mature form of the pesticide by die target pest pathogen For example, formaldehyde will crosslink proteins and could inhibit processing of the proform of a polypeptide pesticide The method of treatment should retain at least a substantial portion of the bio-availability or bioactivity of the toxm Charactenstics of particular mterest m selecting a host cell for purposes of production mclude ease of mtroducmg the B t gene or genes mto the host, availability of expression systems, efficiency of expression, stability of the pesticide m die host, and die presence of auxiliary genetic capabilities Charactenstics of mterest for use as a pesticide microcapsule mclude protective qualities for die pesticide, such as duck cell walls, pigmentation, and intracellular packaging or formation of inclusion bodies, survival aqueous environments, lack of mammalian toxicity , attractiveness to pests for lngestion, ease of killmg and fixmg widiout damage to me toxin, and die hke Otiier considerations mclude ease of formulation and handling, economics, storage stability, and die like

Growth of cells The cellular host containing die B t insecticidal gene or genes mav be grown in anv convenient nutnent medium, where the DNA construct provides a selective advantage, providmg for a selective medium so that substantially all or all of the cells retain the B t gene These cells may dien be harvested in accordance widi conventional way s Alternatively , the cells can be treated pπor to harvesting

The B t cells producmg die toxins of the mvention can be cultured usmg standard art media and fermentation techniques Upon compleϋon of the fermentation c cle the bacteπa can be harvested by first separating the B t spores and crystals from the fermentation broth bv means well known m the art The recovered B t spores and crystals can be formulated mto a wettable powder, liquid concentrate, granules or other formulations bv the addition of surfactants, dispersants, inert earners, and otiier components to facilitate handling and application for particular target pests These formulations and application procedures are all ell known in the art Formulations Formulated bait granules containing an attractant and spores, crystals, and toxins of the _? f isolates, or recombinant microbes compnsmg the genes obtainable from die B t isolates disclosed herein, can be applied to die soil Formulated product can also be applied as a seed-coatmg or root treatment or total plant treatment at later stages of the crop cycle Plant and sod treatments of B.t cells may be employed as wettable powders, granules or dusts, by mixing with vanous inert materials, such as inorganic minerals (phyllosilicates, carbonates, sulfates, phosphates, and die like) or botanical materials (powdered corncobs, nee hulls, walnut shells, and die like) The formulations may mclude spreader-sticker adjuvants, stabilizing agents, other pesticidal additives, or surfactants Liquid formulations may be aqueous-based or non-aqueous and employed as foams, gels, suspensions, emulsifiable concentrates, or die like The ingredients may mclude theological agents, surfactants, emulsifiers, dispersants, or polymers

As would be appreciated by a person skilled m die art, the pesticidal concentration will vary widely dependmg upon the nature of the particular formulation, particularly whether it is a concentrate or to be used direcdy The pesticide will be present m at least 1% by weight and may be 100% by weight The dry formulations will hav e from about 1-95% by weight of die pesticide while die liquid formulations will generally be from about 1-60% by weight of the solids m die liquid phase The formulations will generally have from about 10² to about 10⁴ cells/mg These formulations will be administered at about 50 mg (hq d or dry) to 1 kg or more per hectare

The formulations can be applied to die environment of the lepidopteran pest, e g , foliage or sod, by spraying, dusting, sprinkling, or the Id e

Matenals and Mediods NACS (Bediesda Research Labs, Gaithersburg, MD) column chromatography was used for purification of electioeluted DNA It was performed accordmg to die manufacturer's directions, except that the buffers were modified to 0.5X TBE 0 2 M NaCl for binding, and 0 5X TBE 2 0 M NaCl for elution

Random pruning labeling of DNA with α-[^3JP]dATP was done wtth a kit (Boehπnger- Mannheim Biochemicals, Indianapolis, IN) accordmg to the manufacturer's directions

Gel purification refers to sequential application of agarose-TBE gel electrophoresis. electroelutioa and NACS column chromatography for purification of selected DNA fragments, mediods which are well known m the art

Polvmerase chain reaction (PCR) amplification of DNA was done for 25 cycles on a Perkin Elmer (Norwalk. CT) thermal cycler with die following c cle parameters 94 °C for 1 minute, 37 C for

2 minutes, 72 °C for 3 minutes (each 72 °C cycle has a 5 second extension time) PCR DNA products were proteinase K treated to improve clo ng efficiency (Crowe, J S , Cooper, H J , Srmdi. M A Suns,

M J , Parker, D , Gewert, D [1991] Nucl Acids Res 19 184)

Oligodeoxynbonucleotides (oligonucleotides) were synthesized on an Applied Biosystems (Foster City , CA) model 381 A DNA synthesizer Purification was done widi Nensorb columns (New

England Nuclear-Dupont, Wilmington. DE). if necessary, accordmg to die manufacturer's instructions Electroporation of Pseudomonas fluorescens was done widi log-phase cells grown in L-broth (LB) at 30 °C on a rotary shaker. Cells were washed 2 to 3 times with ice-cold sterile distilled water and concentrated to 0.03x starting volume in distilled water. DNA in 1-20 μl was mixed widi 50-300 μl of cells. Parameters selected for the Biorad Gene Pulser (Bio-Rad, Richmond, CA) were 200 ohms, 25 microfarads, and 2.25 kϋovolts in a cuvette widi a 0.2 cm electrode gap. Following electroporation, one milliliter of LB was added and cells were held on ice for at least 2 minutes. Cells were then incubated for 2 hours to overnight at 30°C without shaking.

B. t. toxin expression in P. fluorescens was done in the recommended medium found in the Manual of Methods for General Bacteriology (P. Gerhardt et al., 1981, American Society for Microbiology, Washington, D.C.). Glycerol was substituted for glucose. The recipe was made widi tap water and the pH adjusted to 7.2. Seed flasks were made from L-broth. The following recipes apply:

Base Medium (for 1 liter) glycerol 65 g (NH₄)₂S0₄ 1.0 g

Na₂HP0₄ 5.24 g

KH₂P0₄ 2.77 g

Yeast extract 5.0 g

Casamino acids 1.0 g

Metals 44 (for 100 ml)

EDTA 250 mg

ZnSO_-7H_:0 1095 mg

FeS0₄-7H_0 500 mg MnS0₄ H₂0 154 mg

CuS0₄-5H₂0 39.2 mg

Co(N0₃)₂-6H₂0 24.8 mg

Na₂B,O. 10H,O 17.7 mg

Add a few drops of 6 N H₂S0₄ to retard precipitation. Huntner's M eral Mix (for 1 liter)

Nitnloacetic acid (dissolved and neutrahzed with KOH) 10 g

MgS0₄-7H₂0 14 45 g

CaCl₂-2H₂0 3.33 g

(NH₄)₆Mo₇0₂₄-4H₂0 9 25 g

FeS0₄-7H₂0 99 mg

Metals 44 50 ml pH adjusted to 6 6-6 8

At inoculation for analysis of B t toxm expression, 4 ml of Huntner's Mmeral Mix was added per 200 ml of broth Flasks were then given a 2% inoculum, by volume, of an overnight culture Cultures were allowed to grow for 24 hours at 32 °C at _ 200 m At this pomt, they were mduced widi 075 mM IPTG and supplemented widi 2 g east extract Protem gels were run on samples pulled at 48 and 72 hours The 130 kDa protem was quantified by laser densitometry

Following are examples which dlustrate procedures, including die 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 otiierwise noted

Example 1 - Expression Vector Modification bv Splice Overlap Extension (SOE)

A clomng vector can be constructed based on pTJS260 a broad host-range plasmid deπved from RSF1010 (pTJS260 can be obtamed from Dr Donald Helinski, U C San Diego) An example of the svstem used in die vector construction can be found m EPO patent application 0 471 564 A cry lA(c)/crvIA(b) gene, refeπed to herem as the 436 gene and toxm, are descnbed m U S Patent No

5 055,294 A plasmid designated pMYC1050 contains the 436 gene pMYC1050 was constructed

re-cloning the toxm gene and promoter of pM3.130-7 (disclosed m U S Patent No 5 055,294) mto a pTJS260-based vector such as pMYC467 (disclosed m U S Patent No 5,169.760) bv mediods well known in die art In particular, the pM3, 130-7 promoter and toxm gene can be obtamed as a BamHl to Ndcl fragment and placed mto die pMYC467 plasmid replacmg a fragment bounded bv die same sites

(Bam HI near base 12100 and Ndel near base 8000)

The unproved vector ideally contains a unique BamHl clomng site The plasmid BamHl site located upstream from die lac promoter (Ptac), can be removed by blunting witii Klenow and re gating (Figure 1) Absence of the site can be confirmed b restnction digestion A plasmid produced accordmg to tins procedure was called pMYC1050Δ__αmHI The construct can now have a BamHl site added to die plasmid by SOE mutagenesis SOE mutagenesis can be facilitated bv subcloning an Nsil tox n- containing DNA fragment mto the smaller pGEM5 (Promega Coφ Madison WI) vector which uses die ampicillin resistance (b la) gene as a selectable marker (Figure 1). The fragment can be oriented by restriction digestion. A plasmid produced according to diis procedure was called pGEMtox.

DNA in die toxin coding region can be mutated by die PCR-mediated technique of SOE to introduce restriction enzyme cloning sites as shown in Figure 2. Oligonucleotides useful as primers are shown below:

"A" (SEQ ID NO. 1) 5' GCATACTAGTAGGAGATTTCCATGGATAACAATCCGAAC 3'

"B" (SEQ ID NO. 2) 5' GGATCCGCTTCCCAGTCT 3' "C" (SEQ ID NO. 3)

5' AGAGAGTGGGAAGCGGATCCTACTAATCC 3'

"D" (SEQ ID NO. 4) 5 ' TGGATACTCGATCGATATGATAATCCGT 3 '

"E" (SEQ ID NO. 5) 5 ' TAATAAGAGCTCCTATGT 3 '

"F" (SEQ ID NO. 6) 5 ' TATCATATCGATCGAGTATCC AATTTAG 3 '

"G" (SEQ ID NO. 7) 5 ' GTC ACATAGCCAGCTGGT 3 '

pMYClOSO DNA was used as the template for PCR amplification usmg primer sets A/B, C D, E D. and F/G Amplified DNA fragments were named AB, CD, ED, and FG Amplified DNAs were purified by agarose-TBE gel electrophoresis, electroelution, and NACS column chromatography. mediods all well- known in the art. Purified template DNAs were used in a second set of PCR reactions Fragments AB and CD were mixed and amplified widi primers A and D. In a separate reaction, fragments ED and FG were mixed and amplified widi primers E and G Amplified DNA was resolved by agarose-TBE gel electrophoresis and die fragments widi the corresponding increase m size were excised, electioeluted. and purified over NACS columns by means well known in die art Amplified DNA fragments are called AD or EG for reference. DNA fragments AD or EG with die new restriction enzyme sites were incoφorated mto the toxin-containing DNA by several subcloning procedures (Figures 2 and 3). pGEMtox was digested with Clal or Hindlll Vector-containing DNA was gel-purified. Fragment AD was digested widi Clal and ligated to C/αl-digested pGEMtox vector DNA. Fragment EG was digested widi Hindlll and ligated to -V.rtdlll-digested pGEMtox vector DNA. E. coli strain NM522 was transformed widi gation mixes Correctly assembled constructs were identified by restriction enzyme digestion of plasmid DNA from isolated colonies. The plasmid widi die new BamHl site was called pGEM tox BamHl The plasmid widi die new Pvul site was called pGEMtox Pvul. The Clal fragment containing die BamHl site from plasmid pGEMtox BamHl was ligated to die phosphatased Clal vector-containing fragment from pGEMtox Pvul. E. coli strain NM522 was transformed widi ligation mixes. Correctly assembled constructs were identified by PCR analysis with primer set C/D, and by restriction digestion. The plasmid widi bodi new restriction enzyme sites was called pGEMtox BamHl/Pvul. A completed expression vector was assembled widi insert from pGEMtox BamHl/Pvul and vector from pMYC1050Δ_5omHI (Figures 3 and 4). Gel-purified insert was prepared from pGEMtox BamHl/Pvul by Nsil digestion, and S al digestion (to remove contaminating vector). It was ligated to gel-purified Ns/I-digested vector-containing pMYC1050Δ5αmHI DΝA. E. coli strain ΝM522 was transformed widi die ligation mixes, and transformation mixes were plated on LB agar containing tetracycline at 12 μg/ml. Colonies containing the Nsil insert were identified by colony hybridization and autoradiography. Inserts were oriented by PCR, using primer set A D, which bridges an Nsil cloning site, and agarose-TBE gel electrophoresis. The correcdy assembled plasmid is called pMYC2224. DNA and protein sequences of the toxin are found in SEQ ID NOS. 18 and 19, respectively. A lactose-inducible P. fluorescens strain was electroporated with correctly assembled plasmid DN Transformation mixes were plated on LB agar containing tetracycline at 20 μg ml. Plasmid DNA was prepared from P. fluorescens for use in subsequent cloning experiments.

Example 2 - Subcloning die crvIF Hvpervariable Region into pMYC2224

A DNA fragment containing die hypervariable region from cryEF (pMYC 1260) was exchanged for the BamHl-Pnil toxin-containing DNA fragment from pMYC2224 (Figure 4). Since the coding sequence contains a preexisting BamHl site, BgUl was chosen for cloning. The 4-base overhangs of BamHl and BgUl are compatible, permitting ligation while eliminating bodi sites from die junction. It was necessary to synthesize a new primer for PCR: "L" (SEQ ID NO. 8) 5 ' GAGTGGGAAGC AGATCTTAATAATGCAC AATTAAGG 3 '

A toxin-containing DNA fragment was generated by PCR widi primers L/D on template pMYC 1260. The DNA was digested widi BglU and Pvul for subcloning. Since the telAR locus contains multiple

sites, it was necessary to isolate the vector-containing DNA on two separate fragments. To obtain die first fragment, pMYC2224 was digested widi BamHl x BstEll. and die large DNA fragment containing the Ptac-tetAR locus-rep functions was gel-purified. To obtain die second fragment. pMYC2224 was digested widi BstEll x Pvul, and die DNA fragment containing the vector-proto in module was gel-purified. A diree-piece ligation was set up and used for E. coli strain NM522 transformation. Grossly conect plasmids were identified by PCR analysis and agarose-TBE gel electrophoresis using die primer set N/O, which bridges die BamHl/Bgfll fusion junction.

"N" (tac promoter) (SEQ ID NO. 9) 5 ' TTAATCATCGGCTCGTA 3 ' "O" (SEQ ID NO. 10) 5' ACTCGATCGATATGATA(GA)TCCGT 3'

The correct plasmid was named pMYC2239. It consists of cιyIA(c) at the ammo-terminus, cryEF up to die toxinprotoxin junction, and cryIA(b) through the protoxm segment. The toxm DNA and protem sequences are in SEQ ID NOS. 20 and 21, respectively.

Example 3 - Construction of the P. fluorescens Expression Plasmids pMYC1260 and pMYC2047

The cloned toxm gene crylF can be modified for expression in P fluorescens in the following way:

1 A plasmid containing the pKK223-3 rrnB termination sequences in the pTJS260- deπved vector (Dr Donald Helinsla, U C. San Diego) can be made by ligating die BamHl-Scal fragment containing die Ptac promoter and rrnB terminator from pKK223-3 (Pharmacia E. coli vector) mto the

BamHl to blunted Kpnl vector fragment of pMYCl 197 (descnbed in EP 0 417 564). The assembled plasmid is recovered following transformation of E. coli and growth under tetracycline selection

2. A plasmid containing die Ptoc-promoted cryEF toxm gene can be made by ligating toxin gene-containing Ndel-Nde-l fragment (with ends blunted using DNA polymerase and dNTPs) of about 3800 bp from pMYC1603 (from NRRL B-18517) into the blunted EcoRI and Hindlll sites of pKK223- 3 The /toe-promoted crylF toxm plasmid can be recovered following transformation of E. coli, grown under ampicilhn selection, and screening for plasmids widi inserts in die proper oπentation for expression from die Ptac promoter by techniques well known m the art

3 The / .ore-promoted crylF toxin can be assembled mto die pTJS260-deπved vector m a three-piece ligation usmg the 2 4 kb DNA fragment having BamHl and Apal ends from die plasmid pTJS260, Apal to Hmd l fragment of 8.5 kb containmg the replication region of die plasmid from step 1 above, and a Hindlll to partial BamHl fragment containing die Ptac promoter and cry IF toxm gene from step 2 above

The resulting pTJS260-denved crylF toxm expression plasmid (pMYC1260) can be introduced mto P. fluorescens by electroporation.

4 pMYC2047 can be constructed by ligating an Spel to Kpnl fragment obtamed dirough PCR of a suitable cry IF template with primers H and K followed by digestion widi Spel and Kpnl and gel purification, an Apal to Kpnl fragment of a 10 kb from die plasmid of step 3. and die Apal to Spe I fragment of ca 2600 bp from pMYC1197 containing the Ptac promoter The coπect cry IF toxin expression plasmids are determmed by restπction enzyme digestion of plasmids following electroporation mto Pseudomonas fluorescens

Example 4 - Construction of a crvIF/crvIA(b) Chimera The cryIA(c) segment at the aπuno-teπrunus can be replaced by die crylF coding sequence by a simple, straightforward swap (Figure 5) Both the tetAR locus and crylF coding sequence contain an Apal site A small Apal fragment containing a portion of the tetAR genes and die anuno-termmus of crylF can be isolated from pMYC2047 and ligated to die large Apal vector-containing fragment from pMYC2239 A P fluorescens lactose-inducible strain can be electroporated with die ligaαon mix and plated on LB agar containmg tetracycline at 20 μg/ml. Lactose-inducible strains are known to diose skilled m the art and are descnbed, for example, m U.S Patent No. 5,169,760. Correct onentation of the Apal fragment reconstitutes tetracycline resistance. A clone produced m tins manner was shown to be grossly coπect by restnction enzyme digestion, and it was named pMYC2244 The toxm DNA sequence is shown in SEQ ID NO 22, and die predicted protem sequence is shown m SEQ ID NO 23

Example 5 - Construction of a Limited Codon Rework of crvIF

Codon usage m Pseudomonas spp favors G or C m the wobble position of triplet codons, as determmed by analysis of genes in die GenBankEMBL sequence libranes A limited region of the crylF gene was reworked by SOE to incoφorate favored wobble position changes diat were sdent (Figure 6)

Oligos used are shown below

"H" (SEQ ID NO 11) 5' GGACTAGTAAAAAGGAGATAACCATGGAAAATAATATTCAAAATC 3'

"I" (SEQ ID NO. 12) 5' TCCAC GGCAC -CGGCCGGTGCTGCGTTCTTCGTTCAGTATTTCTACT

TCAGGATTATTTAAAC 3'

"J" (SEQ ID NO. 13) 5 ' AACGCAGCACCGGCCGCCTGCCGCTGGACATCAGCCTGAGCCTTACAC

GTTTCCTTTTGAGTGAA 3' "K" (SEQ ID NO. 14)

5 CATCAAAGGTACCTGGT 3'

Two separate PCR reactions were done on pMYC2047 template with primer sets H/l or J/K Amplified DNA fragments were called HI or JK A second PCR reaction was set up by mixing fragments HI and JK and PCR amplifying w di pπmer set H/K The larger SOE DNA was gel-purified and digested widi Spel x Kpnl A tiiree-piece ligation was set up vvitii Spel-Apal Plac-teiAR locus DNA Apal-Kpnl vector-protoxin module DNA and Spel-Kpnl PCR DNA A P fluorescens lactose-inducible strain can be electroporated with die ligation mix Grossly' coπect clones can be identified by PCR analv sis using die primer set P/Q and agarose-TBE gel electrophoresis Oligo P (SEQ ID NO 15) was designed to discriminate between the v\ lid-type and codon-reworked gene

"P" (SEQ ID NO 15) 5 ' TGCCGCTGGACATCAGCCTGAG 3 ' "Q" (SEQ ID NO. 16) 5 ' TCTAGAGCGGCCGCTTATAC(CT)CGATCGATATGATA(GA)TCCGT 3 '

The complete plasmid was named pMYC2243 The toxm DNA sequence is shown m SEQ ID NO 24 The toxm protem sequence is predicted to be unchanged, and is shown m SEQ ID NO 25

Example 6 - Construction of the cryEF/cryIA(b) Chimera Containing the Limited Codon Rework

The construct was assembled (Figure 7) using die same Apal fragment exchange strategy as for pMYC2244 (cιyIF/c_yIA(b)) above The small, toxm-tetAR locus Apal DNA fragment was gel-purified from pMYC2243 The larger vector-protoxin module Apal DNA fragment was gel-purified from pMYC2244 The completed plasmid was named pMYC2523 Predicted DNA and protem sequences are m SEQ ID NOS 26 and 27, respectively

Example 7 - Comparative Expression of Toxms from pMYC2244 and pMYC2523 Toxm expression m P fluorescens was analyzed as descnbed above At 24 and 48 hours post- induction, die pMYC2523-contaιnιng strain produced more toxm dian die pMYC2244-contaιnιng strain Toxin specific activity on Spodoptera exigua was statistically unchanged

Example 8 - Construction of the cryIF/436 Chimera Containing die Limited Codon Rework A second type of chimeπc toxm was assembled by substituting the 436 protoxm module for the cryIA(b) protoxm m pMYC2523 (Figure 8) The 436 protoxm sequence consists of crylA(c) sequence except at the very C-termmus (See U S Patent Nos 5,128,130 and 5,169,760, mcoφorated herem by reference m their entirety) Proto m DNA for clo ng was generated by PCR widi die pnmer set F M using a plasmid such as pMYC467 (U S Patent No 5,169,760) as a template "M" (SEQ ID NO 17)

5' AGGCTTCCATAGATACCTTGTGCG 3'

PCR DNA was digested widi Pvul x jSs.EII A three-piece ligation was set up widi Spe -Pvul toxm DNA from pMYC2523, Spel-BstEU vector DNA from pMYC2523. and Pvul-BstEll PCR protoxin module DNA A lactose-inducible P fluorescens strain was electroporated widi die ligation mix

Grossly coπect plasmids were identified by PCR witii pnmer set F/G and screenmg for slight size mcrease by agarose-TBE gel electrophoresis The construct was named pMYC2254 Predicted DNA and protem sequences are found m SEQ ID NOS 28 and 29, respectively Example 9 - Comparative Expression of Toxins from pMYC2243 and PMYC2254

Toxin expression in P. fluorescens was analyzed as described above. Toxin expression from pMYC2254 was improved over pMYC2243 expression.

Example 10 - Analysis for Synergy Between CrvIF Chimeric Toxin and CryIA(c) Chimeric Toxin Against the Corn Earworm. Heliothis zea

Twenty-four Heliothis zea first instar larvae were exposed to agar diet containing various concentiations of toxin- At 7 days post treatment, assays were graded for growth inhibition. Larvae were inhibited if the molt from first to second instar was inhibited. Calculations for estimating synergy factor

(SF) and expected activity (E[exp]) are shown below.

SF = E(obs)fE(exp) where,

SF = synergy factor E(ob s) = observed mortality

E(exp) - expected mortality

E(exp) = a + b - (ab/100) where, a = activity from compound A b = activity from compound B

Table 2.

% INHIBITION

crylF/ cryΙA(c)/

Rate cryIA(b) cryIA(b) 1 1 mix of the two chimenc toxins μg toxin/ g diet a b E(exp) E(obs) SF

50 0 - - 50 78 1 6

25 0 13 23 22 62 2 8

12 5 9 14 22 31 1 4

6 25 9 14 ~ - -

An SF greater than 1 indicates synergy fl_evy, Y , M Benderly, Y Cohen, U Gisi. D Bassard [1986]

Bulletin OEPP/EPPO Bulletin 16 651-657) Abbott. W S (1925) J Economic Entomology 18.265-267

Example 11 - Analysis for Synergy Between CrvIF Chimenc Toxm and CryΙA(c) Chimenc Toxm Agamst die Corn Earworm. Heliothis zea

Twenty-four Heliothis zea first mstar larvae were exposed to agar diet containmg vanous concentrations of toxm At 7 days post treatment, assays were graded for growth inhibition Larv ae were inhibited rfdie molt from first to second mstar was mhibited The dosage reqiured to inhibit 50 percent of die populations (ΕD_X) was estimated usmg standard probit analysis techmques Calculations for estimating svnergy factor (SF) and expected effective dosages (ED[exp]) are shown below

SF = ED(exp) ED(obs) where,

ED(exp) = expected effective dose of a mixture ED(obs) = observed effective dose of a mixture

ED(exp) = (a + b)/a ED_A + b ED_B where, a = proportion of compound A m mixture b = proportion of compound B m mixture

ED_A and ED_B = equally effective doses of A and B m mixture Table 3.

ED(obs) ED(exp)

Treatment (μg toxin g diet) SF cryIA(c)/cryIA(b) (A) 36 - - cryIF/cryIA(b) (B) 135 - -

A:B (1:1) 21 57 2.6

A:B (3:1) 14 44 3.1

A:B (1:3) 35 80 2.3

A SF greater than 1 indicates synergy (Levy et al. [1986], supra).

[CITE for Wadley method]

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

SEQUENCE LISTING

( 1 ) GENERAL INFORMATION :

( i ) APPLICANT :

Applicant Name ( s ) : MYCOGEN CORPORATION

Street address : 5501 Oberlin Drive

City : San Diego

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(ii) TITLE OF INVENTION: Novel Pesticidal Compositions (iii) NUMBER OF SEQUENCES: 34

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: David R. Saliwanchik

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(Vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: US

(B) FILING DATE:

(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Saliwanchik, David R.

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(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 904-375-8100

(B) TELEFAX: 904-372-5800

(2) INFORMATION FOR SEQ ID Nθ:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 39 bases

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID Nθ:l: GCATACTAGT AGGAGATTTC CATGGATAAC AATCCGAAC 39

(2) INFORMATION FOR SEQ ID Nθ:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 bases

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: GGATCCGCTT CCCAGTCT 18

(2) INFORMATION FOR SEQ ID NO:3:

(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:3: AGAGAGTGGG AAGCGGATCC TACTAATCC 29

(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 28 bases

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: TGGATACTCG ATCGATATGA TAATCCGT 28

(2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 bases

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: TAATAAGAGC TCCTATGT 18

(2) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 28 bases

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: TATCATATCG ATCGAGTATC CAATTTAG 28

(2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 bases

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (synthetic) ( i) SEQUENCE DESCRIPTION: SEQ ID NO:7: GTCACATAGC CAGCTGGT 18

(2) INFORMATION FOR SEQ ID NO:8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 36 bases

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: GAGTGGGAAG CAGATCTTAA TAATGCACAA TTAAGG 36

(2) INFORMATION FOR SEQ ID NO:9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 bases

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: TTAATCATCG GCTCGTA 17

(2) INFORMATION FOR SEQ ID NO:10:

(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:10: ACTCGATCGA TATGATARTC CGT 23

(2) INFORMATION FOR SEQ ID NO:11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 45 bases

(B) TYPE: nucleic acid

(C) STRANDEDNESS: Single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: GGACTAGTAA AAAGGAGATA ACCATGGAAA ATAATATTCA AAATC 45

(2) INFORMATION FOR SEQ ID NO: 12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 64 bases

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (synthetic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: TCCAGCGGCA GGCGGCCGGT GCTGCGTTCT TCGTTCAGTA TTTCTACTTC AGGATTATTT 60 AAAC 64 (2) INFORMATION FOR SEQ ID NO:13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 65 bases

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (synthetic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: AACGCAGCAC CGGCCGCCTG CCGCTGGACA TCAGCCTGAG CCTTACACGT TTCCTTTTGA 60 GTGAA 65

(2) INFORMATION FOR SEQ ID NO:14:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 bases

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: CATCAAAGGT ACCTGGT 17

(2) INFORMATION FOR SEQ ID NO: 15:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 bases

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (synthetic) (Xi) SEQUENCE DESCRIPTION: SEQ ID Nθ:15: TGCCGCTGGA CATCAGCCTG AG 22

(2) INFORMATION FOR SEQ ID NO:16:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 41 bases

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: TCTAGAGCGG CCGCTTATAC YCGATCGATA TGATARTCCG T 41

(2) INFORMATION FOR SEQ ID NO:17:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 bases

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (synthetic) (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: AGGCTTCCAT AGATACCTTG TGCG 24

(2) INFORMATION FOR SEQ ID NO:18:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 3465 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:

ATGGATAACA ATCCGAACAT CAATGAATGC ATTCCTTATA ATTGTTTAAG TAACCCTGAA 60

GTAGAAGTAT TAGGTGGAGA AAGAATAGAA ACTGGTTACA CCCCAATCGA TATTTCCTTG 120

TCGCTAACGC AATTTCTTTT GAGTGAATTT GTTCCCGGTG CTGGATTTGT GTTAGGACTA 180

GTTGATATAA TATGGGGAAT TTTTGGTCCC TCTCAATGGG ACGCATTTCT TGTACAAATT 240

GAACAGTTAA TTAACCAAAG AATAGAAGAA TTCGCTAGGA ACCAAGCCAT TTCTAGATTA 300

GAAGGACTAA GCAATCTTTA TCAAATTTAC GCAGAATCTT TTAGAGAGTG GGAAGCGGAT 360

CCTACTAATC CAGCATTAAG AGAAGAGATG CGTATTCAAT TCAATGACAT GAACAGTGCC 420

CTTACAACCG CTATTCCTCT TTTTGCAGTT CAAAATTATC AAGTTCCTCT TTTATCAGTA 480

TATGTTCAAG CTGCAAATTT ACATTTATCA GTTTTGAGAG ATGTTTCAGT GTTTGGACAA 540

AGGTGGGGAT TTGATGCCGC GACTATCAAT AGTCGTTATA ATGATTTAAC TAGGCTTATT 600

GGCAACTATA CAGATTATGC TGTACGCTGG TACAATACGG GATTAGAACG TGTATGGGGA 660

CCGGATTCTA GAGATTGGGT AAGGTATAAT CAATTTAGAA GAGAATTAAC ACTAACTGTA 720

TTAGATATCG TTGCTCTGTT CCCGAATTAT GATAGTAGAA GATATCCAAT TCGAACAGTT 780 TCCCAATTAA CAAGAGAAAT TTATACAAAC CCAGTATTAG AAAATTTTGA TGGTAGTTTT 840

CGAGGCTCGG CTCAGGGCAT AGAAAGAAGT ATTAGGAGTC CACATTTGAT GGATATACTT 900

AACAGTATAA CCATCTATAC GGATGCTCAT AGGGGTTATT ATTATTGGTC AGGGCATCAA 960

ATAATGGCTT CTCCTGTAGG GTTTTCGGGG CCAGAATTCA CTTTTCCGCT ATATGGAACT 1020

ATGGGAAATG CAGCTCCACA ACAACGTATT GTTGCTCAAC TAGGTCAGGG CGTGTATAGA 1080

ACATTATCGT CCACTTTATA TAGAAGACCT TTTAATATAG GGATAAATAA TCAACAACTA 11 0

TCTGTTCTTG ACGGGACAGA ATTTGCTTAT GGAACCTCCT CAAATTTGCC ATCCGCTGTA 1200

TACAGAAAAA GCGGAACGGT AGATTCGCTG GATGAAATAC CGCCACAGAA TAACAACGTG 1260

CCACCTAGGC AAGGATTTAG TCATCGATTA AGCCATGTTT CAATGTTTCG TTCAGGCTTT 1320

AGTAATAGTA GTGTAAGTAT AATAAGAGCT CCTATGTTCT CTTGGATACA TCGTAGTGCT 1380

GAATTTAATA ATATAATTCC TTCATCACAA ATTACACAAA TACCTTTAAC AAAATCTACT 1440

AATCTTGGCT CTGGAACTTC TGTCGTTAAA GGACCAGGAT TTACAGGAGG AGATATTCTT 1500

CGAAGAACTT CACCTGGCCA GATTTCAACC TTAAGAGTAA ATATTACTGC ACCATTATCA 1560

CAAAGATATC GGGTAAGAAT TCGCTACGCT TCTACCACAA ATTTACAATT CCATACATCA 1620

ATTGACGGAA GACCTATTAA TCAGGGGAAT TTTTCAGCAA CTATGAGTAG TGGGAGTAAT 1680

TTACAGTCCG GAAGCTTTAG GACTGTAGGT TTTACTACTC CGTTTAACTT TTCAAATGGA 1740

TCAAGTGTAT TTACGTTAAG TGCTCATGTC TTCAATTCAG GCAATGAAGT TTATATAGAT 1800

CGAATTGAAT TTGTTCCGGC AGAAGTAACC TTTGAGGCAG AATATGATTT AGAAAGAGCA 1860

CAAAAGGCGG TGAATGAGCT GTTTACTTCT TCCAATCAAA TCGGGTTAAA AACAGATGTG 1920

ACGGATTATC ATATCGATCG AGTATCCAAT TTAGTTGAGT GTTTATCTGA TGAATTTTGT 1980

CTGGATGAAA AAAAAGAATT GTCCGAGAAA GTCAAACATG CGAAGCGACT TAGTGATGAG 2040

CGGAATTTAC TTCAAGATCC AAACTTTAGA GGGATCAATA GACAACTAGA CCGTGGCTGG 2100

AGAGGAAGTA CGGATATTAC CATCCAAGGA GGCGATGACG TATTCAAAGA GAATTACGTT 2160

ACGCTATTGG GTACCTTTGA TGAGTGCTAT CCAACGTATT TATATCAAAA AATAGATGAG 2220

TCGAAATTAA AAGCCTATAC CCGTTACCAA TTAAGAGGGT ATATCGAAGA TAGTCAAGAC 2280

TTAGAAATCT ATTTAATTCG CTACAATGCC AAACACGAAA CAGTAAATGT GCCAGGTACG 2340

GGTTCCTTAT GGCCGCTTTC AGCCCCAAGT CCAATCGGAA AATGTGCCCA TCATTCCCAT 2400

CATTTCTCCT TGGACATTGA TGTTGGATGT ACAGACTTAA ATGAGGACTT AGGTGTATGG 2460

GTGATATTCA AGATTAAGAC GCAAGATGGC CATGCAAGAC TAGGAAATCT AGAATTTCTC 2520 GAAGAGAAAC CATTAGTAGG AGAAGCACTA GCTCGTGTGA AAAGAGCGGA GAAAAAATGG 2580

AGAGACAAAC GTGAAAAATT GGAATGGGAA ACAAATATTG TTTATAAAGA GGCAAAAGAA 2640

TCTGTAGATG CTTTATTTGT AAACTCTCAA TATGATAGAT TACAAGCGGA TACCAACATC 2700

GCGATGATTC ATGCGGCAGA TAAACGCGTT CATAGCATTC GAGAAGCTTA TCTGCCTGAG 2760

CTGTCTGTGA TTCCGGGTGT CAATGCGGCT ATTTTTGAAG AATTAGAAGG GCGTATTTTC 2820

ACTGCATTCT CCCTATATGA TGCGAGAAAT GTCATTAAAA ATGGTGATTT TAATAATGGC 2880

TTATCCTGCT GGAACGTGAA AGGGCATGTA GATGTAGAAG AACAAAACAA CCACCGTTCG 2940

GTCCTTGTTG TTCCGGAATG GGAAGCAGAA GTGTCACAAG AAGTTCGTGT CTGTCCGGGT 3000

CGTGGCTATA TCCTTCGTGT CACAGCGTAC AAGGAGGGAT ATGGAGAAGG TTGCGTAACC 3060

ATTCATGAGA TCGAGAACAA TACAGACGAA CTGAAGTTTA GCAACTGTGT AGAAGAGGAA 3120

GTATATCCAA ACAACACGGT AACGTGTAAT GATTATACTG CGACTCAAGA AGAATATGAG 3180

GGTACGTACA CTTCTCGTAA TCGAGGATAT GACGGAGCCT ATGAAAGCAA TTCTTCTGTA 3240

CCAGCTGATT ATGCATCAGC CTATGAAGAA AAAGCATATA CAGATGGACG AAGAGACAAT 3300

CCTTGTGAAT CTAACAGAGG ATATGGGGAT TACACACCAC TACCAGCTGG CTATGTGACA 3360

AAAGAATTAG AGTACTTCCC AGAAACCGAT AAGGTATGGA TTGAGATCGG AGAAACGGAA 3420

GGAACATTCA TCGTGGACAG CGTGGAATTA CTTCTTATGG AGGAA 3465

(2) INFORMATION FOR SEQ ID NO:19:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1155 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:

Met Asp Asn Asn Pro Asn lie Asn Glu Cys lie Pro Tyr Asn Cys Leu 1 5 10 15

Ser Asn Pro Glu Val Glu Val Leu Gly Gly Glu Arg lie Glu Thr Gly 20 25 30

Tyr Thr Pro lie Asp lie Ser Leu ser Leu Thr Gin Phe Leu Leu Ser 35 40 45

Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gly Leu Val Asp lie He 50 55 60 Trp Gly He Phe Gly Pro Ser Gin Trp Asp Ala Phe Leu Val Gin He 65 70 75 80

Glu Gin Leu He Asn Gin Arg He Glu Glu Phe Ala Arg Asn Gin Ala 85 90 95

He Ser Arg Leu Glu Gly Leu Ser Asn Leu Tyr Gin He Tyr Ala Glu 100 105 110

Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr Asn Pro Ala Leu Arg Glu

115 120 125

Glu Met Arg He Gin Phe Asn Asp Met Asn Ser Ala Leu Thr Thr Ala 130 135 140

He Pro Leu Phe Ala Val Gin Asn Tyr Gin Val Pro Leu Leu Ser Val 145 150 155 160

Tyr Val Gin Ala Ala Asn Leu His Leu ser Val Leu Arg Asp Val Ser 165 170 175

Val Phe Gly Gin Arg Trp Gly Phe Asp Ala Ala Thr He Asn Ser Arg 180 185 190

Tyr Asn Asp Leu Thr Arg Leu He Gly Asn Tyr Thr Asp Tyr Ala Val 195 200 205

Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Trp Gly Pro Asp Ser Arg 210 215 220

Asp Trp Val Arg Tyr Asn Gin Phe Arg Arg Glu Leu Thr Leu Thr Val 225 230 235 240

Leu Asp He Val Ala Leu Phe Pro Asn Tyr Asp Ser Arg Arg Tyr Pro 245 250 255

He Arg Thr Val Ser Gin Leu Thr Arg Glu He Tyr Thr Asn Pro Val 260 265 270

Leu Glu Asn Phe Asp Gly Ser Phe Arg Gly Ser Ala Gin Gly He Glu 275 280 285

Arg Ser He Arg ser Pro His Leu Met Asp He Leu Asn Ser He Thr 290 295 300

He Tyr Thr Asp Ala His Arg Gly Tyr Tyr Tyr Trp Ser Gly His Gin 305 310 315 320

He Met Ala Ser Pro Val Gly Phe Ser Gly Pro Glu Phe Thr Phe Pro 325 330 335

Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro Gin Gin Arg He Val Ala 340 345 350

Gin Leu Gly Gin Gly Val Tyr Arg Thr Leu Ser ser Thr Leu Tyr Arg 355 360 365 Arg Pro Phe Asn He Gly He Asn Asn Gin Gin Leu Ser Val Leu Asp 370 375 380

Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser Asn Leu Pro Ser Ala Val 385 390 395 400

Tyr Arg Lys Ser Gly Thr val Asp Ser Leu Asp Glu He Pro Pro Gin 405 410 415

Asn Asn Asn Val Pro Pro Arg Gin Gly Phe ser His Arg Leu Ser His 420 425 430

Val Ser Met Phe Arg Ser Gly Phe Ser Asn Ser Ser Val Ser He He

435 440 445

Arg Ala Pro Met Phe Ser Trp He His Arg Ser Ala Glu Phe Asn Asn 450 455 460

He He Pro Ser Ser Gin He Thr Gin He Pro Leu Thr Lys Ser Thr 465 470 475 480

Asn Leu Gly Ser Gly Thr Ser Val Val Lys Gly Pro Gly Phe Thr Gly 485 490 495

Gly Asp He Leu Arg Arg Thr Ser Pro Gly Gin He Ser Thr Leu Arg 500 505 510

Val Asn He Thr Ala Pro Leu Ser Gin Arg Tyr Arg Val Arg He Arg 515 520 525

Tyr Ala Ser Thr Thr Asn Leu Gin Phe His Thr Ser He Asp Gly Arg 530 535 540

Pro He Asn Gin Gly Asn Phe Ser Ala Thr Met Ser Ser Gly Ser Asn 545 550 555 560

Leu Gin Ser Gly Ser Phe Arg Thr Val Gly Phe Thr Thr Pro Phe Asn 565 570 575

Phe Ser Asn Gly Ser Ser Val Phe Thr Leu ser Ala His Val Phe Asn 580 585 590

Ser Gly Asn Glu val Tyr He Asp Arg He Glu Phe Val Pro Ala Glu 595 600 605

Val Thr Phe Glu Ala Glu Tyr Asp Leu Glu Arg Ala Gin Lys Ala Val 610 615 620

Asn Glu Leu Phe Thr Ser Ser Asn Gin He Gly Leu Lys Thr Asp Val 625 630 635 640

Thr Asp Tyr His He Asp Arg Val ser Asn Leu Val Glu Cys Leu Ser 645 650 655

Asp Glu Phe cys Leu Asp Glu Lys Lys Glu Leu Ser Glu Lys val Lys 660 665 670 His Ala Lys Arg Leu Ser Asp Glu Arg Asn Leu Leu Gin Asp Pro Asn 675 680 685

Phe Arg Gly He Asn Arg Gin Leu Asp Arg Gly Trp Arg Gly Ser Thr 690 695 700

Asp He Thr He Gin Gly Gly Asp Asp Val Phe Lys Glu Asn Tyr Val 705 710 715 720

Thr Leu Leu Gly Thr Phe Asp Glu Cys Tyr Pro Thr Tyr Leu Tyr Gin 725 730 735

Lys He Asp Glu Ser Lys Leu Lys Ala Tyr Thr Arg Tyr Gin Leu Arg 740 745 750

Gly Tyr He Glu Asp Ser Gin Asp Leu Glu He Tyr Leu He Arg Tyr 755 760 765

Asn Ala Lys His Glu Thr Val Asn Val Pro Gly Thr Gly Ser Leu Trp 770 775 780

Pro Leu Ser Ala Pro Ser Pro He Gly Lys Cys Ala His His Ser His 785 790 795 800

His Phe Ser Leu Asp He Asp Val Gly Cys Thr Asp Leu Asn Glu Asp 805 810 815

Leu Gly Val Trp Val He Phe Lys He Lys Thr Gin Asp Gly His Ala 820 825 830

Arg Leu Gly Asn Leu Glu Phe Leu Glu Glu Lys Pro Leu Val Gly Glu 835 840 845

Ala Leu Ala Arg Val Lys Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg 850 855 860

Glu Lys Leu Glu Trp Glu Thr Asn He Val Tyr Lys Glu Ala Lys Glu 865 870 875 880

Ser Val Asp Ala Leu Phe Val Asn Ser Gin Tyr Asp Arg Leu Gin Ala 885 890 895

Asp Thr Asn He Ala Met He His Ala Ala Asp Lys Arg Val His Ser 900 905 910

He Arg Glu Ala Tyr Leu Pro Glu Leu Ser Val He Pro Gly Val Asn 915 920 925

Ala Ala He Phe Glu Glu Leu Glu Gly Arg He Phe Thr Ala Phe Ser 930 935 940

Leu Tyr Asp Ala Arg Asn Val He Lys Asn Gly Asp Phe Asn Asn Gly 945 950 955 960

Leu Ser Cys Trp Asn Val Lys Gly His Val Asp Val Glu Glu Gin Asn 965 970 975 Asn His Arg Ser Val Leu Val Val Pro Glu Trp Glu Ala Glu Val Ser 980 985 990

Gin Glu Val Arg Val Cys Pro Gly Arg Gly Tyr He Leu Arg Val Thr 995 1000 1005

Ala Tyr Lys Glu Gly Tyr Gly Glu Gly Cys Val Thr He His Glu He 1010 1015 1020

Glu Asn Asn Thr Asp Glu Leu Lys Phe Ser Asn cys Val Glu Glu Glu 1025 1030 1035 1040

Val Tyr Pro Asn Asn Thr Val Thr Cys Asn Asp Tyr Thr Ala Thr Gin 1045 1050 1055

Glu Glu Tyr Glu Gly Thr Tyr Thr Ser Arg Asn Arg Gly Tyr Asp Gly 1060 1065 1070

Ala Tyr Glu Ser Asn Ser Ser Val Pro Ala Asp Tyr Ala ser Ala Tyr 1075 1080 1085

Glu Glu Lys Ala Tyr Thr Asp Gly Arg Arg Asp Asn Pro Cys Glu Ser 1090 1095 1100

Asn Arg Gly Tyr Gly Asp Tyr Thr Pro Leu Pro Ala Gly Tyr Val Thr 1105 1110 1115 1120

Lys Glu Leu Glu Tyr Phe Pro Glu Thr Asp Lys Val Trp He Glu He 1125 1130 1135

Gly Glu Thr Glu Gly Thr Phe He Val Asp Ser Val Glu Leu Leu Leu 1140 1145 1150

Met Glu Glu 1155

(2) INFORMATION FOR SEQ ID NO:20:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 3450 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:

ATGGATAACA ATCCGAACAT CAATGAATGC ATTCCTTATA ATTGTTTAAG TAACCCTGAA 60

GTAGAAGTAT TAGGTGGAGA AAGAATAGAA ACTGGTTACA CCCCAATCGA TATTTCCTTG 120

TCGCTAACGC AATTTCTTTT GAGTGAATTT GTTCCCGGTG CTGGATTTGT GTTAGGACTA 180

GTTGATATAA TATGGGGAAT TTTTGGTCCC TCTCAATGGG ACGCATTTCT TGTACAAATT 240 GAACAGTTAA TTAACCAAAG AATAGAAGAA TTCGCTAGGA ACCAAGCCAT TTCTAGATTA 300

GAAGGACTAA GCAATCTTTA TCAAATTTAC GCAGAATCTT TTAGAGAGTG GGAAGCGGAT 360

CTTAATAATG CACAATTAAG GGAAGATGTG CGTATTCGAT TTGCTAATAC AGACGACGCT 420

TTAATAACAG CAATAAATAA TTTTACACTT ACAAGTTTTG AAATCCCTCT TTTATCGGTC 480

TATGTTCAAG CGGCGAATTT ACATTTATCA CTATTAAGAG ACGCTGTATC GTTTGGGCAG 540

GGTTGGGGAC TGGATATAGC TACTGTTAAT AATCATTATA ATAGATTAAT AAATCTTATT 600

CATAGATATA CGAAACATTG TTTGGACACA TACAATCAAG GATTAGAAAA CTTAAGAGGT 660

ACTAATACTC GACAATGGGC AAGATTCAAT CAGTTTAGGA GAGATTTAAC ACTTACTGTA 720

TTAGATATCG TTGCTCTTTT TCCGAACTAC GATGTTAGAA CATATCCAAT TCAAACGTCA 780

TCCCAATTAA CAAGGGAAAT TTATACAAGT TCAGTAATTG AGGATTCTCC AGTTTCTGCT 840

AATATACCTA ATGGTTTTAA TAGGGCGGAA TTTGGAGTTA GACCGCCCCA TCTTATGGAC 900

TTTATGAATT CTTTGTTTGT AACTGCAGAG ACTGTTAGAA GTCAAACTGT GTGGGGAGGA 960

CACTTAGTTA GTTCACGAAA TACGGCTGGT AACCGTATAA ATTTCCCTAG TTACGGGGTC 1020

TTCAATCCTG GTGGCGCCAT TTGGATTGCA GATGAGGATC CACGTCCTTT TTATCGGACA 1080

TTATCAGATC CTGTTTTTGT CCGAGGAGGA TTTGGGAATC CTCATTATGT ACTGGGGCTT 1140

AGGGGAGTAG CATTTCAACA AACTGGTACG AACCACACCC GAACATTTAG AAATAGTGGG 1200

ACCATAGATT CTCTAGATGA AATCCCACCT CAGGATAATA GTGGGGCACC TTGGAATGAT 1260

TATAGTCATG TATTAAATCA TGTTACATTT GTACGATGGC CAGGTGAGAT TTCAGGAAGT 1320

GATTCATGGA GAGCTCCAAT GTTTTCTTGG ACGCACCGTA GTGCAACCCC TACAAATACA 1380

ATTGATCCGG AGAGGATTAC TCAAATACCA TTGGTAAAAG CACATACACT TCAGTCAGGT 1440

ACTACTGTTG TAAGAGGGCC CGGGTTTACG GGAGGAGATA TTCTTCGACG AACAAGTGGA 1500

GGACCATTTG CTTATACTAT TGTTAATATA AATGGGCAAT TACCCCAAAG GTATCGTGCA 1560

AGAATACGCT ATGCCTCTAC TACAAATCTA AGAATTTACG TAACGGTTGC AGGTGAACGG 1620

ATTTTTGCTG GTCAATTTAA CAAAACAATG GATACCGGTG ACCCATTAAC ATTCCAATCT 1680

TTTAGTTACG CAACTATTAA TACAGCTTTT ACATTCCCAA TGAGCCAGAG TAGTTTCACA 1740

GTAGGTGCTG ATACTTTTAG TTCAGGGAAT GAAGTTTATA TAGACAGATT TGAATTGATT 1800

CCAGTTACTG CAACATTTGA AGCAGAATAT GATTTAGAAA GAGCACAAAA GGCGGTGAAT 1860

GCGCTGTTTA CTTCTATAAA CCAAATAGGG ATAAAAACAG ATGTGACGGA TTATCATATC 1920

GATCGAGTAT CCAATTTAGT TGAGTGTTTA TCTGATGAAT TTTGTCTGGA TGAAAAAAAA 1980 GAATTGTCCG AGAAAGTCAA ACATGCGAAG CGACTTAGTG ATGAGCGGAA TTTACTTCAA 2040

GATCCAAACT TTAGAGGGAT CAATAGACAA CTAGACCGTG GCTGGAGAGG AAGTACGGAT 2100

ATTACCATCC AAGGAGGCGA TGACGTATTC AAAGAGAATT ACGTTACGCT ATTGGGTACC 2160

TTTGATGAGT GCTATCCAAC GTATTTATAT CAAAAAATAG ATGAGTCGAA ATTAAAAGCC 2220

TATACCCGTT ACCAATTAAG AGGGTATATC GAAGATAGTC AAGACTTAGA AATCTATTTA 2280

ATTCGCTACA ATGCCAAACA CGAAACAGTA AATGTGCCAG GTACGGGTTC CTTATGGCCG 2340

CTTTCAGCCC CAAGTCCAAT CGGAAAATGT GCCCATCATT CCCATCATTT CTCCTTGGAC 2 00

ATTGATGTTG GATGTACAGA CTTAAATGAG GACTTAGGTG TATGGGTGAT ATTCAAGATT 2460

AAGACGCAAG ATGGCCATGC AAGACTAGGA AATCTAGAAT TTCTCGAAGA GAAACCATTA 2520

GTAGGAGAAG CACTAGCTCG TGTGAAAAGA GCGGAGAAAA AATGGAGAGA CAAACGTGAA 2580

AAATTGGAAT GGGAAACAAA TATTGTTTAT AAAGAGGCAA AAGAATCTGT AGATGCTTTA 2640

TTTGTAAACT CTCAATATGA TAGATTACAA GCGGATACCA ACATCGCGAT GATTCATGCG 2700

GCAGATAAAC GCGTTCATAG CATTCGAGAA GCTTATCTGC CTGAGCTGTC TGTGATTCCG 2760

GGTGTCAATG CGGCTATTTT TGAAGAATTA GAAGGGCGTA TTTTCACTGC ATTCTCCCTA 2820

TATGATGCGA GAAATGTCAT TAAAAATGGT GATTTTAATA ATGGCTTATC CTGCTGGAAC 2880

GTGAAAGGGC ATGTAGATGT AGAAGAACAA AACAACCACC GTTCGGTCCT TGTTGTTCCG 2940

GAATGGGAAG CAGAAGTGTC ACAAGAAGTT CGTGTCTGTC CGGGTCGTGG CTATATCCTT 3000

CGTGTCACAG CGTACAAGGA GGGATATGGA GAAGGTTGCG TAACCATTCA TGAGATCGAG 3060

AACAATACAG ACGAACTGAA GTTTAGCAAC TGTGTAGAAG AGGAAGTATA TCCAAACAAC 3120

ACGGTAACGT GTAATGATTA TACTGCGACT CAAGAAGAAT ATGAGGGTAC GTACACTTCT 3180

CGTAATCGAG GATATGACGG AGCCTATGAA AGCAATTCTT CTGTACCAGC TGATTATGCA 3240

TCAGCCTATG AAGAAAAAGC ATATACAGAT GGACGAAGAG ACAATCCTTG TGAATCTAAC 3300

AGAGGATATG GGGATTACAC ACCACTACCA GCTGGCTATG TGACAAAAGA ATTAGAGTAC 3360

TTCCCAGAAA CCGATAAGGT ATGGATTGAG ATCGGAGAAA CGGAAGGAAC ATTCATCGTG 3420

GACAGCGTGG AATTACTTCT TATGGAGGAA 3450

(2) INFORMATION FOR SEQ ID Nθ:21:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1150 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single ( D ) TOPOLOGY : linear ( ii ) MOLECULE TYPE : protein ( i ) SEQUENCE DESCRIPTION : SEQ ID NO : 21 :

Met Asp Asn Asn Pro Asn He Asn Glu Cys He Pro Tyr Asn Cys Leu

1 5 10 15

Ser Asn Pro Glu Val Glu Val Leu Gly Gly Glu Arg He Glu Thr Gly 20 25 30

Tyr Thr Pro He Asp He Ser Leu ser Leu Thr Gin Phe Leu Leu ser 35 40 45

Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gly Leu Val Asp He He 50 55 60

Trp Gly He Phe Gly Pro Ser Gin Trp Asp Ala Phe Leu Val Gin He 65 70 75 80

Glu Gin Leu He Asn Gin Arg He Glu Glu Phe Ala Arg Asn Gin Ala 85 90 95

He Ser Arg Leu Glu Gly Leu Ser Asn Leu Tyr Gin He Tyr Ala Glu 100 105 110

Ser Phe Arg Glu Trp Glu Ala Asp Leu Asn Asn Ala Gin Leu Arg Glu 115 120 125

Asp Val Arg He Arg Phe Ala Asn Thr Asp Asp Ala Leu He Thr Ala

130 135 140

He Asn Asn Phe Thr Leu Thr Ser Phe Glu He Pro Leu Leu Ser Val

145 150 155 160

Tyr Val Gin Ala Ala Asn Leu His Leu Ser Leu Leu Arg Asp Ala Val 165 170 175

Ser Phe Gly Gin Gly Trp Gly Leu Asp He Ala Thr Val Asn Asn His 180 185 190

Tyr Asn Arg Leu He Asn Leu He His Arg Tyr Thr Lys His Cys Leu 195 200 205

Asp Thr Tyr Asn Gin Gly Leu Glu Asn Leu Arg Gly Thr Asn Thr Arg 210 215 220

Gin Trp Ala Arg Phe Asn Gin Phe Arg Arg Asp Leu Thr Leu Thr Val 225 230 235 240

Leu Asp He Val Ala Leu Phe Pro Asn Tyr Asp Val Arg Thr Tyr Pro 245 250 255

He Gin Thr Ser Ser Gin Leu Thr Arg Glu He Tyr Thr Ser Ser Val 260 265 270 He Glu Asp Ser Pro Val ser Ala Asn He Pro Asn Gly Phe Asn Arg 275 280 285

Ala Glu Phe Gly Val Arg Pro Pro His Leu Met Asp Phe Met Asn Ser 290 295 300

Leu Phe Val Thr Ala Glu Thr Val Arg Ser Gin Thr Val Trp Gly Gly 305 310 315 320

His Leu Val Ser Ser Arg Asn Thr Ala Gly Asn Arg He Asn Phe Pro 325 330 335 ser Tyr Gly Val Phe Asn Pro Gly Gly Ala He Trp He Ala Asp Glu 340 345 350

Asp Pro Arg Pro Phe Tyr Arg Thr Leu Ser Asp Pro Val Phe Val Arg 355 360 365

Gly Gly Phe Gly Asn Pro His Tyr Val Leu Gly Leu Arg Gly Val Ala 370 375 380

Phe Gin Gin Thr Gly Thr Asn His Thr Arg Thr Phe Arg Asn ser Gly 385 390 395 400

Thr He Asp Ser Leu Asp Glu He Pro Pro Gin Asp Asn Ser Gly Ala 405 410 415

Pro Trp Asn Asp Tyr Ser His Val Leu Asn His Val Thr Phe Val Arg 420 425 430

Trp Pro Gly Glu He Ser Gly Ser Asp Ser Trp Arg Ala Pro Met Phe 435 440 445

Ser Trp Thr His Arg Ser Ala Thr Pro Thr Asn Thr He Asp Pro Glu 450 455 460

Arg He Thr Gin He Pro Leu Val Lys Ala His Thr Leu Gin Ser Gly 465 470 475 480

Thr Thr Val Val Arg Gly Pro Gly Phe Thr Gly Gly Asp He Leu Arg 485 490 495

Arg Thr Ser Gly Gly Pro Phe Ala Tyr Thr He Val Asn He Asn Gly 500 505 510

Gin Leu Pro Gin Arg Tyr Arg Ala Arg He Arg Tyr Ala Ser Thr Thr 515 520 525

Asn Leu Arg He Tyr Val Thr Val Ala Gly Glu Arg He Phe Ala Gly 530 535 540

Gin Phe Asn Lys Thr Met Asp Thr Gly Asp Pro Leu Thr Phe Gin Ser 545 550 555 560

Phe ser Tyr Ala Thr He Asn Thr Ala Phe Thr Phe Pro Met Ser Gin 565 570 575 Ser Ser Phe Thr Val Gly Ala Asp Thr Phe Ser Ser Gly Asn Glu Val 580 585 590

Tyr He Asp Arg Phe Glu Leu He Pro Val Thr Ala Thr Phe Glu Ala 595 600 605

Glu Tyr Asp Leu Glu Arg Ala Gin Lys Ala Val Asn Ala Leu Phe Thr 610 615 620

Ser He Asn Gin He Gly He Lys Thr Asp Val Thr Asp Tyr His He 625 630 635 640

Asp Arg Val Ser Asn Leu Val Glu Cys Leu Ser Asp Glu Phe Cys Leu 645 650 655

Asp Glu Lys Lys Glu Leu Ser Glu Lys Val Lys His Ala Lys Arg Leu 660 665 670

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

Arg Gin Leu Asp Arg Gly Trp Arg Gly. Ser Thr Asp He Thr He Gin 690 695 700

Gly Gly Asp Asp Val Phe Lys Glu Asn Tyr Val Thr Leu Leu Gly Thr 705 710 715 720

Phe Asp Glu Cys Tyr Pro Thr Tyr Leu Tyr Gin Lys He Asp Glu Ser 725 730 735

Lys Leu Lys Ala Tyr Thr Arg Tyr Gin Leu Arg Gly Tyr He Glu Asp 740 745 750

Ser Gin Asp Leu Glu He Tyr Leu He Arg Tyr Asn Ala Lys His Glu 755 760 765

Thr Val Asn Val Pro Gly Thr Gly ser Leu Trp Pro Leu Ser Ala Pro 770 775 780

Ser Pro He Gly Lys Cys Ala His His Ser His His Phe Ser Leu Asp 785 790 795 800

He Asp val Gly Cys Thr Asp Leu Asn Glu Asp Leu Gly Val Trp Val 805 810 815

He Phe Lys He Lys Thr Gin Asp Gly His Ala Arg Leu Gly Asn Leu 820 825 830

Glu Phe Leu Glu Glu Lys Pro Leu Val Gly Glu Ala Leu Ala Arg Val 835 840 845

Lys Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Glu Lys Leu Glu Trp 850 855 860

Glu Thr Asn He Val Tyr Lys Glu Ala Lys Glu Ser Val Asp Ala Leu 865 870 875 880

Phe Val Asn Ser Gin Tyr Asp Arg Leu Gin Ala Asp Thr Asn He Ala 885 890 895

Met He His Ala Ala Asp Lys Arg Val His Ser He Arg Glu Ala Tyr 900 905 910

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

Glu Leu Glu Gly Arg He Phe Thr Ala Phe Ser Leu Tyr Asp Ala Arg 930 935 940

Asn Val He Lys Asn Gly Asp Phe Asn Asn Gly Leu Ser Cys Trp Asn 945 950 955 960

Val Lys Gly His Val Asp Val Glu Glu Gin Asn Asn His Arg ser Val 965 970 975 Leu Val Val Pro Glu Trp Glu Ala Glu val Ser Gin Glu Val Arg Val 980 985 990

Cys Pro Gly Arg Gly Tyr He Leu Arg Val Thr Ala Tyr Lys Glu Gly 995 1000 1005

Tyr Gly Glu Gly Cys Val Thr He His Glu He Glu Asn Asn Thr Asp 1010 1015 1020

Glu Leu Lys Phe Ser Asn Cys Val Glu Glu Glu Val Tyr Pro Asn Asn 1025 1030 1035 1040

Thr Val Thr cys Asn Asp Tyr Thr Ala Thr Gin Glu Glu Tyr Glu Gly 1045 1050 1055

Thr Tyr Thr Ser Arg Asn Arg Gly Tyr Asp Gly Ala Tyr Glu Ser Asn 1060 1065 1070

Ser Ser Val Pro Ala Asp Tyr Ala ser Ala Tyr Glu Glu Lys Ala Tyr 1075 1080 1085

Thr Asp Gly Arg Arg Asp Asn Pro Cys Glu ser Asn Arg Gly Tyr Gly 1090 1095 1100

Asp Tyr Thr Pro Leu Pro Ala Gly Tyr Val Thr Lys Glu Leu Glu Tyr 1105 1110 1115 1120

Phe Pro Glu Thr Asp Lys Val Trp He Glu He Gly Glu Thr Glu Gly 1125 1130 1135

Thr Phe He Val Asp Ser Val Glu Leu Leu Leu Met Glu Glu 1140 1145 1150

(2) INFORMATION FOR SEQ ID NO:22:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 3444 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:

ATGGAGAATA ATATTCAAAA TCAATGCGTA CCTTACAATT GTTTAAATAA TCCTGAAGTA 60

GAAATATTAA ATGAAGAAAG AAGTACTGGC AGATTACCGT TAGATATATC CTTATCGCTT 120

ACACGTTTCC TTTTGAGTGA ATTTGTTCCA GGTGTGGGAG TTGCGTTTGG ATTATTTGAT 180

TTAATATGGG GTTTTATAAC TCCTTCTGAT TGGAGCTTAT TTCTTTTACA GATTGAACAA 240

TTGATTGAGC AAAGAATAGA AACATTGGAA AGGAACCGGG CAATTACTAC ATTACGAGGG 300

TTAGCAGATA GCTATGAAAT TTATATTGAA GCACTAAGAG AGTGGGAAGC AAATCCTAAT 360 AATGCACAAT TAAGGGAAGA TGTGCGTATT CGATTTGCTA ATACAGACGA CGCTTTAATA 420

ACAGCAATAA ATAATTTTAC ACTTACAAGT TTTGAAATCC CTCTTTTATC GGTCTATGTT 480

CAAGCGGCGA ATTTACATTT ATCACTATTA AGAGACGCTG TATCGTTTGG GCAGGGTTGG 540

GGACTGGATA TAGCTACTGT TAATAATCAT TATAATAGAT TAATAAATCT TATTCATAGA 600

TATACGAAAC ATTGTTTGGA CACATACAAT CAAGGATTAG AAAACTTAAG AGGTACTAAT 660

ACTCGACAAT GGGCAAGATT CAATCAGTTT AGGAGAGATT TAACACTTAC TGTATTAGAT 720

ATCGTTGCTC TTTTTCCGAA CTACGATGTT AGAACATATC CAATTCAAAC GTCATCCCAA 780

TTAACAAGGG AAATTTATAC AAGTTCAGTA ATTGAGGATT CTCCAGTTTC TGCTAATATA 840

CCTAATGGTT TTAATAGGGC GGAATTTGGA GTTAGACCGC CCCATCTTAT GGACTTTATG 900

AATTCTTTGT TTGTAACTGC AGAGACTGTT AGAAGTCAAA CTGTGTGGGG AGGACACTTA 960

GTTAGTTCAC GAAATACGGC TGGTAACCGT ATAAATTTCC CTAGTTACGG GGTCTTCAAT 1020

CCTGGTGGCG CCATTTGGAT TGCAGATGAG GATCCACGTC CTTTTTATCG GACATTATCA 1080

GATCCTGTTT TTGTCCGAGG AGGATTTGGG AATCCTCATT ATGTACTGGG GCTTAGGGGA 1140

GTAGCATTTC AACAAACTGG TACGAACCAC ACCCGAACAT TTAGAAATAG TGGGACCATA 1200

GATTCTCTAG ATGAAATCCC ACCTCAGGAT AATAGTGGGG CACCTTGGAA TGATTATAGT 1260

CATGTATTAA ATCATGTTAC ATTTGTACGA TGGCCAGGTG AGATTTCAGG AAGTGATTCA 1320

TGGAGAGCTC CAATGTTTTC TTGGACGCAC CGTAGTGCAA CCCCTACAAA TACAATTGAT 1380

CCGGAGAGGA TTACTCAAAT ACCATTGGTA AAAGCACATA CACTTCAGTC AGGTACTACT 1440

GTTGTAAGAG GGCCCGGGTT TACGGGAGGA GATATTCTTC GACGAACAAG TGGAGGACCA 1500

TTTGCTTATA CTATTGTTAA TATAAATGGG CAATTACCCC AAAGGTATCG TGCAAGAATA 1560

CGCTATGCCT CTACTACAAA TCTAAGAATT TACGTAACGG TTGCAGGTGA ACGGATTTTT 1620

GCTGGTCAAT TTAACAAAAC AATGGATACC GGTGACCCAT TAACATTCCA ATCTTTTAGT 1680

TACGCAACTA TTAATACAGC TTTTACATTC CCAATGAGCC AGAGTAGTTT CACAGTAGGT 1740

GCTGATACTT TTAGTTCAGG GAATGAAGTT TATATAGACA GATTTGAATT GATTCCAGTT 1800

ACTGCAACAT TTGAAGCAGA ATATGATTTA GAAAGAGCAC AAAAGGCGGT GAATGCGCTG 1860

TTTACTTCTA TAAACCAAAT AGGGATAAAA ACAGATGTGA CGGATTATCA TATCGATCGA 1920

GTATCCAATT TAGTTGAGTG TTTATCTGAT GAATTTTGTC TGGATGAAAA AAAAGAATTG 1980

TCCGAGAAAG TCAAACATGC GAAGCGACTT AGTGATGAGC GGAATTTACT TCAAGATCCA 2040

AACTTTAGAG GGATCAATAG ACAACTAGAC CGTGGCTGGA GAGGAAGTAC GGATATTACC 2100 ATCCAAGGAG GCGATGACGT ATTCAAAGAG AATTACGTTA CGCTATTGGG TACCTTTGAT 2160

GAGTGCTATC CAACGTATTT ATATCAAAAA ATAGATGAGT CGAAATTAAA AGCCTATACC 2220

CGTTACCAAT TAAGAGGGTA TATCGAAGAT AGTCAAGACT TAGAAATCTA TTTAATTCGC 2280

TACAATGCCA AACACGAAAC AGTAAATGTG CCAGGTACGG GTTCCTTATG GCCGCTTTCA 2340

GCCCCAAGTC CAATCGGAAA ATGTGCCCAT CATTCCCATC ATTTCTCCTT GGACATTGAT 2400

GTTGGATGTA CAGACTTAAA TGAGGACTTA GGTGTATGGG TGATATTCAA GATTAAGACG 2460

CAAGATGGCC ATGCAAGACT AGGAAATCTA GAATTTCTCG AAGAGAAACC ATTAGTAGGA 2520

GAAGCACTAG CTCGTGTGAA AAGAGCGGAG AAAAAATGGA GAGACAAACG TGAAAAATTG 2580

GAATGGGAAA CAAATATTGT TTATAAAGAG GCAAAAGAAT CTGTAGATGC TTTATTTGTA 2640

AACTCTCAAT ATGATAGATT ACAAGCGGAT ACCAACATCG CGATGATTCA TGCGGCAGAT 2700

AAACGCGTTC ATAGCATTCG AGAAGCTTAT CTGCCTGAGC TGTCTGTGAT TCCGGGTGTC 2760

AATGCGGCTA TTTTTGAAGA ATTAGAAGGG CGTATTTTCA CTGCATTCTC CCTATATGAT 2820

GCGAGAAATG TCATTAAAAA TGGTGATTTT AATAATGGCT TATCCTGCTG GAACGTGAAA 2880

GGGCATGTAG ATGTAGAAGA ACAAAACAAC CACCGTTCGG TCCTTGTTGT TCCGGAATGG 2940

GAAGCAGAAG TGTCACAAGA AGTTCGTGTC TGTCCGGGTC GTGGCTATAT CCTTCGTGTC 3000

ACAGCGTACA AGGAGGGATA TGGAGAAGGT TGCGTAACCA TTCATGAGAT CGAGAACAAT 3060

ACAGACGAAC TGAAGTTTAG CAACTGTGTA GAAGAGGAAG TATATCCAAA CAACACGGTA 3120

ACGTGTAATG ATTATACTGC GACTCAAGAA GAATATGAGG GTACGTACAC TTCTCGTAAT 3180

CGAGGATATG ACGGAGCCTA TGAAAGCAAT TCTTCTGTAC CAGCTGATTA TGCATCAGCC 3240

TATGAAGAAA AAGCATATAC AGATGGACGA AGAGACAATC CTTGTGAATC TAACAGAGGA 3300

TATGGGGATT ACACACCACT ACCAGCTGGC TATGTGACAA AAGAATTAGA GTACTTCCCA 3360

GAAACCGATA AGGTATGGAT TGAGATCGGA GAAACGGAAG GAACATTCAT CGTGGACAGC 3420

GTGGAATTAC TTCTTATGGA GGAA 3444

[2) INFORMATION FOR SEQ ID NO:23:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1148 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:

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

Asn Pro Glu Val Glu He Leu Asn Glu Glu Arg Ser Thr Gly Arg Leu 20 25 30

Pro Leu Asp lie Ser Leu Ser Leu Thr Arg Phe Leu Leu Ser Glu Phe 35 40 45

Val Pro Gly Val Gly Val Ala Phe Gly Leu Phe Asp Leu He Trp Gly 50 55 60

Phe He Thr Pro Ser Asp Trp Ser Leu Phe Leu Leu Gin He Glu Gin 65 70 75 80

Leu He Glu Gin Arg He Glu Thr Leu Glu Arg Asn Arg Ala He Thr 85 90 95

Thr Leu Arg Gly Leu Ala Asp Ser Tyr Glu He Tyr He Glu Ala Leu

100 105 110

Arg Glu Trp Glu Ala Asn Pro Asn Asn Ala Gin Leu Arg Glu Asp Val 115 120 125

Arg He Arg Phe Ala Asn Thr Asp Asp Ala Leu He Thr Ala He Asn

130 135 140

Asn Phe Thr Leu Thr Ser Phe Glu He Pro Leu Leu Ser Val Tyr Val 145 150 155 160

Gin Ala Ala Asn Leu His Leu Ser Leu Leu Arg Asp Ala Val Ser Phe 165 170 175

Gly Gin Gly Trp Gly Leu Asp He Ala Thr Val Asn Asn His Tyr Asn 180 185 190

Arg Leu He Asn Leu He His Arg Tyr Thr Lys His Cys Leu Asp Thr 195 200 205

Tyr Asn Gin Gly Leu Glu Asn Leu Arg Gly Thr Asn Thr Arg Gin Trp 210 215 220

Ala Arg Phe Asn Gin Phe Arg Arg Asp Leu Thr Leu Thr Val Leu Asp 225 230 235 240

He Val Ala Leu Phe Pro Asn Tyr Asp Val Arg Thr Tyr Pro He Gin 245 250 255

Thr Ser Ser Gin Leu Thr Arg Glu He Tyr Thr Ser ser Val He Glu 260 265 270

Asp ser Pro Val Ser Ala Asn He Pro Asn Gly Phe Asn Arg Ala Glu 275 280 285 Phe Gly Val Arg Pro Pro His Leu Met Asp Phe Met Asn Ser Leu Phe 290 295 300

Val Thr Ala Glu Thr Val Arg Ser Gin Thr Val Trp Gly Gly His Leu 305 310 315 320

Val Ser Ser Arg Asn Thr Ala Gly Asn Arg He Asn Phe Pro Ser Tyr 325 330 335

Gly Val Phe Asn Pro Gly Gly Ala He Trp He Ala Asp Glu Asp Pro 340 345 350

Arg Pro Phe Tyr Arg Thr Leu Ser Asp Pro Val Phe Val Arg Gly Gly

355 360 365

Phe Gly Asn Pro His Tyr Val Leu Gly Leu Arg Gly Val Ala Phe Gin 370 375 380

Gin Thr Gly Thr Asn His Thr Arg Thr Phe Arg Asn Ser Gly Thr He 385 390 395 400

Asp Ser Leu Asp Glu He Pro Pro Gin Asp Asn Ser Gly Ala Pro Trp 405 410 415

Asn Asp Tyr Ser His Val Leu Asn His val Thr Phe Val Arg Trp Pro 420 425 430

Gly Glu lie Ser Gly Ser Asp Ser Trp Arg Ala Pro Met Phe Ser Trp 435 440 445

Thr His Arg Ser Ala Thr Pro Thr Asn Thr He Asp Pro Glu Arg He 450 455 460

Thr Gin He Pro Leu Val Lys Ala His Thr Leu Gin Ser Gly Thr Thr 465 470 475 480

Val Val Arg Gly Pro Gly Phe Thr Gly Gly Asp He Leu Arg Arg Thr 485 490 495

Ser Gly Gly Pro Phe Ala Tyr Thr He Val Asn He Asn Gly Gin Leu 500 505 510

Pro Gin Arg Tyr Arg Ala Arg He Arg Tyr Ala ser Thr Thr Asn Leu 515 520 525

Arg He Tyr Val Thr Val Ala Gly Glu Arg He Phe Ala Gly Gin Phe 530 535 540

Asn Lys Thr Met Asp Thr Gly Asp Pro Leu Thr Phe Gin Ser Phe Ser

545 550 555 560

Tyr Ala Thr He Asn Thr Ala Phe Thr Phe Pro Met Ser Gin Ser ser 565 570 575

Phe Thr Val Gly Ala Asp Thr Phe ser ser Gly Asn Glu Val Tyr He 580 585 590 Asp Arg Phe Glu Leu He Pro Val Thr Ala Thr Phe Glu Ala Glu Tyr 595 600 605

Asp Leu Glu Arg Ala Gin Lys Ala Val Asn Ala Leu Phe Thr Ser He

610 615 620

Asn Gin He Gly He Lys Thr Asp Val Thr Asp Tyr His He Asp Arg 625 630 635 640

Val Ser Asn Leu Val Glu Cys Leu Ser Asp Glu Phe Cys Leu Asp Glu 645 650 655

Lys Lys Glu Leu Ser Glu Lys Val Lys His Ala Lys Arg Leu Ser Asp 660 665 670

Glu Arg Asn Leu Leu Gin Asp Pro Asn Phe Arg Gly He Asn Arg Gin 675 680 685

Leu Asp Arg Gly Trp Arg Gly Ser Thr Asp He Thr He Gin Gly Gly 690 695 700

Asp Asp Val Phe Lys Glu Asn Tyr Val Thr Leu Leu Gly Thr Phe Asp 705 710 715 720

Glu Cys Tyr Pro Thr Tyr Leu Tyr Gin Lys He Asp Glu Ser Lys Leu 725 730 735

Lys Ala Tyr Thr Arg Tyr Gin Leu Arg Gly Tyr He Glu Asp Ser Gin 740 745 750

Asp Leu Glu He Tyr Leu lie Arg Tyr Asn Ala Lys His Glu Thr Val 755 760 765

Asn val Pro Gly Thr Gly ser Leu Trp Pro Leu Ser Ala Pro ser Pro 770 775 780

He Gly Lys Cys Ala His His Ser His His Phe Ser Leu Asp He Asp 785 790 795 800

Val Gly Cys Thr Asp Leu Asn Glu Asp Leu Gly Val Trp val He Phe 805 810 815

Lys He Lys Thr Gin Asp Gly His Ala Arg Leu Gly Asn Leu Glu Phe 820 825 830

Leu Glu Glu Lys Pro Leu Val Gly Glu Ala Leu Ala Arg val Lys Arg 835 840 845

Ala Glu Lys Lys Trp Arg Asp Lys Arg Glu Lys Leu Glu Trp Glu Thr 850 855 860

Asn He Val Tyr Lys Glu Ala Lys Glu Ser Val Asp Ala Leu Phe Val 865 870 875 880

Asn Ser Gin Tyr Asp Arg Leu Gin Ala Asp Thr Asn He Ala Met He 885 890 895 His Ala Ala Asp Lys Arg Val His ser He Arg Glu Ala Tyr Leu Pro 900 905 910

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

Glu Gly Arg He Phe Thr Ala Phe Ser Leu Tyr Asp Ala Arg Asn Val 930 935 940

He Lys Asn Gly Asp Phe Asn Asn Gly Leu ser Cys Trp Asn Val Lys 945 950 955 960

Gly His Val Asp val Glu Glu Gin Asn Asn His Arg Ser Val Leu Val 965 970 975

Val Pro Glu Trp Glu Ala Glu Val ser Gin Glu Val Arg Val Cys Pro 980 985 990

Gly Arg Gly Tyr He Leu Arg Val Thr Ala Tyr Lys Glu Gly Tyr Gly 995 1000 1005

Glu Gly Cys Val Thr He His Glu He Glu Asn Asn Thr Asp Glu Leu 1010 1015 1020

Lys Phe Ser Asn Cys Val Glu Glu Glu Val Tyr Pro Asn Asn Thr Val 1025 1030 1035 1040

Thr Cys Asn Asp Tyr Thr Ala Thr Gin Glu Glu Tyr Glu Gly Thr Tyr 1045 1050 1055

Thr Ser Arg Asn Arg Gly Tyr Asp Gly Ala Tyr Glu Ser Asn Ser ser 1060 1065 1070

Val Pro Ala Asp Tyr Ala ser Ala Tyr Glu Glu Lys Ala Tyr Thr Asp 1075 1080 1085

Gly Arg Arg Asp Asn Pro Cys Glu Ser Asn Arg Gly Tyr Gly Asp Tyr 1090 1095 1100

Thr Pro Leu Pro Ala Gly Tyr Val Thr Lys Glu Leu Glu Tyr Phe Pro 1105 1110 1115 1120

Glu Thr Asp Lys Val Trp He Glu He Gly Glu Thr Glu Gly Thr Phe 1125 1130 1135

He Val Asp Ser Val Glu Leu Leu Leu Met Glu Glu 1140 1145

(2) INFORMATION FOR SEQ ID NO:24:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 3522 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:

ATGGAAAATA ATATTCAAAA TCAATGCGTA CCTTACAATT GTTTAAATAA TCCTGAAGTA 60

GAAATACTGA ACGAAGAACG CAGCACCGGC CGCCTGCCGC TGGACATCAG CCTGAGCCTT 120

ACACGTTTCC TTTTGAGTGA ATTTGTTCCA GGTGTGGGAG TTGCGTTTGG ATTATTTGAT 180

TTAATATGGG GTTTTATAAC TCCTTCTGAT TGGAGCTTAT TTCTTTTACA GATTGAACAA 240

TTGATTGAGC AAAGAATAGA AACATTGGAA AGGAACCGGG CAATTACTAC ATTACGAGGG 300

TTAGCAGATA GCTATGAAAT TTATATTGAA GCACTAAGAG AGTGGGAAGC AAATCCTAAT 360

AATGCACAAT TAAGGGAAGA TGTGCGTATT CGATTTGCTA ATACAGACGA CGCTTTAATA 420

ACAGCAATAA ATAATTTTAC ACTTACAAGT TTTGAAATCC CTCTTTTATC GGTCTATGTT 480

CAAGCGGCGA ATTTACATTT ATCACTATTA AGAGACGCTG TATCGTTTGG GCAGGGTTGG 540

GGACTGGATA TAGCTACTGT TAATAATCAT TATAATAGAT TAATAAATCT TATTCATAGA 600

TATACGAAAC ATTGTTTGGA CACATACAAT CAAGGATTAG AAAACTTAAG AGGTACTAAT 660

ACTCGACAAT GGGCAAGATT CAATCAGTTT AGGAGAGATT TAACACTTAC TGTATTAGAT 720

ATCGTTGCTC TTTTTCCGAA CTACGATGTT AGAACATATC CAATTCAAAC GTCATCCCAA 780

TTAACAAGGG AAATTTATAC AAGTTCAGTA ATTGAGGATT CTCCAGTTTC TGCTAATATA 840

CCTAATGGTT TTAATAGGGC GGAATTTGGA GTTAGACCGC CCCATCTTAT GGACTTTATG 900

AATTCTTTGT TTGTAACTGC AGAGACTGTT AGAAGTCAAA CTGTGTGGGG AGGACACTTA 960

GTTAGTTCAC GAAATACGGC TGGTAACCGT ATAAATTTCC CTAGTTACGG GGTCTTCAAT 1020

CCTGGTGGCG CCATTTGGAT TGCAGATGAG GATCCACGTC CTTTTTATCG GACATTATCA 1080

GATCCTGTTT TTGTCCGAGG AGGATTTGGG AATCCTCATT ATGTACTGGG GCTTAGGGGA 1140

GTAGCATTTC AACAAACTGG TACGAACCAC ACCCGAACAT TTAGAAATAG TGGGACCATA 1200

GATTCTCTAG ATGAAATCCC ACCTCAGGAT AATAGTGGGG CACCTTGGAA TGATTATAGT 1260

CATGTATTAA ATCATGTTAC ATTTGTACGA TGGCCAGGTG AGATTTCAGG AAGTGATTCA 1320

TGGAGAGCTC CAATGTTTTC TTGGACGCAC CGTAGTGCAA CCCCTACAAA TACAATTGAT 1380

CCGGAGAGGA TTACTCAAAT ACCATTGGTA AAAGCACATA CACTTCAGTC AGGTACTACT 1440

GTTGTAAGAG GGCCCGGGTT TACGGGAGGA GATATTCTTC GACGAACAAG TGGAGGACCA 1500

TTTGCTTATA CTATTGTTAA TATAAATGGG CAATTACCCC AAAGGTATCG TGCAAGAATA 1560

CGCTATGCCT CTACTACAAA TCTAAGAATT TACGTAACGG TTGCAGGTGA ACGGATTTTT 1620 GCTGGTCAAT TTAACAAAAC AATGGATACC GGTGACCCAT TAACATTCCA ATCTTTTAGT 1680

TACGCAACTA TTAATACAGC TTTTACATTC CCAATGAGCC AGAGTAGTTT CACAGTAGGT 1740

GCTGATACTT TTAGTTCAGG GAATGAAGTT TATATAGACA GATTTGAATT GATTCCAGTT 1800

ACTGCAACAT TTGAAGCAGA ATATGATTTA GAAAGAGCAC AAAAGGCGGT GAATGCGCTG 1860

TTTACTTCTA TAAACCAAAT AGGGATAAAA ACAGATGTGA CGGATTATCA TATTGATCAA 1920

GTATCCAATT TAGTGGATTG TTTATCAGAT GAATTTTGTC TGGATGAAAA GCGAGAATTG 1980

TCCGAGAAAG TCAAACATGC GAAGCGACTC AGTGATGAGC GGAATTTACT TCAAGATCCA 040

AACTTCAAAG GCATCAATAG GCAACTAGAC CGTGGTTGGA GAGGAAGTAC GGATATTACC 2100

ATCCAAAGAG GAGATGACGT ATTCAAAGAA AATTATGTCA CACTACCAGG TACCTTTGAT 2160

GAGTGCTATC CAACGTATTT ATATCAAAAA ATAGATGAGT CGAAATTAAA ACCCTATACT 2220

CGTTATCAAT TAAGAGGGTA TATCGAGGAT AGTCAAGACT TAGAAATCTA TTTGATCCGC 2280

TATAATGCAA AACACGAAAC AGTAAATGTG CTAGGTACGG GTTCTTTATG GCCGCTTTCA 2340

GTCCAAAGTC CAATCAGAAA GTGTGGAGAA CCGAATCGAT GCGCGCCACA CCTTGAATGG 2400

AATCCTGATC TAGATTGTTC CTGCAGAGAC GGGGAAAAAT GTGCACATCA TTCGCATCAT 2460

TTCTCCTTGG ACATTGATGT TGGATGTACA GACTTAAATG AGGACTTAGA TGTATGGGTG 2520

ATATTCAAGA TTAAGACGCA AGATGGCCAT GCAAGACTAG GAAATCTAGA GTTTCTCGAA 2580

GAGAAACCAT TAGTCGGGGA AGCACTAGCT CGTGTGAAAA GAGCAGAGAA AAAATGGAGA 2640

GATAAACGTG AAAAATTGGA ATTGGAAACA AATATTGTTT ATAAAGAGGC AAAAGAATCT 2700

GTAGATGCTT TATTTGTAAA CTCTCAATAT GATCAATTAC AAGCGGATAC GAATATTGCC 2760

ATGATTCATG CGGCAGATAA ACGTGTTCAT AGAATTCGGG AAGCGTATCT TCCAGAGTTA 2820

TCTGTGATTC CGGGTGTAAA TGTAGACATT TTCGAAGAAT TAAAAGGGCG TATTTTCACT 2880

GCATTCTTCC TATATGATGC GAGAAATGTC ATTAAAAACG GTGATTTCAA TAATGGCTTA 2940

TCATGCTGGA ACGTGAAAGG GCATGTAGAT GTAGAAGAAC AAAACAACCA CCGTTCGGTC 3000

CTTGTTGTTC CGGAATGGGA AGCAGAAGTG TCACAAGAAG TTCGTGTCTG TCCGGGTCGT 3060

GGCTATATCC TTCGTGTCAC AGCGTACAAG GAGGGATATG GAGAAGGTTG CGTAACCATT 3120

CATGAGATCG AGAACAATAC AGACGAACTG AAGTTTAGCA ACTGCGTAGA AGAGGAAGTC 3180

TATCCAAACA ACACGGTAAC GTGTAATGAT TATACTGCAA ATCAAGAAGA ATACGGGGGT 32 0

GCGTACACTT CCCGTAATCG TGGATATGAC GAAACTTATG GAAGCAATTC TTCTGTACCA 3300

GCTGATTATG CGTCAGTCTA TGAAGAAAAA TCGTATACAG ATGGACGAAG AGACAATCCT 3360 TGTGAATCTA ACAGAGGATA TGGGGATTAC ACACCACTAC CAGCTGGCTA TGTGACAAAA 3420

GAATTAGAGT ACTTCCCAGA AACCGATAAG GTATGGATTG AGATCGGAGA AACGGAAGGA 3480

ACATTCATCG TGGACAGCGT GGAATTACTC CTTATGGAGG AA 3522

(2) INFORMATION FOR SEQ ID NO:25:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1174 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:

Met Glu Asn Asn He Gin Asn Gin cys Val Pro Tyr Asn cys Leu Asn 1 5 10 15

Asn Pro Glu Val Glu lie Leu Asn Glu Glu Arg Ser Thr Gly Arg Leu 20 25 30

Pro Leu Asp lie Ser Leu Ser Leu Thr Arg Phe Leu Leu Ser Glu Phe 35 40 45

Val Pro Gly Val Gly Val Ala Phe Gly Leu Phe Asp Leu He Trp Gly 50 55 60

Phe He Thr Pro Ser Asp Trp Ser Leu Phe Leu Leu Gin He Glu Gin 65 70 75 80

Leu He Glu Gin Arg He Glu Thr Leu Glu Arg Asn Arg Ala He Thr 85 90 95

Thr Leu Arg Gly Leu Ala Asp Ser Tyr Glu He Tyr He Glu Ala Leu 100 105 110

Arg Glu Trp Glu Ala Asn Pro Asn Asn Ala Gin Leu Arg Glu Asp Val 115 120 125

Arg He Arg Phe Ala Asn Thr Asp Asp Ala Leu He Thr Ala He Asn 130 135 140

Asn Phe Thr Leu Thr ser Phe Glu He Pro Leu Leu Ser Val Tyr Val

145 150 155 160

Gin Ala Ala Asn Leu His Leu Ser Leu Leu Arg Asp Ala Val Ser Phe 165 170 175

Gly Gin Gly Trp Gly Leu Asp He Ala Thr Val Asn Asn His Tyr Asn 180 185 190

Arg Leu He Asn Leu He His Arg Tyr Thr Lys His Cys Leu Asp Thr 195 200 205 Tyr Asn Gin Gly Leu Glu Asn Leu Arg Gly Thr Asn Thr Arg Gin Trp 210 215 220

Ala Arg Phe Asn Gin Phe Arg Arg Asp Leu Thr Leu Thr Val Leu Asp 225 230 235 240

He Val Ala Leu Phe Pro Asn Tyr Asp Val Arg Thr Tyr Pro He Gin 245 250 255

Thr Ser Ser Gin Leu Thr Arg Glu He Tyr Thr Ser Ser Val He Glu 260 265 270

Asp Ser Pro Val Ser Ala Asn He Pro Asn Gly Phe Asn Arg Ala Glu 275 280 285

Phe Gly Val Arg Pro Pro His Leu Met Asp Phe Met Asn Ser Leu Phe 290 295 300

Val Thr Ala Glu Thr Val Arg Ser Gin Thr Val Trp Gly Gly His Leu 305 310 315 320

Val Ser Ser Arg Asn Thr Ala Gly Asn Arg He Asn Phe Pro Ser Tyr 325 330 335

Gly Val Phe Asn Pro Gly Gly Ala He Trp He Ala Asp Glu Asp Pro 340 345 350

Arg Pro Phe Tyr Arg Thr Leu ser Asp Pro Val Phe Val Arg Gly Gly 355 360 365

Phe Gly Asn Pro His Tyr Val Leu Gly Leu Arg Gly Val Ala Phe Gin 370 375 380

Gin Thr Gly Thr Asn His Thr Arg Thr Phe Arg Asn Ser Gly Thr lie 385 390 395 400

Asp Ser Leu Asp Glu He Pro Pro Gin Asp Asn Ser Gly Ala Pro Trp

405 410 415

Asn Asp Tyr Ser His Val Leu Asn His Val Thr Phe Val Arg Trp Pro 420 425 430

Gly Glu He Ser Gly Ser Asp Ser Trp Arg Ala Pro Met Phe Ser Trp

435 440 445

Thr His Arg Ser Ala Thr Pro Thr Asn Thr He Asp Pro Glu Arg He 450 455 460

Thr Gin He Pro Leu Val Lys Ala His Thr Leu Gin Ser Gly Thr Thr 465 470 475 480

Val Val Arg Gly Pro Gly Phe Thr Gly Gly Asp He Leu Arg Arg Thr 485 490 495

Ser Gly Gly Pro Phe Ala Tyr Thr He Val Asn lie Asn Gly Gin Leu

500 505 510 Pro Gin Arg Tyr Arg Ala Arg He Arg Tyr Ala Ser Thr Thr Asn Leu 515 520 525

Arg He Tyr Val Thr Val Ala Gly Glu Arg He Phe Ala Gly Gin Phe 530 535 540

Asn Lys Thr Met Asp Thr Gly Asp Pro Leu Thr Phe Gin Ser Phe Ser 545 550 555 560

Tyr Ala Thr He Asn Thr Ala Phe Thr Phe Pro Met Ser Gin Ser Ser 565 570 575

Phe Thr Val Gly Ala Asp Thr Phe Ser Ser Gly Asn Glu Val Tyr He 580 585 590

Asp Arg Phe Glu Leu He Pro Val Thr Ala Thr Phe Glu Ala Glu Tyr 595 600 605

Asp Leu Glu Arg Ala Gin Lys Ala Val Asn Ala Leu Phe Thr Ser He 610 615 620

Asn Gin He Gly lie Lys Thr Asp Val Thr Asp Tyr His lie Asp Gin 625 630 635 640

Val Ser Asn Leu Val Asp cys Leu Ser Asp Glu Phe Cys Leu Asp Glu 645 650 655

Lys Arg Glu Leu Ser Glu Lys Val Lys His Ala Lys Arg Leu Ser Asp 660 665 670

Glu Arg Asn Leu Leu Gin Asp Pro Asn Phe Lys Gly He Asn Arg Gin 675 680 685

Leu Asp Arg Gly Trp Arg Gly ser Thr Asp He Thr He Gin Arg Gly 690 695 700

Asp Asp Val Phe Lys Glu Asn Tyr Val Thr Leu Pro Gly Thr Phe Asp 705 710 715 720

Glu Cys Tyr Pro Thr Tyr Leu Tyr Gin Lys He Asp Glu Ser Lys Leu 725 730 735

Lys Pro Tyr Thr Arg Tyr Gin Leu Arg Gly Tyr He Glu Asp Ser Gin 740 745 750

Asp Leu Glu He Tyr Leu He Arg Tyr Asn Ala Lys His Glu Thr Val 755 760 765

Asn Val Leu Gly Thr Gly ser Leu Trp Pro Leu Ser Val Gin Ser Pro

770 775 780

He Arg Lys Cys Gly Glu Pro Asn Arg Cys Ala Pro His Leu Glu Trp 785 790 795 800

Asn Pro Asp Leu Asp Cys Ser Cys Arg Asp Gly Glu Lys Cys Ala His

805 810 815 His ser His His Phe Ser Leu Asp He Asp Val Gly Cys Thr Asp Leu 820 825 830

Asn Glu Asp Leu Asp Val Trp Val He Phe Lys He Lys Thr Gin Asp 835 840 845

Gly His Ala Arg Leu Gly Asn Leu Glu Phe Leu Glu Glu Lys Pro Leu 850 855 860

Val Gly Glu Ala Leu Ala Arg Val Lys Arg Ala Glu Lys Lys Trp Arg 865 870 875 880

Asp Lys Arg Glu Lys Leu Glu Leu Glu Thr Asn He Val Tyr Lys Glu 885 890 895

Ala Lys Glu Ser Val Asp Ala Leu Phe Val Asn Ser Gin Tyr Asp Gin 900 905 910

Leu Gin Ala Asp Thr Asn He Ala Met He His Ala Ala Asp Lys Arg 915 920 925

Val His Arg He Arg Glu Ala Tyr Leu Pro Glu Leu ser Val He Pro 930 935 940

Gly Val Asn Val Asp He Phe Glu Glu Leu Lys Gly Arg He Phe Thr 945 950 955 960

Ala Phe Phe Leu Tyr Asp Ala Arg Asn Val He Lys Asn Gly Asp Phe 965 970 975

Asn Asn Gly Leu Ser Cys Trp Asn Val Lys Gly His Val Asp Val Glu 980 985 990

Glu Gin Asn Asn His Arg Ser Val Leu Val Val Pro Glu Trp Glu Ala 995 1000 1005

Glu Val Ser Gin Glu Val Arg Val Cys Pro Gly Arg Gly Tyr He Leu 1010 1015 1020

Arg Val Thr Ala Tyr Lys Glu Gly Tyr Gly Glu Gly Cys Val Thr He 1025 1030 1035 1040

His Glu He Glu Asn Asn Thr Asp Glu Leu Lys Phe Ser Asn Cys Val 1045 1050 1055

Glu Glu Glu Val Tyr Pro Asn Asn Thr Val Thr cys Asn Asp Tyr Thr 1060 1065 1070

Ala Asn Gin Glu Glu Tyr Gly Gly Ala Tyr Thr Ser Arg Asn Arg Gly 1075 1080 1085

Tyr Asp Glu Thr Tyr Gly Ser Asn Ser ser Val Pro Ala Asp Tyr Ala 1090 1095 1100

Ser Val Tyr Glu Glu Lys Ser Tyr Thr Asp Gly Arg Arg Asp Asn Pro

1105 1110 1115 1120 Cys Glu ser Asn Arg Gly Tyr Gly Asp Tyr Thr Pro Leu Pro Ala Gly 1125 1130 1135

Tyr Val Thr Lys Glu Leu Glu Tyr Phe Pro Glu Thr Asp Lys Val Trp 1140 1145 1150

He Glu He Gly Glu Thr Glu Gly Thr Phe He Val Asp Ser Val Glu 1155 1160 1165

Leu Leu Leu Met Glu Glu 1170

(2) INFORMATION FOR SEQ ID NO:26:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 3444 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:

ATGGAAAATA ATATTCAAAA TCAATGCGTA CCTTACAATT GTTTAAATAA TCCTGAAGTA 60

GAAATACTGA ACGAAGAACG CAGCACCGGC CGCCTGCCGC TGGACATCAG CCTGAGCCTT 120

ACACGTTTCC TTTTGAGTGA ATTTGTTCCA GGTGTGGGAG TTGCGTTTGG ATTATTTGAT 180

TTAATATGGG GTTTTATAAC TCCTTCTGAT TGGAGCTTAT TTCTTTTACA GATTGAACAA 240

TTGATTGAGC AAAGAATAGA AACATTGGAA AGGAACCGGG CAATTACTAC ATTACGAGGG 300

TTAGCAGATA GCTATGAAAT TTATATTGAA GCACTAAGAG AGTGGGAAGC AAATCCTAAT 360

AATGCACAAT TAAGGGAAGA TGTGCGTATT CGATTTGCTA ATACAGACGA CGCTTTAATA 420

ACAGCAATAA ATAATTTTAC ACTTACAAGT TTTGAAATCC CTCTTTTATC GGTCTATGTT 480

CAAGCGGCGA ATTTACATTT ATCACTATTA AGAGACGCTG TATCGTTTGG GCAGGGTTGG 540

GGACTGGATA TAGCTACTGT TAATAATCAT TATAATAGAT TAATAAATCT TATTCATAGA 600

TATACGAAAC ATTGTTTGGA CACATACAAT CAAGGATTAG AAAACTTAAG AGGTACTAAT 660

ACTCGACAAT GGGCAAGATT CAATCAGTTT AGGAGAGATT TAACACTTAC TGTATTAGAT 720

ATCGTTGCTC TTTTTCCGAA CTACGATGTT AGAACATATC CAATTCAAAC GTCATCCCAA 780

TTAACAAGGG AAATTTATAC AAGTTCAGTA ATTGAGGATT CTCCAGTTTC TGCTAATATA 8 0

CCTAATGGTT TTAATAGGGC GGAATTTGGA GTTAGACCGC CCCATCTTAT GGACTTTATG 900

AATTCTTTGT TTGTAACTGC AGAGACTGTT AGAAGTCAAA CTGTGTGGGG AGGACACTTA 960 GTTAGTTCAC GAAATACGGC TGGTAACCGT ATAAATTTCC CTAGTTACGG GGTCTTCAAT 1020

CCTGGTGGCG CCATTTGGAT TGCAGATGAG GATCCACGTC CTTTTTATCG GACATTATCA 1080

GATCCTGTTT TTGTCCGAGG AGGATTTGGG AATCCTCATT ATGTACTGGG GCTTAGGGGA 1140

GTAGCATTTC AACAAACTGG TACGAACCAC ACCCGAACAT TTAGAAATAG TGGGACCATA 1200

GATTCTCTAG ATGAAATCCC ACCTCAGGAT AATAGTGGGG CACCTTGGAA TGATTATAGT 1260

CATGTATTAA ATCATGTTAC ATTTGTACGA TGGCCAGGTG AGATTTCAGG AAGTGATTCA 1320

TGGAGAGCTC CAATGTTTTC TTGGACGCAC CGTAGTGCAA CCCCTACAAA TACAATTGAT 1380

CCGGAGAGGA TTACTCAAAT ACCATTGGTA AAAGCACATA CACTTCAGTC AGGTACTACT 1440

GTTGTAAGAG GGCCCGGGTT TACGGGAGGA GATATTCTTC GACGAACAAG TGGAGGACCA 1500

TTTGCTTATA CTATTGTTAA TATAAATGGG CAATTACCCC AAAGGTATCG TGCAAGAATA 1560

CGCTATGCCT CTACTACAAA TCTAAGAATT TACGTAACGG TTGCAGGTGA ACGGATTTTT 1620

GCTGGTCAAT TTAACAAAAC AATGGATACC GGTGACCCAT TAACATTCCA ATCTTTTAGT 1680

TACGCAACTA TTAATACAGC TTTTACATTC CCAATGAGCC AGAGTAGTTT CACAGTAGGT 1740

GCTGATACTT TTAGTTCAGG GAATGAAGTT TATATAGACA GATTTGAATT GATTCCAGTT 1800

ACTGCAACAT TTGAAGCAGA ATATGATTTA GAAAGAGCAC AAAAGGCGGT GAATGCGCTG 1860

TTTACTTCTA TAAACCAAAT AGGGATAAAA ACAGATGTGA CGGATTATCA TATCGATCGA 1920

GTATCCAATT TAGTTGAGTG TTTATCTGAT GAATTTTGTC TGGATGAAAA AAAAGAATTG 1980

TCCGAGAAAG TCAAACATGC GAAGCGACTT AGTGATGAGC GGAATTTACT TCAAGATCCA 2040

AACTTTAGAG GGATCAATAG ACAACTAGAC CGTGGCTGGA GAGGAAGTAC GGATATTACC 2100

ATCCAAGGAG GCGATGACGT ATTCAAAGAG AATTACGTTA CGCTATTGGG TACCTTTGAT 2160

GAGTGCTATC CAACGTATTT ATATCAAAAA ATAGATGAGT CGAAATTAAA AGCCTATACC 2220

CGTTACCAAT TAAGAGGGTA TATCGAAGAT AGTCAAGACT TAGAAATCTA TTTAATTCGC 2280

TACAATGCCA AACACGAAAC AGTAAATGTG CCAGGTACGG GTTCCTTATG GCCGCTTTCA 2340

GCCCCAAGTC CAATCGGAAA ATGTGCCCAT CATTCCCATC ATTTCTCCTT GGACATTGAT 2400

GTTGGATGTA CAGACTTAAA TGAGGACTTA GGTGTATGGG TGATATTCAA GATTAAGACG 2460

CAAGATGGCC ATGCAAGACT AGGAAATCTA GAATTTCTCG AAGAGAAACC ATTAGTAGGA 2520

GAAGCACTAG CTCGTGTGAA AAGAGCGGAG AAAAAATGGA GAGACAAACG TGAAAAATTG 2580

GAATGGGAAA CAAATATTGT TTATAAAGAG GCAAAAGAAT CTGTAGATGC TTTATTTGTA 2640

AACTCTCAAT ATGATAGATT ACAAGCGGAT ACCAACATCG CGATGATTCA TGCGGCAGAT 2700 AAACGCGTTC ATAGCATTCG AGAAGCTTAT CTGCCTGAGC TGTCTGTGAT TCCGGGTGTC 2760

AATGCGGCTA TTTTTGAAGA ATTAGAAGGG CGTATTTTCA CTGCATTCTC CCTATATGAT 2820

GCGAGAAATG TCATTAAAAA TGGTGATTTT AATAATGGCT TATCCTGCTG GAACGTGAAA 2880

GGGCATGTAG ATGTAGAAGA ACAAAACAAC CACCGTTCGG TCCTTGTTGT TCCGGAATGG 2940

GAAGCAGAAG TGTCACAAGA AGTTCGTGTC TGTCCGGGTC GTGGCTATAT CCTTCGTGTC 3000

ACAGCGTACA AGGAGGGATA TGGAGAAGGT TGCGTAACCA TTCATGAGAT CGAGAACAAT 3060

ACAGACGAAC TGAAGTTTAG CAACTGTGTA GAAGAGGAAG TATATCCAAA CAACACGGTA 3120

ACGTGTAATG ATTATACTGC GACTCAAGAA GAATATGAGG GTACGTACAC TTCTCGTAAT 3180

CGAGGATATG ACGGAGCCTA TGAAAGCAAT TCTTCTGTAC CAGCTGATTA TGCATCAGCC 3240

TATGAAGAAA AAGCATATAC AGATGGACGA AGAGACAATC CTTGTGAATC TAACAGAGGA 3300

TATGGGGATT ACACACCACT ACCAGCTGGC TATGTGACAA AAGAATTAGA GTACTTCCCA 3360

GAAACCGATA AGGTATGGAT TGAGATCGGA GAAACGGAAG GAACATTCAT CGTGGACAGC 3420

GTGGAATTAC TTCTTATGGA GGAA 3444

(2) INFORMATION FOR SEQ ID NO:27:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1148 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:

Met Glu Asn Asn He Gin Asn Gin Cys Val Pro Tyr Asn Cys Leu Asn 1 5 10 15

Asn Pro Glu Val Glu He Leu Asn Glu Glu Arg Ser Thr Gly Arg Leu 20 25 30

Pro Leu Asp He Ser Leu Ser Leu Thr Arg Phe Leu Leu Ser Glu Phe 35 40 45

Val Pro Gly Val Gly Val Ala Phe Gly Leu Phe Asp Leu He Trp Gly 50 55 60

Phe He Thr Pro Ser Asp Trp ser Leu Phe Leu Leu Gin He Glu Gin 65 70 75 80

Leu He Glu Gin Arg He Glu Thr Leu Glu Arg Asn Arg Ala He Thr 85 90 95 Thr Leu Arg Gly Leu Ala Asp Ser Tyr Glu He Tyr He Glu Ala Leu 100 105 110

Arg Glu Trp Glu Ala Asn Pro Asn Asn Ala Gin Leu Arg Glu Asp Val 115 120 125

Arg He Arg Phe Ala Asn Thr Asp Asp Ala Leu He Thr Ala He Asn 130 135 140

Asn Phe Thr Leu Thr Ser Phe Glu lie Pro Leu Leu Ser Val Tyr Val 145 150 155 160

Gin Ala Ala Asn Leu His Leu Ser Leu Leu Arg Asp Ala Val Ser Phe 165 170 175

Gly Gin Gly Trp Gly Leu Asp He Ala Thr Val Asn Asn His Tyr Asn 180 185 190

Arg Leu He Asn Leu He His Arg Tyr Thr Lys His Cys Leu Asp Thr 195 200 205

Tyr Asn Gin Gly Leu Glu Asn Leu Arg Gly Thr Asn Thr Arg Gin Trp

210 215 220

Ala Arg Phe Asn Gin Phe Arg Arg Asp Leu Thr Leu Thr Val Leu Asp 225 230 235 240

He Val Ala Leu Phe Pro Asn Tyr Asp Val Arg Thr Tyr Pro He Gin 245 250 255

Thr Ser ser Gin Leu Thr Arg Glu He Tyr Thr Ser Ser Val He Glu 260 265 270

Asp Ser Pro val Ser Ala Asn He Pro Asn Gly Phe Asn Arg Ala Glu 275 280 285

Phe Gly Val Arg Pro Pro His Leu Met Asp Phe Met Asn Ser Leu Phe 290 295 300

Val Thr Ala Glu Thr Val Arg Ser Gin Thr Val Trp Gly Gly His Leu 305 310 315 320

Val Ser Ser Arg Asn Thr Ala Gly Asn Arg He Asn Phe Pro Ser Tyr 325 330 335

Gly Val Phe Asn Pro Gly Gly Ala He Trp He Ala Asp Glu Asp Pro 340 345 350

Arg Pro Phe Tyr Arg Thr Leu Ser Asp Pro Val Phe Val Arg Gly Gly 355 360 365

Phe Gly Asn Pro His Tyr Val Leu Gly Leu Arg Gly Val Ala Phe Gin 370 375 380

Gin Thr Gly Thr Asn His Thr Arg Thr Phe Arg Asn Ser Gly Thr He 385 390 395 400

Asp Ser Leu Asp Glu He Pro Pro Gin Asp Asn Ser Gly Ala Pro Trp 405 410 415

Asn Asp Tyr Ser His Val Leu Asn His Val Thr Phe Val Arg Trp Pro 420 425 430

Gly Glu He Ser Gly Ser Asp Ser Trp Arg Ala Pro Met Phe Ser Trp 435 440 445

Thr His Arg Ser Ala Thr Pro Thr Asn Thr He Asp Pro Glu Arg He 450 455 460

Thr Gin He Pro Leu Val Lys Ala His Thr Leu Gin ser Gly Thr Thr 465 470 475 480

Val Val Arg Gly Pro Gly Phe Thr Gly Gly Asp He Leu Arg Arg Thr 485 490 495

Ser Gly Gly Pro Phe Ala Tyr Thr He Val Asn He Asn Gly Gin Leu

500 505 510

Pro Gin Arg Tyr Arg Ala Arg He Arg Tyr Ala Ser Thr Thr Asn Leu 515 520 525

Arg He Tyr Val Thr Val Ala Gly Glu Arg He Phe Ala Gly Gin Phe 530 535 540 Asn Lys Thr Met Asp Thr Gly Asp Pro Leu Thr Phe Gin Ser Phe Ser 545 550 555 560

Tyr Ala Thr He Asn Thr Ala Phe Thr Phe Pro Met Ser Gin Ser Ser 565 570 575

Phe Thr Val Gly Ala Asp Thr Phe Ser Ser Gly Asn Glu Val Tyr He 580 585 590

Asp Arg Phe Glu Leu He Pro Val Thr Ala Thr Phe Glu Ala Glu Tyr 595 600 605

Asp Leu Glu Arg Ala Gin Lys Ala Val Asn Ala Leu Phe Thr Ser He 610 615 620

Asn Gin He Gly He Lys Thr Asp Val Thr Asp Tyr His He Asp Arg 625 630 635 640

Val Ser Asn Leu Val Glu Cys Leu ser Asp Glu Phe cys Leu Asp Glu 645 650 655

Lys Lys Glu Leu Ser Glu Lys Val Lys His Ala Lys Arg Leu Ser Asp 660 665 670

Glu Arg Asn Leu Leu Gin Asp Pro Asn Phe Arg Gly He Asn Arg Gin 675 680 685

Leu Asp Arg Gly Trp Arg Gly ser Thr Asp He Thr He Gin Gly Gly 690 695 700

Asp Asp Val Phe Lys Glu Asn Tyr Val Thr Leu Leu Gly Thr Phe Asp 705 710 715 720

Glu Cys Tyr Pro Thr Tyr Leu Tyr Gin Lys He Asp Glu Ser Lys Leu 725 730 735

Lys Ala Tyr Thr Arg Tyr Gin Leu Arg Gly Tyr He Glu Asp Ser Gin 740 745 750

Asp Leu Glu He Tyr Leu He Arg Tyr Asn Ala Lys His Glu Thr Val 755 760 765

Asn Val Pro Gly Thr Gly ser Leu Trp Pro Leu Ser Ala Pro Ser Pro 770 775 780

He Gly Lys Cys Ala His His Ser His His Phe Ser Leu Asp He Asp 785 790 795 800

Val Gly Cys Thr Asp Leu Asn Glu Asp Leu Gly Val Trp Val He Phe 805 810 815

Lys He Lys Thr Gin Asp Gly His Ala Arg Leu Gly Asn Leu Glu Phe 820 825 830

Leu Glu Glu Lys Pro Leu Val Gly Glu Ala Leu Ala Arg Val Lys Arg 835 840 845 Ala Glu Lys Lys Trp Arg Asp Lys Arg Glu Lys Leu Glu Trp Glu Thr 850 855 860

Asn He Val Tyr Lys Glu Ala Lys Glu Ser Val Asp Ala Leu Phe Val 865 870 875 880

Asn ser Gin Tyr Asp Arg Leu Gin Ala Asp Thr Asn He Ala Met He 885 890 895

His Ala Ala Asp Lys Arg Val His Ser He Arg Glu Ala Tyr Leu Pro 900 905 910

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

Glu Gly Arg He Phe Thr Ala Phe Ser Leu Tyr Asp Ala Arg Asn Val 930 935 940

He Lys Asn Gly Asp Phe Asn Asn Gly Leu Ser Cys Trp Asn Val Lys 945 950 955 960

Gly His Val Asp Val Glu Glu Gin Asn Asn His Arg Ser Val Leu Val 965 970 975

Val Pro Glu Trp Glu Ala Glu Val Ser Gin Glu Val Arg Val Cys Pro 980 985 990

Gly Arg Gly Tyr He Leu Arg Val Thr Ala Tyr Lys Glu Gly Tyr Gly 995 1000 1005

Glu Gly Cys Val Thr He His Glu He Glu Asn Asn Thr Asp Glu Leu 1010 1015 1020

Lys Phe Ser Asn cys Val Glu Glu Glu Val Tyr Pro Asn Asn Thr Val 1025 1030 1035 1040

Thr Cys Asn Asp Tyr Thr Ala Thr Gin Glu Glu Tyr Glu Gly Thr Tyr 1045 1050 1055

Thr Ser Arg Asn Arg Gly Tyr Asp Gly Ala Tyr Glu ser Asn Ser ser 1060 1065 1070

Val Pro Ala Asp Tyr Ala Ser Ala Tyr Glu Glu Lys Ala Tyr Thr Asp 1075 1080 1085

Gly Arg Arg Asp Asn Pro Cys Glu Ser Asn Arg Gly Tyr Gly Asp Tyr 1090 1095 1100

Thr Pro Leu Pro Ala Gly Tyr Val Thr Lys Glu Leu Glu Tyr Phe Pro 1105 1110 1115 1120

Glu Thr Asp Lys Val Trp He Glu He Gly Glu Thr Glu Gly Thr Phe 1125 1130 1135

He Val Asp Ser Val Glu Leu Leu Leu Met Glu Glu 1140 1145

(2) INFORMATION FOR SEQ ID NO:28:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 3522 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: ATGGAAAATA ATATTCAAAA TCAATGCGTA CCTTACAATT GTTTAAATAA TCCTGAAGTA 60 GAAATACTGA ACGAAGAACG CAGCACCGGC CGCCTGCCGC TGGACATCAG CCTGAGCCTT 120 ACACGTTTCC TTTTGAGTGA ATTTGTTCCA GGTGTGGGAG TTGCGTTTGG ATTATTTGAT 180 TTAATATGGG GTTTTATAAC TCCTTCTGAT TGGAGCTTAT TTCTTTTACA GATTGAACAA 240

TTGATTGAGC AAAGAATAGA AACATTGGAA AGGAACCGGG CAATTACTAC ATTACGAGGG 300

TTAGCAGATA GCTATGAAAT TTATATTGAA GCACTAAGAG AGTGGGAAGC AAATCCTAAT 360

AATGCACAAT TAAGGGAAGA TGTGCGTATT CGATTTGCTA ATACAGACGA CGCTTTAATA 420

ACAGCAATAA ATAATTTTAC ACTTACAAGT TTTGAAATCC CTCTTTTATC GGTCTATGTT 480

CAAGCGGCGA ATTTACATTT ATCACTATTA AGAGACGCTG TATCGTTTGG GCAGGGTTGG 5 0

GGACTGGATA TAGCTACTGT TAATAATCAT TATAATAGAT TAATAAATCT TATTCATAGA 600

TATACGAAAC ATTGTTTGGA CACATACAAT CAAGGATTAG AAAACTTAAG AGGTACTAAT 660

ACTCGACAAT GGGCAAGATT CAATCAGTTT AGGAGAGATT TAACACTTAC TGTATTAGAT 720

ATCGTTGCTC TTTTTCCGAA CTACGATGTT AGAACATATC CAATTCAAAC GTCATCCCAA 780

TTAACAAGGG AAATTTATAC AAGTTCAGTA ATTGAGGATT CTCCAGTTTC TGCTAATATA 840

CCTAATGGTT TTAATAGGGC GGAATTTGGA GTTAGACCGC CCCATCTTAT GGACTTTATG 900

AATTCTTTGT TTGTAACTGC AGAGACTGTT AGAAGTCAAA CTGTGTGGGG AGGACACTTA 960

GTTAGTTCAC GAAATACGGC TGGTAACCGT ATAAATTTCC CTAGTTACGG GGTCTTCAAT 1020

CCTGGTGGCG CCATTTGGAT TGCAGATGAG GATCCACGTC CTTTTTATCG GACATTATCA 1080

GATCCTGTTT TTGTCCGAGG AGGATTTGGG AATCCTCATT ATGTACTGGG GCTTAGGGGA 1140

GTAGCATTTC AACAAACTGG TACGAACCAC ACCCGAACAT TTAGAAATAG TGGGACCATA 1200

GATTCTCTAG ATGAAATCCC ACCTCAGGAT AATAGTGGGG CACCTTGGAA TGATTATAGT 1260

CATGTATTAA ATCATGTTAC ATTTGTACGA TGGCCAGGTG AGATTTCAGG AAGTGATTCA 1320

TGGAGAGCTC CAATGTTTTC TTGGACGCAC CGTAGTGCAA CCCCTACAAA TACAATTGAT 1380

CCGGAGAGGA TTACTCAAAT ACCATTGGTA AAAGCACATA CACTTCAGTC AGGTACTACT 1440

GTTGTAAGAG GGCCCGGGTT TACGGGAGGA GATATTCTTC GACGAACAAG TGGAGGACCA 1500

TTTGCTTATA CTATTGTTAA TATAAATGGG CAATTACCCC AAAGGTATCG TGCAAGAATA 1560

CGCTATGCCT CTACTACAAA TCTAAGAATT TACGTAACGG TTGCAGGTGA ACGGATTTTT 1620

GCTGGTCAAT TTAACAAAAC AATGGATACC GGTGACCCAT TAACATTCCA ATCTTTTAGT 1680

TACGCAACTA TTAATACAGC TTTTACATTC CCAATGAGCC AGAGTAGTTT CACAGTAGGT 1740

GCTGATACTT TTAGTTCAGG GAATGAAGTT TATATAGACA GATTTGAATT GATTCCAGTT 1800

ACTGCAACAT TTGAAGCAGA ATATGATTTA GAAAGAGCAC AAAAGGCGGT GAATGCGCTG 1860

TTTACTTCTA TAAACCAAAT AGGGATAAAA ACAGATGTGA CGGATTATCA TATCGATCGA 1920 GTGTCCAATT TAGTTACGTA TTTATCGGAT GAATTTTGTC TGGATGAAAA GCGAGAATTG 1980

TCCGAGAAAG TCAAACATGC GAAGCGACTC AGTGATGAAC GCAATTTACT CCAAGATTCA 2040

AATTTCAAAG ACATTAATAG GCAACCAGAA CGTGGGTGGG GCGGAAGTAC AGGGATTACC 2100

ATCCAAGGAG GGGATGACGT ATTTAAAGAA AATTACGTCA CACTATCAGG TACCTTTGAT 2160

GAGTGCTATC CAACATATTT GTATCAAAAA ATCGATGAAT CAAAATTAAA AGCCTTTACC 2220

CGTTATCAAT TAAGAGGGTA TATCGAAGAT AGTCAAGACT TAGAAATCTA TTTAATTCGC 2280

TACAATGCAA AACATGAAAC AGTAAATGTG CCAGGTACGG GTTCCTTATG GCCGCTTTCA 2340

GCCCAAAGTC CAATCGGAAA GTGTGGAGAG CCGAATCGAT GCGCGCCACA CCTTGAATGG 2400

AATCCTGACT TAGATTGTTC GTGTAGGGAT GGAGAAAAGT GTGCCCATCA TTCGCATCAT 2460

TTCTCCTTAG ACATTGATGT AGGATGTACA GACTTAAATG AGGACCTAGG TGTATGGGTG 2520

ATCTTTAAGA TTAAGACGCA AGATGGGCAC GCAAGACTAG GGAATCTAGA GTTTCTCGAA 2580

GAGAAACCAT TAGTAGGAGA AGCGCTAGCT CGTGTGAAAA GAGCGGAGAA AAAATGGAGA 2640

GACAAACGTG AAAAATTGGA ATGGGAAACA AATATCGTTT ATAAAGAGGC AAAAGAATCT 2700

GTAGATGCTT TATTTGTAAA CTCTCAATAT GATCAATTAC AAGCGGATAC GAATATTGCC 2760

ATGATTCATG CGGCAGATAA ACGTGTTCAT AGCATTCGAG AAGCTTATCT GCCTGAGCTG 2820

TCTGTGATTC CGGGTGTCAA TGCGGCTATT TTTGAAGAAT TAGAAGGGCG TATTTTCACT 2880

GCATTCTCCC TATATGATGC GAGAAATGTC ATTAAAAATG GTGATTTTAA TAATGGCTTA 2940

TCCTGCTGGA ACGTGAAAGG GCATGTAGAT GTAGAAGAAC AAAACAACCA CCGTTCGGTC 3000

CTTGTTGTTC CGGAATGGGA AGCAGAAGTG TCACAAGAAG TTCGTGTCTG TCCGGGTCGT 3060

GGCTATATCC TTCGTGTCAC AGCGTACAAG GAGGGATATG GAGAAGGTTG CGTAACCATT 3120

CATGAGATCG AGAACAATAC AGACGAACTG AAGTTTAGCA ACTGTGTAGA AGAGGAAGTA 3180

TATCCAAACA ACACGGTAAC GTGTAATGAT TATACTGCGA CTCAAGAAGA ATATGAGGGT 3240

ACGTACACTT CTCGTAATCG AGGATATGAC GGAGCCTATG AAAGCAATTC TTCTGTACCA 3300

GCTGATTATG CATCAGCCTA TGAAGAAAAA GCATATACAG ATGGACGAAG AGACAATCCT 3360

TGTGAATCTA ACAGAGGATA TGGGGATTAC ACACCACTAC CAGCTGGCTA TGTGACAAAA 3420

GAATTAGAGT ACTTCCCAGA AACCGATAAG GTATGGATTG AGATCGGAGA AACGGAAGGA 3480

ACATTCATCG TGGACAGCGT GGAATTACTT CTTATGGAGG AA 3522

(2) INFORMATION FOR SEQ ID NO:29: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1174 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:

Met Glu Asn Asn He Gin Asn Gin Cys Val Pro Tyr Asn Cys Leu Asn

1 5 10 15

Asn Pro Glu Val Glu He Leu Asn Glu Glu Arg Ser Thr Gly Arg Leu 20 25 30

Pro Leu Asp He Ser Leu Ser Leu Thr Arg Phe Leu Leu Ser Glu Phe 35 40 45

Val Pro Gly Val Gly Val Ala Phe Gly Leu Phe Asp Leu He Trp Gly 50 55 60

Phe He Thr Pro Ser Asp Trp Ser Leu Phe Leu Leu Gin He Glu Gin 65 70 75 80

Leu He Glu Gin Arg He Glu Thr Leu Glu Arg Asn Arg Ala He Thr 85 90 95

Thr Leu Arg Gly Leu Ala Asp Ser Tyr Glu He Tyr He Glu Ala Leu 100 105 110

Arg Glu Trp Glu Ala Asn Pro Asn Asn Ala Gin Leu Arg Glu Asp Val 115 120 125

Arg He Arg Phe Ala Asn Thr Asp Asp Ala Leu He Thr Ala He Asn

130 135 140

Asn Phe Thr Leu Thr Ser Phe Glu He Pro Leu Leu Ser Val Tyr Val 145 150 155 160

Gin Ala Ala Asn Leu His Leu Ser Leu Leu Arg Asp Ala Val Ser Phe 165 170 175

Gly Gin Gly Trp Gly Leu Asp He Ala Thr Val Asn Asn His Tyr Asn 180 185 190

Arg Leu He Asn Leu He His Arg Tyr Thr Lys His Cys Leu Asp Thr 195 200 205

Tyr Asn Gin Gly Leu Glu Asn Leu Arg Gly Thr Asn Thr Arg Gin Trp 210 215 220

Ala Arg Phe Asn Gin Phe Arg Arg Asp Leu Thr Leu Thr Val Leu Asp 225 230 235 240

He Val Ala Leu Phe Pro Asn Tyr Asp Val Arg Thr Tyr Pro He Gin 245 250 255 Thr Ser Ser Gin Leu Thr Arg Glu He Tyr Thr ser ser Val He Glu 260 265 270

Asp Ser Pro Val Ser Ala Asn He Pro Asn Gly Phe Asn Arg Ala Glu 275 280 285

Phe Gly Val Arg Pro Pro His Leu Met Asp Phe Met Asn Ser Leu Phe 290 295 300

Val Thr Ala Glu Thr Val Arg Ser Gin Thr Val Trp Gly Gly His Leu 305 310 315 320

Val ser ser Arg Asn Thr Ala Gly Asn Arg He Asn Phe Pro Ser Tyr 325 330 335

Gly Val Phe Asn Pro Gly Gly Ala He Trp He Ala Asp Glu Asp Pro 340 345 350

Arg Pro Phe Tyr Arg Thr Leu Ser Asp Pro Val Phe Val Arg Gly Gly 355 360 365

Phe Gly Asn Pro His Tyr Val Leu Gly Leu Arg Gly Val Ala Phe Gin 370 375 380

Gin Thr Gly Thr Asn His Thr Arg Thr Phe Arg Asn Ser Gly Thr He 385 390 395 400

Asp Ser Leu Asp Glu He Pro Pro Gin Asp Asn Ser Gly Ala Pro Trp

405 410 415

Asn Asp Tyr Ser His Val Leu Asn His Val Thr Phe Val Arg Trp Pro 420 425 430

Gly Glu He Ser Gly ser Asp ser Trp Arg Ala Pro Met Phe Ser Trp 435 440 445

Thr His Arg Ser Ala Thr Pro Thr Asn Thr He Asp Pro Glu Arg He 450 455 460

Thr Gin He Pro Leu Val Lys Ala His Thr Leu Gin Ser Gly Thr Thr 465 470 475 480

Val Val Arg Gly Pro Gly Phe Thr Gly Gly Asp He Leu Arg Arg Thr 485 490 495

Ser Gly Gly Pro Phe Ala Tyr Thr He Val Asn He Asn Gly Gin Leu 500 505 510

Pro Gin Arg Tyr Arg Ala Arg He Arg Tyr Ala Ser Thr Thr Asn Leu 515 520 525

Arg He Tyr Val Thr Val Ala Gly Glu Arg He Phe Ala Gly Gin Phe 530 535 540

Asn Lys Thr Met Asp Thr Gly Asp Pro Leu Thr Phe Gin Ser Phe Ser

545 550 555 560 Tyr Ala Thr He Asn Thr Ala Phe Thr Phe Pro Met Ser Gin Ser Ser 565 570 575

Phe Thr Val Gly Ala Asp Thr Phe Ser Ser Gly Asn Glu Val Tyr He 580 585 590

Asp Arg Phe Glu Leu He Pro Val Thr Ala Thr Phe Glu Ala Glu Tyr 595 600 605

Asp Leu Glu Arg Ala Gin Lys Ala Val Asn Ala Leu Phe Thr Ser He 610 615 620

Asn Gin He Gly He Lys Thr Asp Val Thr Asp Tyr His He Asp Arg 625 630 635 640

Val Ser Asn Leu Val Thr Tyr Leu Ser Asp Glu Phe cys Leu Asp Glu 645 650 655

Lys Arg Glu Leu Ser Glu Lys Val Lys His Ala Lys Arg Leu Ser Asp 660 665 670

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

Pro Glu Arg Gly Trp Gly Gly Ser Thr Gly He Thr He Gin Gly Gly 690 695 700

Asp Asp Val Phe Lys Glu Asn Tyr Val Thr Leu Ser Gly Thr Phe Asp 705 710 715 720

Glu Cys Tyr Pro Thr Tyr Leu Tyr Gin Lys He Asp Glu Ser Lys Leu 725 730 735

Lys Ala Phe Thr Arg Tyr Gin Leu Arg Gly Tyr He Glu Asp Ser Gin 740 745 750

Asp Leu Glu He Tyr Leu He Arg Tyr Asn Ala Lys His Glu Thr Val 755 760 765

Asn Val Pro Gly Thr Gly ser Leu Trp Pro Leu Ser Ala Gin Ser Pro 770 775 780

He Gly Lys Cys Gly Glu Pro Asn Arg Cys Ala Pro His Leu Glu Trp 785 790 795 800

Asn Pro Asp Leu Asp Cys Ser Cys Arg Asp Gly Glu Lys Cys Ala His 805 810 815

His Ser His His Phe Ser Leu Asp He Asp Val Gly Cys Thr Asp Leu 820 825 830

Asn Glu Asp Leu Gly Val Trp Val He Phe Lys He Lys Thr Gin Asp 835 840 845

Gly His Ala Arg Leu Gly Asn Leu Glu Phe Leu Glu Glu Lys Pro Leu 850 855 860 Val Gly Glu Ala Leu Ala Arg Val Lys Arg Ala Glu Lys Lys Trp Arg 865 870 875 880

Asp Lys Arg Glu Lys Leu Glu Trp Glu Thr Asn He Val Tyr Lys Glu 885 890 895

Ala Lys Glu Ser Val Asp Ala Leu Phe Val Asn Ser Gin Tyr Asp Gin 900 905 910

Leu Gin Ala Asp Thr Asn He Ala Met He His Ala Ala Asp Lys Arg 915 920 925

Val His Ser He Arg Glu Ala Tyr Leu Pro Glu Leu Ser Val He Pro 930 935 940

Gly Val Asn Ala Ala He Phe Glu Glu Leu Glu Gly Arg He Phe Thr 945 950 955 960

Ala Phe Ser Leu Tyr Asp Ala Arg Asn Val He Lys Asn Gly Asp Phe 965 970 975

Asn Asn Gly Leu Ser cys Trp Asn Val Lys Gly His Val Asp Val Glu 980 985 990

Glu Gin Asn Asn His Arg Ser Val Leu Val Val Pro Glu Trp Glu Ala 995 1000 1005

Glu Val Ser Gin Glu Val Arg Val Cys Pro Gly Arg Gly Tyr He Leu 1010 1015 1020

Arg Val Thr Ala Tyr Lys Glu Gly Tyr Gly Glu Gly cys Val Thr He 1025 1030 1035 1040

His Glu He Glu Asn Asn Thr Asp Glu Leu Lys Phe Ser Asn Cys Val 1045 1050 1055

Glu Glu Glu Val Tyr Pro Asn Asn Thr Val Thr Cys Asn Asp Tyr Thr 1060 1065 1070

Ala Thr Gin Glu Glu Tyr Glu Gly Thr Tyr Thr Ser Arg Asn Arg Gly 1075 1080 1085

Tyr Asp Gly Ala Tyr Glu Ser Asn Ser Ser Val Pro Ala Asp Tyr Ala 1090 1095 1100

Ser Ala Tyr Glu Glu Lys Ala Tyr Thr Asp Gly Arg Arg Asp Asn Pro 1105 1110 1115 1120

Cys Glu Ser Asn Arg Gly Tyr Gly Asp Tyr Thr Pro Leu Pro Ala Gly

1125 1130 1135

Tyr val Thr Lys Glu Leu Glu Tyr Phe Pro Glu Thr Asp Lys Val Trp 1140 1145 1150

He Glu He Gly Glu Thr Glu Gly Thr Phe He Val Asp Ser Val Glu 1155 1160 1165 Leu Leu Leu Met Glu Glu

1170

(2) INFORMATION FOR SEQ ID NO:30:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:

Xaa Xaa lie Asp Xaa Xaa Glu Xaa Xaa Xaa Xaa Xaa

5 10

(2) INFORMATION FOR SEQ ID NO:31:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 8 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:

Tyr Pro Asn Asn Thr Val Thr cys

5

(2) INFORMATION FOR SEQ ID NO:32:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1174 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:

Met Glu Asn Asn He Gin Asn Gin cys Val Pro Tyr Asn Cys Leu Asn

1 5 10 15

Asn Pro Glu Val Glu He Leu Asn Glu Glu Arg ser Thr Gly Arg Leu 20 25 30

Pro Leu Asp He Ser Leu ser Leu Thr Arg Phe Leu Leu Ser Glu Phe 35 40 45 Val Pro Gly Val Gly Val Ala Phe Gly Leu Phe Asp Leu He Trp Gly 50 55 60

Phe He Thr Pro Ser Asp Trp Ser Leu Phe Leu Leu Gin He Glu Gin 65 70 75 80

Leu He Glu Gin Arg He Glu Thr Leu Glu Arg Asn Arg Ala He Thr 85 90 95

Thr Leu Arg Gly Leu Ala Asp Ser Tyr Glu He Tyr He Glu Ala Leu 100 105 110

Arg Glu Trp Glu Ala Asn Pro Asn Asn Ala Gin Leu Arg Glu Asp Val

115 120 125

Arg He Arg Phe Ala Asn Thr Asp Asp Ala Leu He Thr Ala He Asn

130 135 140

Asn Phe Thr Leu Thr Ser Phe Glu He Pro Leu Leu ser Val Tyr Val 145 150 155 160

Gin Ala Ala Asn Leu His Leu Ser Leu Leu Arg Asp Ala Val Ser Phe 165 170 175

Gly Gin Gly Trp Gly Leu Asp He Ala Thr Val Asn Asn His Tyr Asn 180 185 190

Arg Leu He Asn Leu He His Arg Tyr Thr Lys His cys Leu Asp Thr 195 200 205

Tyr Asn Gin Gly Leu Glu Asn Leu Arg Gly Thr Asn Thr Arg Gin Trp 210 215 220

Ala Arg Phe Asn Gin Phe Arg Arg Asp Leu Thr Leu Thr Val Leu Asp 225 230 235 240

He Val Ala Leu Phe Pro Asn Tyr Asp Val Arg Thr Tyr Pro He Gin 245 250 255

Thr ser Ser Gin Leu Thr Arg Glu He Tyr Thr Ser Ser Val He Glu 260 265 270

Asp Ser Pro Val Ser Ala Asn He Pro Asn Gly Phe Asn Arg Ala Glu 275 280 285

Phe Gly Val Arg Pro Pro His Leu Met Asp Phe Met Asn Ser Leu Phe 290 295 300

Val Thr Ala Glu Thr Val Arg Ser Gin Thr Val Trp Gly Gly His Leu 305 310 315 320

Val Ser Ser Arg Asn Thr Ala Gly Asn Arg He Asn Phe Pro Ser Tyr 325 330 335

Gly Val Phe Asn Pro Gly Gly Ala He Trp He Ala Asp Glu Asp Pro 340 345 350 Arg Pro Phe Tyr Arg Thr Leu Ser Asp Pro Val Phe Val Arg Gly Gly 355 360 365

Phe Gly Asn Pro His Tyr Val Leu Gly Leu Arg Gly Val Ala Phe Gin 370 375 380

Gin Thr Gly Thr Asn His Thr Arg Thr Phe Arg Asn ser Gly Thr He 385 390 395 400

Asp ser Leu Asp Glu He Pro Pro Gin Asp Asn Ser Gly Ala Pro Trp

405 410 415

Asn Asp Tyr Ser His Val Leu Asn His Val Thr Phe Val Arg Trp Pro 420 425 430

Gly Glu He ser Gly Ser Asp Ser Trp Arg Ala Pro Met Phe Ser Trp

435 440 445

Thr His Arg Ser Ala Thr Pro Thr Asn Thr He Asp Pro Glu Arg He 450 455 460

Thr Gin He Pro Leu Val Lys Ala His Thr Leu Gin Ser Gly Thr Thr 465 470 475 480

Val Val Arg Gly Pro Gly Phe Thr Gly Gly Asp He Leu Arg Arg Thr 485 490 495

Ser Gly Gly Pro Phe Ala Tyr Thr He Val Asn He Asn Gly Gin Leu 500 505 510

Pro Gin Arg Tyr Arg Ala Arg He Arg Tyr Ala Ser Thr Thr Asn Leu 515 520 525

Arg He Tyr Val Thr Val Ala Gly Glu Arg He Phe Ala Gly Gin Phe 530 535 540

Asn Lys Thr Met Asp Thr Gly Asp Pro Leu Thr Phe Gin Ser Phe ser 545 550 555 560

Tyr Ala Thr He Asn Thr Ala Phe Thr Phe Pro Met Ser Gin ser Ser 565 570 575

Phe Thr Val Gly Ala Asp Thr Phe Ser Ser Gly Asn Glu Val Tyr He 580 585 590

Asp Arg Phe Glu Leu lie Pro Val Thr Ala Thr Phe Glu Ala Glu Tyr 595 600 605

Asp Leu Glu Arg Ala Gin Lys Ala Val Asn Ala Leu Phe Thr ser He 610 615 620

Asn Gin He Gly He Lys Thr Asp Val Thr Asp Tyr His He Asp Gin 625 630 635 640

Val Ser Asn Leu Val Asp Cys Leu Ser Asp Glu Phe Cys Leu Asp Glu 645 650 655 Lys Arg Glu Leu ser Glu Lys Val Lys His Ala Lys Arg Leu Ser Asp 660 665 670

Glu Arg Asn Leu Leu Gin Asp Pro Asn Phe Lys Gly He Asn Arg Gin 675 680 685

Leu Asp Arg Gly Trp Arg Gly Ser Thr Asp He Thr He Gin Arg Gly 690 695 700

Asp Asp Val Phe Lys Glu Asn Tyr Val Thr Leu Pro Gly Thr Phe Asp 705 710 715 720

Glu Cys Tyr Pro Thr Tyr Leu Tyr Gin Lys He Asp Glu Ser Lys Leu 725 730 735

Lys Pro Tyr Thr Arg Tyr Gin Leu Arg Gly Tyr He Glu Asp Ser Gin 740 745 750

Asp Leu Glu He Tyr Leu He Arg Tyr Asn Ala Lys His Glu Thr Val 755 760 765

Asn Val Leu Gly Thr Gly Ser Leu Trp Pro Leu Ser Val Gin ser Pro 770 775 780

He Arg Lys Cys Gly Glu Pro Asn Arg Cys Ala Pro His Leu Glu Trp 785 790 795 800

Asn Pro Asp Leu Asp Cys Ser Cys Arg Asp Gly Glu Lys Cys Ala His 805 810 815

His Ser His His Phe Ser Leu Asp He Asp Val Gly Cys Thr Asp Leu 820 825 830

Asn Glu Asp Leu Asp Val Trp Val He Phe Lys He Lys Thr Gin Asp 835 840 845

Gly His Ala Arg Leu Gly Asn Leu Glu Phe Leu Glu Glu Lys Pro Leu 850 855 860

Val Gly Glu Ala Leu Ala Arg Val Lys Arg Ala Glu Lys Lys Trp Arg 865 870 875 880

Asp Lys Arg Glu Lys Leu Glu Leu Glu Thr Asn He Val Tyr Lys Glu 885 890 895

Ala Lys Glu Ser Val Asp Ala Leu Phe Val Asn Ser Gin Tyr Asp Gin 900 905 910

Leu Gin Ala Asp Thr Asn He Ala Met He His Ala Ala Asp Lys Arg 915 920 925 val His Arg He Arg Glu Ala Tyr Leu Pro Glu Leu ser Val He Pro 930 935 940

Gly Val Asn Val Asp He Phe Glu Glu Leu Lys Gly Arg He Phe Thr 945 950 955 960 Ala Phe Phe Leu Tyr Asp Ala Arg Asn Val He Lys Asn Gly Asp Phe 965 970 975

Asn Asn Gly Leu ser Cys Trp Asn Val Lys Gly His Val Asp Val Glu 980 985 990

Glu Gin Asn Asn His Arg Ser Val Leu Val Val Pro Glu Trp Glu Ala 995 1000 1005

Glu Val Ser Gin Glu val Arg Val Cys Pro Gly Arg Gly Tyr He Leu 1010 1015 1020

Arg Val Thr Ala Tyr Lys Glu Gly Tyr Gly Glu Gly Cys Val Thr He 1025 1030 1035 1040

His Glu He Glu Asn Asn Thr Asp Glu Leu Lys Phe Ser Asn Cys Val 1045 1050 1055

Glu Glu Glu Val Tyr Pro Asn Asn Thr Val Thr Cys Asn Asp Tyr Thr 1060 1065 1070

Ala Asn Gin Glu Glu Tyr Gly Gly Ala Tyr Thr ser Arg Asn Arg Gly 1075 1080 1085

Tyr Asp Glu Thr Tyr Gly Ser Asn Ser Ser Val Pro Ala Asp Tyr Ala 1090 1095 1100

Ser Val Tyr Glu Glu Lys Ser Tyr Thr Asp Gly Arg Arg Asp Asn Pro 1105 1110 1115 1120

Cys Glu Ser Asn Arg Gly Tyr Gly Asp Tyr Thr Pro Leu Pro Ala Gly 1125 1130 1135

Tyr Val Thr Lys Glu Leu Glu Tyr Phe Pro Glu Thr Asp Lys Val Trp 1140 1145 1150

He Glu He Gly Glu Thr Glu Gly Thr Phe He Val Asp Ser Val Glu 1155 1160 1165

Leu Leu Leu Met Glu Glu 1170

(2) INFORMATION FOR SEQ ID NO:33:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1155 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:

Met Asp Asn Asn Pro Asn He Asn Glu Cys He Pro Tyr Asn cys Leu 1 5 10 15 Ser Asn Pro Glu Val Glu Val Leu Gly Gly Glu Arg He Glu Thr Gly 20 25 30

Tyr Thr Pro He Asp He Ser Leu Ser Leu Thr Gin Phe Leu Leu Ser 35 40 45

Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gly Leu Val Asp He He 50 55 60

Trp Gly He Phe Gly Pro Ser Gin Trp Asp Ala Phe Leu Val Gin He 65 70 75 80

Glu Gin Leu He Asn Gin Arg He Glu Glu Phe Ala Arg Asn Gin Ala 85 90 95

He Ser Arg Leu Glu Gly Leu ser Asn Leu Tyr Gin He Tyr Ala Glu

100 105 110

Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr Asn Pro Ala Leu Arg Glu 115 120 125

Glu Met Arg He Gin Phe Asn Asp Met Asn Ser Ala Leu Thr Thr Ala 130 135 140

He Pro Leu Phe Ala Val Gin Asn Tyr Gin Val Pro Leu Leu Ser Val 145 150 155 160

Tyr Val Gin Ala Ala Asn Leu His Leu Ser Val Leu Arg Asp Val Ser 165 170 175

Val Phe Gly Gin Arg Trp Gly Phe Asp Ala Ala Thr He Asn Ser Arg 180 185 190

Tyr Asn Asp Leu Thr Arg Leu He Gly Asn Tyr Thr Asp His Ala Val 195 200 205

Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Trp Gly Pro Asp Ser Arg 210 215 220

Asp Trp He Arg Tyr Asn Gin Phe Arg Arg Glu Leu Thr Leu Thr Val 225 230 235 240

Leu Asp He Val Ser Leu Phe Pro Asn Tyr Asp Ser Arg Thr Tyr Pro 245 250 255

He Arg Thr Val Ser Gin Leu Thr Arg Glu He Tyr Thr Asn Pro Val 260 265 270

Leu Glu Asn Phe Asp Gly Ser Phe Arg Gly Ser Ala Gin Gly He Glu 275 280 285

Gly Ser He Arg Ser Pro His Leu Met Asp He Leu Asn Ser He Thr 290 295 300

He Tyr Thr Asp Ala His Arg Gly Glu Tyr Tyr Trp Ser Gly His Gin 305 310 315 320 He Met Ala ser Pro Val Gly Phe Ser Gly Pro Glu Phe Thr Phe Pro 325 330 335

Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro Gin Gin Arg He Val Ala 340 345 350

Gin Leu Gly Gin Gly Val Tyr Arg Thr Leu Ser Ser Thr Leu Tyr Arg 355 360 365

Arg Pro Phe Asn He Gly He Asn Asn Gin Gin Leu Ser Val Leu Asp

370 375 380

Gly Thr Glu Phe Ala Tyr Gly Thr Ser ser Asn Leu Pro Ser Ala Val 385 390 395 400

Tyr Arg Lys ser Gly Thr Val Asp Ser Leu Asp Glu He Pro Pro Gin 405 410 415

Asn Asn Asn Val Pro Pro Arg Gin Gly Phe ser His Arg Leu Ser His 420 425 430

Val ser Met Phe Arg Ser Gly Phe Ser Asn Ser ser Val Ser He He

435 440 445

Arg Ala Pro Met Phe Ser Trp He His Arg Ser Ala Glu Phe Asn Asn 450 455 460

He He Pro ser ser Gin lie Thr Gin He Pro Leu Thr Lys ser Thr 465 470 475 480

Asn Leu Gly Ser Gly Thr Ser Val Val Lys Gly Pro Gly Phe Thr Gly 485 490 495

Gly Asp lie Leu Arg Arg Thr Ser Pro Gly Gin He ser Thr Leu Arg 500 505 510

Val Asn He Thr Ala Pro Leu Ser Gin Arg Tyr Arg Val Arg He Arg 515 520 525

Tyr Ala ser Thr Thr Asn Leu Gin Phe His Thr Ser He Asp Gly Arg 530 535 540

Pro He Asn Gin Gly Asn Phe Ser Ala Thr Met ser Ser Gly Ser Asn 545 550 555 560

Leu Gin Ser Gly Ser Phe Arg Thr Val Gly Phe Thr Thr Pro Phe Asn 565 570 575

Phe Ser Asn Gly Ser Ser Val Phe Thr Leu Ser Ala His Val Phe Asn 580 585 590

Ser Gly Asn Glu Val Tyr He Asp Arg He Glu Phe Val Pro Ala Glu 595 600 605

Val Thr Phe Glu Ala Glu Tyr Asp Leu Glu Arg Ala Gin Lys Ala Val

610 615 620 Asn Glu Leu Phe Thr Ser Ser Asn Gin He Gly Leu Lys Thr Asp Val 625 630 635 640

Thr Asp Tyr His He Asp Gin Val Ser Asn Leu Val Glu Cys Leu Ser 645 650 655

Asp Glu Phe Cys Leu Asp Glu Lys Lys Glu Leu Ser Glu Lys Val Lys 660 665 670

His Ala Lys Arg Leu Ser Asp Glu Arg Asn Leu Leu Gin Asp Pro Asn 675 680 685

Phe Arg Gly He Asn Arg Gin Leu Asp Arg Gly Trp Arg Gly Ser Thr 690 695 700

Asp He Thr He Gin Gly Gly Asp Asp Val Phe Lys Glu Asn Tyr Val 705 710 715 720

Thr Leu Leu Gly Thr Phe Asp Glu Cys Tyr Pro Thr Tyr Leu Tyr Gin 725 730 735

Lys He Asp Glu Ser Lys Leu Lys Ala Tyr Thr Arg Tyr Gin Leu Arg 740 745 750

Gly Tyr He Glu Asp Ser Gin Asp Leu Glu He Tyr Leu He Arg Tyr 755 760 765

Asn Ala Lys His Glu Thr Val Asn Val Pro Gly Thr Gly Ser Leu Trp 770 775 780

Pro Leu Ser Ala Pro Ser Pro He Gly Lys Cys Ala His His Ser His 785 790 795 800

His Phe Ser Leu Asp He Asp Val Gly Cys Thr Asp Leu Asn Glu Asp 805 810 815

Leu Gly Val Trp Val He Phe Lys He Lys Thr Gin Asp Gly His Ala 820 825 830

Arg Leu Gly Asn Leu Glu Phe Leu Glu Glu Lys Pro Leu Val Gly Glu 835 840 845

Ala Leu Ala Arg Val Lys Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg 850 855 860

Glu Lys Leu Glu Trp Glu Thr Asn He Val Tyr Lys Glu Ala Lys Glu 865 870 875 880

Ser Val Asp Ala Leu Phe Val Asn ser Gin Tyr Asp Arg Leu Gin Ala 885 890 895

Asp Thr Asn He Ala Met He His Ala Ala Asp Lys Arg Val His Ser 900 905 910

He Arg Glu Ala Tyr Leu Pro Glu Leu Ser Val He Pro Gly Val Asn 915 920 925 Ala Ala He Phe Glu Glu Leu Glu Gly Arg He Phe Thr Ala Phe Ser 930 935 940

Leu Tyr Asp Ala Arg Asn Val He Lys Asn Gly Asp Phe Asn Asn Gly 945 950 955 960

Leu Ser Cys Trp Asn Val Lys Gly His Val Asp Val Glu Glu Gin Asn 965 970 975

Asn His Arg ser Val Leu Val Val Pro Glu Trp Glu Ala Glu Val Ser 980 985 990

Gin Glu Val Arg Val Cys Pro Gly Arg Gly Tyr He Leu Arg Val Thr 995 1000 1005

Ala Tyr Lys Glu Gly Tyr Gly Glu Gly Cys Val Thr He His Glu He 1010 1015 1020

Glu Asn Asn Thr Asp Glu Leu Lys Phe ser Asn Cys Val Glu Glu Glu 1025 1030 1035 1040

Val Tyr Pro Asn Asn Thr val Thr Cys Asn Asp Tyr Thr Ala Thr Gin 1045 1050 1055

Glu Glu Tyr Glu Gly Thr Tyr Thr Ser Arg Asn Arg Gly Tyr Asp Gly 1060 1065 1070

Ala Tyr Glu Ser Asn Ser Ser Val Pro Ala Asp Tyr Ala Ser Ala Tyr 1075 1080 1085

Glu Glu Lys Ala Tyr Thr Asp Gly Arg Arg Asp Asn Pro Cys Glu Ser 1090 1095 1100

Asn Arg Gly Tyr Gly Asp Tyr Thr Pro Leu Pro Ala Gly Tyr Val Thr 1105 1110 1115 1120

Lys Glu Leu Glu Tyr Phe Pro Glu Thr Asp Lys Val Trp He Glu He 1125 1130 1135

Gly Glu Thr Glu Gly Thr Phe He Val Asp ser Val Glu Leu Leu Leu 1140 1145 1150

Met Glu Glu

1155

(2) INFORMATION FOR SEQ ID NO:34:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1182 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: 1inear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: Met Asp Asn Asn Pro Asn He Asn Glu Cys He Pro Tyr Asn Cys Leu

1 5 10 15

Ser Asn Pro Glu Val Glu Val Leu Gly Gly Glu Arg He Glu Thr Gly 20 25 30

Tyr Thr Pro He Asp He Ser Leu Ser Leu Thr Gin Phe Leu Leu Ser 35 40 45

Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gly Leu Val Asp He He 50 55 60

Trp Gly He Phe Gly Pro Ser Gin Trp Asp Ala Phe Leu Val Gin He 65 70 75 80

Glu Gin Leu He Asn Gin Arg He Glu Glu Phe Ala Arg Asn Gin Ala 85 90 95

He Ser Arg Leu Glu Gly Leu Ser Asn Leu Tyr Gin He Tyr Ala Glu

100 105 110

Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr Asn Pro Ala Leu Arg Glu 115 120 125

Glu Met Arg He Gin Phe Asn Asp Met Asn ser Ala Leu Thr Thr Ala 130 135 140

He Pro Leu Phe Ala Val Gin Asn Tyr Gin Val Pro Leu Leu ser Val 145 150 155 160

Tyr Val Gin Ala Ala Asn Leu His Leu Ser Val Leu Arg Asp Val Ser 165 170 175

Val Phe Gly Gin Arg Trp Gly Phe Asp Ala Ala Thr He Asn Ser Arg 180 185 190

Tyr Asn Asp Leu Thr Arg Leu He Gly Asn Tyr Thr Asp Tyr Ala Val 195 200 205

Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Trp Gly Pro Asp Ser Arg

210 215 220

Asp Trp Val Arg Tyr Asn Gin Phe Arg Arg Glu Leu Thr Leu Thr Val 225 230 235 240

Leu Asp He Val Ala Leu Phe Pro Asn Tyr Asp Ser Arg Arg Tyr Pro 245 250 255

He Arg Thr Val Ser Gin Leu Thr Arg Glu He Tyr Thr Asn Pro Val 260 265 270

Leu Glu Asn Phe Asp Gly Ser Phe Arg Gly Ser Ala Gin Gly He Glu 275 280 285

Arg Ser He Arg Ser Pro His Leu Met Asp He Leu Asn Ser He Thr

290 295 300 He Tyr Thr Asp Ala His Arg Gly Tyr Tyr Tyr Trp ser Gly His Gin 305 310 315 320

He Met Ala Ser Pro Val Gly Phe ser Gly Pro Glu Phe Thr Phe Pro 325 330 335

Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro Gin Gin Arg He Val Ala 340 345 350

Gin Leu Gly Gin Gly Val Tyr Arg Thr Leu ser ser Thr Leu Tyr Arg 355 360 365

Arg Pro Phe Asn He Gly He Asn Asn Gin Gin Leu Ser Val Leu Asp 370 375 380

Gly Thr Glu Phe Ala Tyr Gly Thr Ser ser Asn Leu Pro Ser Ala Val 385 390 395 400

Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu Asp Glu He Pro Pro Gin 405 410 415

Asn Asn Asn Val Pro Pro Arg Gin Gly Phe Ser His Arg Leu Ser His 420 425 430

Val Ser Met Phe Arg Ser Gly Phe Ser Asn Ser Ser Val Ser lie He

435 440 445

Arg Ala Pro Met Phe Ser Trp He His Arg Ser Ala Glu Phe Asn Asn 450 455 460

He He Ala Ser Asp Ser He Thr Gin lie Pro Ala Val Lys Gly Asn 465 470 475 480

Phe Leu Phe Asn Gly Ser Val He Ser Gly Pro Gly Phe Thr Gly Gly 485 490 495

Asp Leu Val Arg Leu Asn Ser Ser Gly Asn Asn He Gin Asn Arg Gly

500 505 510

Tyr He Glu Val Pro He His Phe Pro Ser Thr Ser Thr Arg Tyr Arg 515 520 525

Val Arg Val Arg Tyr Ala ser Val Thr Pro He His Leu Asn Val Asn 530 535 540

Trp Gly Asn Ser Ser He Phe Ser Asn Thr Val Pro Ala Thr Ala Thr 545 550 555 560

Ser Leu Asp Asn Leu Gin Ser Ser Asp Phe Gly Tyr Phe Glu Ser Ala 565 570 575

Asn Ala Phe Thr Ser Ser Leu Gly Asn He Val Gly Val Arg Asn Phe 580 585 590

Ser Gly Thr Ala Gly Val He He Asp Arg Phe Glu Phe He Pro Val 595 600 605 Thr Ala Thr Leu Glu Ala Glu Tyr Asn Leu Glu Arg Ala Gin Lys Ala 610 615 620

Val Asn Ala Leu Phe Thr Ser Thr Asn Gin Leu Gly Leu Lys Thr Asn 625 630 635 640

Val Thr Asp Tyr His He Asp Gin Val Ser Asn Leu Val Thr Tyr Leu 645 650 655

Ser Asp Glu Phe Cys Leu Asp Glu Lys Arg Glu Leu Ser Glu Lys Val 660 665 670

Lys His Ala Lys Arg Leu Ser Asp Glu Arg Asn Leu Leu Gin Asp Ser 675 680 685

Asn Phe Lys Asp He Asn Arg Gin Pro Glu Arg Gly Trp Gly Gly Ser 690 695 700

Thr Gly He Thr He Gin Gly Gly Asp Asp Val Phe Lys Glu Asn Tyr 705 710 715 720

Val Thr Leu Ser Gly Thr Phe Asp Glu Cys Tyr Pro Thr Tyr Leu Tyr 725 730 735

Gin Lys He Asp Glu ser Lys Leu Lys Ala Phe Thr Arg Tyr Gin Leu 740 745 750

Arg Gly Tyr He Glu Asp Ser Gin Asp Leu Glu He Tyr Leu He Arg 755 760 765

Tyr Asn Ala Lys His Glu Thr Val Asn Val Pro Gly Thr Gly Ser Leu 770 775 780

Trp Pro Leu Ser Ala Gin ser Pro He Gly Lys Cys Gly Glu Pro Asn 785 790 795 800

Arg cys Ala Pro His Leu Glu Trp Asn Pro Asp Leu Asp Cys Ser Cys 805 810 815

Arg Asp Gly Glu Lys Cys Ala His His Ser His His Phe Ser Leu Asp 820 825 830

He Asp Val Gly Cys Thr Asp Leu Asn Glu Asp Leu Gly Val Trp Val 835 840 845

He Phe Lys He Lys Thr Gin Asp Gly His Ala Arg Leu Gly Asn Leu 850 855 860

Glu Phe Leu Glu Glu Lys Pro Leu Val Gly Glu Ala Leu Ala Arg Val 865 870 875 880

Lys Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Glu Lys Leu Glu Trp 885 890 895

Glu Thr Asn He Val Tyr Lys Glu Ala Lys Glu Ser Val Asp Ala Leu 900 905 910 Phe Val Asn Ser Gin Tyr Asp Gin Leu Gin Ala Asp Thr Asn He Ala 915 920 925

Met He His Ala Ala Asp Lys Arg Val His Ser He Arg Glu Ala Tyr 930 935 940

Leu Pro Glu Leu ser Val He Pro Gly Val Asn Ala Ala He Phe Glu 945 950 955 960

Glu Leu Glu Gly Arg He Phe Thr Ala Phe Ser Leu Tyr Asp Ala Arg 965 970 975

Asn Val He Lys Asn Gly Asp Phe Asn Asn Gly Leu Ser cys Trp Asn 980 985 990

Val Lys Gly His Val Asp Val Glu Glu Gin Asn Asn His Arg Ser Val 995 1000 1005

Leu Val Val Pro Glu Trp Glu Ala Glu val Ser Gin Glu val Arg Val 1010 1015 1020

Cys Pro Gly Arg Gly Tyr He Leu Arg Val Thr Ala Tyr Lys Glu Gly

1025 1030 1035 1040

Tyr Gly Glu Gly Cys Val Thr lie His Glu He Glu Asn Asn Thr Asp 1045 1050 1055

Glu Leu Lys Phe Ser Asn cys val Glu Glu Glu Val Tyr Pro Asn Asn 1060 1065 1070

Thr Val Thr Cys Asn Asp Tyr Thr Ala Thr Gin Glu Glu Tyr Glu Gly 1075 1080 1085

Thr Tyr Thr Ser Arg Asn Arg Gly Tyr Asp Gly Ala Tyr Glu Ser Asn 1090 1095 1100

Ser Ser Val Pro Ala Asp Tyr Ala Ser Ala Tyr Glu Glu Lys Ala Tyr

1105 1110 1115 1120

Thr Asp Gly Arg Arg Asp Asn Pro Cys Glu Ser Asn Arg Gly Tyr Gly 1125 1130 1135

Asp Tyr Thr Pro Leu Pro Ala Gly Tyr Val Thr Lys Glu Leu Glu Tyr 1140 1145 1150

Phe Pro Glu Thr Asp Lys Val Trp He Glu He Gly Glu Thr Glu Gly 1155 1160 1165

Thr Phe He Val Asp Ser Val Glu Leu Leu Leu Met Glu Glu 1170 1175 1180

Claims

Claims

1 A composition for controlling lepidopteran pests compnsmg a CrylF chimenc core toxin- containmg protem and a CryLA(c) chimenc core toxin-containing protem, or cells which express these proteins

2 The composition, accordmg to claim 1 , compnsmg a cell expressmg a CrylF chimenc core toxin-containing protem and a cell expressmg a CryΙA(c) chimenc core toxin-containing protem

3 The composition, accordmg to claim 1, compnsmg a cell expressmg a CrylF chimenc core to vui-containing protem and a CryΙA(c) chimenc core toxin-containing protem

4 The composition, accordmg to claim 1, wherem said CrylF chimenc core toxin-containing protem compnses a Cr lF core N-terminal protem portion and a heterologous C-terminal toxm portion from a CrvIA(b) toxm or CryIA(b)/CryIA(c) chimeπc toxm

5 The composition, accordmg to claim 4, wherem said CrylF chimenc core toxin-containing protem has approximately 1150 to 1200 ammo acids and compnses a CrylF core N-terminal sequence of at least about 590 ammo acids and no more than about 1100 ammo acids, wherem said CryIA(b) or CryIA(c)/CrvIA(b) proto m portion compnses at least 100 ammo acids at die C-termmus of said protem

6 The composition, accordmg to claim 4, wherem the transition from Cry IF core N-terminal toxm portion to heterologous protoxm portion occurs after the sequence shown m SEQ ID NO 30 and before the end of die peptide sequence of SEQ ID NO 31

7 The composition, accordmg to claim 6, wherem said core toxm portion compnses the first about 601 ammo acids of a CrylF toxm and wherem said C-terminal protoxm portion compnses the CrvIA(b) or CrvIA(c)/CrvIA(b) ammo acid sequence which follows the peptide sequence shown m SEQ ID NO 31

8 The composition, accordmg to claim 6, wherem said core toxin-containing protem consists essentially of the ammo acid sequence shown m SEQ ID NO 23

9 The composition, accordmg to claim 6. wherem said core to in-containing protem consists essentially of the ammo acid sequence shown m SEQ ID NO 29

10. The composition, according to claim 6, wherein said CrylF chimeric core toxin-containing protein consists essentially of die amino acid sequence shown in Figure 9.

11. The composition, according to claim 1, wherein said CryIA(c) chimeric core toxin- containing protein has an amino acid sequence consisting essentially of die sequence shown in SEQ ID NO. 34.

12. The composition, according to claim 3, wherein said CryEF chimeric and CryIA(c) chimeric core toxin-containing proteins are produced by a host cell transformed to express SEQ ID NO. 23 and SEQ ID NO. 34.

13. The composition, according to claim 3, wherein said CrylF chimeric and CryΙA(c) chimeric core toxin-containing proteins are produced by a host cell transformed to express SEQ ID NO. 29 and SEQ ID NO. 34.

14. A host transformed to express both a CrylF chimeric core toxin-containing protein and a CryΙA(c) chimeric core toxin-containing protein.

15. A method for controlling lepidopteran pests comprising contacting said pests, or die environment of said pests, with an effective amount of a composition comprising a CrylF chimeric core toxin-containing protein and a CryΙA(c) chimeric core toxin-containing protein, or cells which produce tiiese proteins.

16. The method, according to claim 15, wherein said composition comprises a cell expressmg a CrvIF chimeric core toxin-containing protein and a cell expressing a CryΙA(c) chimeric core toxin- containing protein.

17. The method, according to claim 15, wherein said composition comprises a cell expressmg a CrvIF chimeric core toxin-containing protein and a CryΙA(c) chimeric core toxin-containing protein

18. The method, according to claim 15, wherein said Cr lF chimeric core toxin-containing protein comprises a CryEF core N-terminal toxin portion and a heterologous C-terminal protoxin portion from a CryIA(b) toxin or CryIA(b)/CryIA(c) chimeric toxin.

19. The method, according to claim 18. wherein said CrylF chimeric core toxin-containing protein has approximately 1150 to 1200 amino acids and comprises a CrylF core N-terminal sequence of at least about 590 ammo acids and no more than about 1100 ammo acids, wherem said CryIA(b) or CryIA(c)/CryIA(b) protoxm portion comprises at least 100 ammo acids at die C-terminus of said protem

20 The method, accordmg to claim 18, wherem die transition from CrylF core N-terminal toxm portion to heterologous protoxm portion occurs after the sequence shown m SEQ ID NO 30 and before the end of die peptide sequence of SEQ ID NO 31

21 The method, accordmg to claim 20, wherem said core toxm portion compnses die first about 601 ammo acids of a CrylF toxm and wherem said C-terminal protoxm portion compnses die CryIA(b) or CryIA(c)/CryIA(b) ammo acid sequence which follows the peptide sequence shown m SEQ ID NO 31

22 The method, accordmg to claim 20, wherem said core toxm-contaimng protem consists essentially of the ammo acid sequence shown in SEQ ID NO 23

23 The method, accordmg to claim 20, wherem said core toxin-containing protem consists essentially of die ammo acid sequence shown m SEQ ID NO 29

24 The method, accordmg to claim 20, wherem said CrylF chimenc core toxin-containing protem consists essentially of an ammo acid sequence shown m Figure 9

25 The mediod, accordmg to claim 18, wherem said CryΙA(c) chimeπc core toxin-containing protem has an ammo acid sequence consisting essentially of die sequence shown m SEQ ID NO 34

26 The mediod, accordmg to claim 18, wherem said Cπ IF chimeπc and CryIA(c) chimenc core toxin-containing protems are from a host cell transformed to express SEQ ID NO 23 and SEQ ID NO 34

27 The mediod. accordmg to claim 13. wherem said CrvIF chimeπc and CryΙA(c) chimenc core

are from a host cell transformed to express SEQ ID NO 29 and SEQ ID NO 34